br Immunofluorescence br Immunofluorescence was performed to stain E
Immunofluorescence was performed to stain E-cadherin and vi-mentin at a primary antibody dilution of 1:200. The slides were in-cubated with Alexa Fluor®488 or Alexa Fluor®594 (Proteintech, IL, USA), and the nucleus was counter-stained with DAPI-mounting media (Sangon Biotech, Shanghai, China). The stained sections were visua-lized under a fluorescence microscope.
2.13. Prussian blue staining
A549 cells were grown in 6-well plate at density of 2 × 105 per well on coverslips. After 24 h’ incubation with treatments, the cells were fixed with 4% paraformaldehyde for 10 min, washed with PBS, and stained with Prussian blue. Reagent A (Perls stain A: Perls stain B = 1:1; 200 μL) was added onto the slides and incubated for 30 min. Their nuclei were stained with reagent B for 30 s, and intracytoplasmic blue granules were observed with a microscope.
2.14. Tumor xenografts in nude mice
All the animal experiments were conducted and approved by the Institutional Animal Care and Use Committee of Weifang Medical University. A549 cells (3 × 106) were injected subcutaneously into the right flank of six-week-old BALB/c male nude mice. When the tumor volume reached 100 mm3, the mice were randomly divided into four groups: control group, iron oxide-based MNP-treated group (0.5 mg/ kg), CAP-treated group, and dual CAP- and iron oxide-based MNPs-treated group. Treatment of iron oxide-based MNPs on mice is 20 μL of iron oxide-based MNPs by intratumoral injection. CAP treatments on mice were performed in open air, reactor head being at a distance of two millimeters from the skin. The established xenografts were treated
Fig. 5. CAP individually or in combination with iron oxide-based MNPs arrest AR-13324 and promote apoptosis: (a) cell apoptosis and necrosis was analyzed by PI and annexin V-FITC staining when A549 treated by CAP and iron oxide-based MNPs, (b) flow cytometry was performed to analyses the cell cycle, and (c) pro-liferation of A549 cell was determined by colony formation assay.
irradiated with CAP directly for 2 min per day (total 10 days). The tumor dimensions were measured using an electronic Vernier caliper every 2 days, and the tumor volume (V) was calculated by using an improved ellipsoid equation: V = (a × b2)/2, (a, longitudinal diameter; b, latitudinal diameter).
2.15. Statistical analysis
Data were presented as mean ± SD, and Student’s t-test was per-formed for comparisons. P values were obtained with SPSS 18.0 (SPSS, Chicago, IL, USA). The level of significance was defined as P < 0.05.
Iron oxide-based MNPs were monodispersed with a nearly spherical shape, and TEM confirmed that the average size of iron oxide-based MNPs was 200 nm with a narrow size distribution (Fig. 2a). Dynamic light scattering assay was used to measure the size of iron oxide-based MNPs when they were monodispersed in the culture medium at ap-proximately 210 nm in diameter (Fig. 2b). In aqueous solutions, iron oxide-based MNPs were stable without aggregating for at least 12 months, and their Zeta potential was −31.3 ± 5.46 mV (Fig. 2c). A
magnetic hysteresis loop at 300 K demonstrated that the saturation magnetization of the iron oxide-based MNPs was approximately 50 emu/g (Fig. 2d). The endocytosis of iron oxide-based MNPs in A549 cells was shown by Prussian blue staining after these cells were in-cubated with MNPs for 12 h. The results showed that A549 cells could phagocytose MNPs with blue-stained cytoplasm (Fig. 2e).
The metabolic viability of A549 cells was determined by MTT assay, and the results indicated that the treatment with CAP significantly decreased the cell viability in a time-dependent manner (Fig. 3a). The dose-dependent inhibitory eﬀects of iron oxide-based MNPs on the cells were also observed, and low doses of iron oxide-based MNPs eﬃciently attenuated cell viability (Fig. 3b). To determine the synergistic eﬀect of iron oxide-based MNPs and to enhance CAP cytotoxicity, we de-termined the viability of A549 cells exposed to dual treatment. The results revealed that the enhancement eﬀect of proliferation inhibition was achieved when the cells were exposed to various concentrations of iron oxide-based MNPs and 150 s CAP, showing a significantly strong synergistic eﬀect (Fig. 3c).
It is known that CAP-treated cancer cells induced increase in in-tracellular ROS . In our study, the fluorescence microscopic images of the A549 cells stained with DCFH-DA showed a high level of ROS intensity after treatment of CAP or iron oxide-based MNPs individually
Fig. 6. Inhibition of cell migration and invasion by treatment of iron oxide-based MNPs and CAP: (a) wound healing assay was performed on A549 cells treated by iron oxide-based MNPs and CAP, and (b) invasive phenotype of A549 cells via treatment of iron oxide-based MNPs and CAP was confirmed with Transwell assay.