Superparamagnetic iron oxide nanoparticles enhance glioma radiosensitivity via inducing cell cycle arrest and apoptosis


 Aim: Superparamagnetic iron oxide nanoparticles (SPIONs) is a widely used biomedical material for imaging and targeting drug delivery. We synthesized SPIONs and tested their effects on the radiosensitization of glioma.Methods: Acetylated 3-aminopropyltrimethoxysilane (APTS)-coated iron oxide nanoparticles (Fe3O4 NPs) were synthesized via a one-step hydrothermal approach and the surface was chemically modified with acetic anhydride to generate surface charge-neutralized NPs. NPs were characterized by TEM and ICP-AES. Radiosensitivity of U87MG glioma cells was evaluated by MTT assay. Cell cycle and apoptosis in glioma cells were examined by flow cytometry. Results: APTS-coated Fe3O4 NPs had a spherical or quasi-spherical shape with average size of 10.5±1.1 nm. NPs had excellent biocompatibility and intracellular uptake of NPs reached the peak 24 hours after treatment. U87 cell viability decreased significantly after treatment with both X-ray and NPs compared to X-ray treatment alone. Compared to X-ray treatment alone, the percentage of cells in G2/M phase (31.83%) significantly increased in APTS-coated Fe3O4 NPs plus X-ray treated group (P<0.05). In addition, the percentage of apoptotic cells was significant higher in APTS-coated Fe3O4 NPs plus X-ray treated group than in X-ray treatment alone group (P<0.05). Conclusion: APTS-coated Fe3O4 NPs achieved excellent biocompatibility and increased radiosensitivity for glioma cells.


Introduction
Glioma is the most common malignant primary brain tumor in the adults and occurs about ve cases per 100,000 population per year 1,2,3 . Despite the advances in surgery therapy, radiotherapy and chemotherapy, the prognosis of glioma is still poor, and glioma recurs in most patients within 1 to 5 year 4,5 . Radiotherapy (RT) could penetrate into tissues deeply and it is important to improve radiation delivery technology to minimize its secondary damage to the surrounding normal tissues 6,7 . The side effects of RT such as granulocytopenia or radiation encephalopathy cause trouble to many patients 8,9 . Therefore, many nanoparticles such as water-soluble carbon nanotubes, fullerene-C60, silver and golden nanoparticle have been developed to decrease the side effects, and at the same time, radiosensitivity is increased [10][11][12][13][14] .
Radiosensitization of high atomic number (Z) metallic nanoparticles such as golden nanoparticle and sliver nanoparticle has been reported. X-ray beams with high energies in the presence of high-atomic number nanoparticles lead to not only secondary electrons including auger electrons but also high-energy electrons produced by coulomb interaction of electron and nuclei eld. Either electrons or photons disperse in the target volume and their absorption will increase in the presence of high-Z metallic nanoparticles, eventually killing tumor cells. Superparamagnetic iron oxide nanoparticles (SPIONs) as a kind of high-Z nanoparticle have attracted broad interest and intense attention because of the potential as contrast agents for magnetic resonance imaging (MRI) and other biomedical applications such as multimodal biomedical imaging and targeting drug delivery 15,16 . SPIONs are mono-crystalline nanoparticles with properties of superparamagnetism and biocompatibility 17,18,19 . Superparamagnetism could guide SPIONs to the targeting area through an external autoclaved inhomogeneous magnetic eld.
In addition, biocompatibility and effective biodistribution of SPIONs allow for their broad medical application 20 . In this study, we aimed to develop SPIONs for glioma radiotherapy. We synthesized SPIONs and tested their effects on the radiosensitization of glioma.

Materials And Methods
NPs synthesis 3-aminopropyltrimethoxysilane (APTS) coated Fe 3 O 4 NPs were synthesized using hydrothermal approach as described previously 21 . Brie y, 1 g FeCl 2 ·4H 2 O was dissolved in 6.2 ml distilled water, then 5 ml ammonium hydroxide was added and the suspension was continuously stirred at room temperature for 10 min. Then, 2 ml APTS was added and the mixture was autoclaved at 134℃ for 3 h. Then the mixture was cooled and puri ed with distilled water, then centrifuged (5,000 rpm, 10 min) to remove excess reactants. The process was repeated for ve times to get APTS-coated Fe 3 O 4 NPs.
The amine groups were connected on the surface of APTS-coated Fe 3 O 4 NPs via a reaction with acetic anhydride. In brief, 6 mg APTS-coated Fe 3 O 4 NPs was dispersed in 5 ml ethanol and mixed with 1 ml of triethylamine. 5 ml dimethyl sulfoxide (DMSO) containing 1 ml acetic anhydride was then added and mixed for 24 h. Then the mixture was puri ed with distilled water, then centrifuged to remove excess reactants. The process was repeated for three times to get acetylated APTS-coated Fe 3 O 4 NPs.

Characterization of NPs
The morphology of NPs was observed under JEO2010 F transmission electron microscope (Akishima-shi, Japan) operated at 200 kV. The size of NPs was measured using Image J image analysis software. The size distribution histogram was obtained via different TEM images. Fe concentration of APTS-coated Fe 3 O 4 NPs was tested with Prodigy inductively coupled plasma-atomic emission spectroscopy (ICP-AES) (PerkinElmer Optima 8000, USA).

The intracellular uptake of NPs
The intracellular uptake of APTS-coated Fe 3 O 4 NPs was quanti ed via ICP-AES analysis with Prodigy ICP-AES system (PerkinElmer Optima 8000, USA). U87 cells were seeded in six-well plates at density of 1×10 6 per well and cultured for 24 h, then incubated with different concentrations of acetylated APTS-coated

MTT assay
The viability of U87 glioma cells was tested via MTT assay as described previously 22 . Brie y, U87 cells were seeded into a 96-well plate with 1×10 4 per well and incubated for 24 h. Next the cells were incubated with different concentrations of acetylated APTS-coated Fe 3 O 4 NPs (0, 1, 10, 20, 40, and 80 μg/ml) for another 24 h, then treated with or without irradiated with different dose of X-ray (0,2,4,6,8,10 Gy) and cultured for 24 h. 20 μl of 5 mg/ml MTT solution was added to each well. After 4 h of incubation, the medium was removed and 200 μl DMSO was added. The absorbance was measured in a BioTek Elx800 (Thermo Scienti c, Waltham, MA, USA) at a wavelength of 490 nm. The inhibition of cell growth was calculated with following formula: Cell viability (%) = (mean of Abs. value of treatment group/mean of Abs. value of control group)×100%. All treatments were carried out in triplicate.

Apoptosis assay
Apoptotic cells were detected by double-staining with annexin V-uorescein isothiocyanate (FITC) and propidium iodide (PI). U87 cells were treated with APTS-coated Fe 3 O 4 NPs or/and X-rays as described

Statistical analysis
Values were expressed as the mean ± standard deviation (SD) and analyzed by SPSS version 13.0.
Statistical analysis was carried out using t test or χ 2 test. P<0.05 was considered signi cant.

Characterization of APTS-coated Fe 3 O 4 NPs
The acetylated APTS-coated Fe 3 O 4 NPs were synthesized, and then were characterized by TEM. TEM images indicated that the particles had a spherical or quasi-spherical shape (Fig. 1A). The size distribution of APTS-coated Fe 3 O 4 NPs characterized by TEM was shown in Fig. 1B. The average size of APTS-coated Fe 3 O 4 NPs was 9.8±1.1 nm (Fig. 1B). In addition, DLS technique was employed to show that dominant fraction of NP diameter was about 13 nm (Fig. 1C). The average particle sizes determined by TEM and DLS were consistent.
The intracellular uptake of NPs ICP-AES analysis showed that intracellular iron content increased in U87 cells with increased concentration of acetylated APTS-coated Fe 3 O 4 NPs in culture medium ( Fig. 2A). In addition, intracellular iron content increased with the prolongation of treatment time and reached peak at 24 h after treatment with acetylated APTS-coated Fe 3 O 4 NPs, and then gradually decreased over time (Fig. 2B). Therefore, in the following experiments 24 h was selected as the appropriate time point.

NPs increased glioma cell radiosensitivity
MTT assay showed that different concentrations of acetylated APTS-coated Fe 3 O 4 NPs had minimal effects on the growth of U87 cells (Fig. 3A). These results indicated good biocompatibility of NPs. On the other hand, X-ray decreased U87 cell viability in a dose dependent manner (Fig. 3B). 4 Gy X-ray inhibited cell viability by about 25%, and was used in the following experiments. As shown in Fig. 3C, cell viability decreased signi cantly in cells treated with both X-ray and NPs compared to cells treated with X-ray alone. These data indicated that NPs increased glioma cell radiosensitivity.  Table 1). These results indicated that NPs promoted radiation induced apoptosis of glioma cells.

Discussion
The presence of macromolecules such as serum proteins may affect the stability of NPs and cause the agglomeration and sedimentation of NPs, impairing biological effects of NPs and even inducing side effects 19,20 . Therefore, it is important to establish compatible physiologically or chemical conditions to improve nanoparticle stability for biomedical applications. The amine groups on the surface of APTS- TEM images showed that the mean diameter of NPs was 10.5 ± 1.1 nm, with a narrow distribution without obvious agglomeration. NPs with diameter in such range can be easily engulfed by cells as demonstrated in other studies 13,18,19 . In this study, cellular uptake of acetylated APTS-coated Fe 3 O 4 NPs was quanti ed via ICP-AES, and iron uptake by glioma cells was concentration dependent. MTT assay showed that acetylated APTS-coated Fe 3 O 4 NPs had no signi cant cytotoxicity to glioma cells, but increased the cytotoxicity of X-ray in glioma cells.
G0/G1 is the cell cycle phase that is most resistant to radiotherapy while G2/M is the phase that is most sensitive to radiotherapy 22,23 . To reveal the mechanism by which acetylated APTS-coated Fe 3 O 4 NPs increased radiosensitivity of glioma cells, we examined cell cycle and apoptosis in different treatment groups. The results showed that G2/M ratio increased signi cantly and G0/G1 ratio decreased signi cantly in NPs and X-ray combined treatment group compared to NPs or X-ray alone treatment group. In addition, apoptosis ratio increased signi cantly in NPs and X-ray combined treatment group compared to NPs or X-ray alone treatment group. These data indicate that APTS-coated    Cell viability tested by MTT assay. A. U87 cell viability after treatment with NPs at different concentrations. B. U87 cell viability after treatment with X-ray at different doses. *P<0.05 compared to 0 Gy. C. U87 cell viability after treatment with NPs or/and X-ray. *P<0.05 compared to X-ray alone.