Synthesis, cytotoxicity and antitumour mechanism investigations of polyoxometalate doped silica nanospheres on breast cancer MCF-7 cells

Polyoxometalates (POMs) have shown the potential anti-bacterial, anti-viral and anti-tumor activities. In order to improve their physiological stability and antitumour activity for medical application, K2Na[AsIIIMo6O21(O2CCH2NH3)3]·6H2O doped silica nanospheres (POM@SiO2) with diameters of ~40 nm have been synthesized by the water-in-oil microemulsion method in this study. The obtained spheres were morphologically uniform nanosized and nearly monodispersed in solution. The nanoparticles had high entrapment efficiency, which was upto 46.2% by the inductively coupled plasma mass spectrometry (ICP-MS) analysis and POMs slowly released from the nanospheres both in the PH 7.4 and 5.5 phosphate buffer saline (PBS) solutions in 60 h. The in vitro MTT assays of particles on MCF-7 cell line (a human breast adenocarcinoma cell line) exhibited enhanced antitumor activity compared to that of plain polyoxometalate. The IC50 value of the POM@SiO2 nanoparticles was 40.0 μg/mL at 24 h calculated by the encapsulated POM concentration, which was much lower comparing to that of 2.0 × 104 μg/mL according to the pure POM. And the SiO2 shells showed low inhibitory effect at the corresponding concentration. Confocal images further indicated the cell morphology changes and necrosis. Flow cytometric analysis showed nanoparticles induced the apoptosis by arresting the cells in S phase and western blot analysis indicated they promoted apoptosis by inhibiting the Bcl-2 protein. Moreover, the study of interactions between human serum albumin (HSA) and the nanoparticles indicated the fluorescence quenching was static, and the nanoparticles were likely to bind to HSA and changed its conformation.


Introduction
Cancer is a serious public health problem which threatens human health in the world due to the increasing incidence and mortality. And breast cancer is one of the most common types a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 various endogenous and exogenous compounds [37,38]. Although the antitumor activity of POMs has been investigated intensively, the binding actions between core/shell POMs decorated silica nanoparticles and HSA remain limited.
In our previous work, the in vitro antitumor activity of K 2 Na[As III Mo 6 O 21 (O 2 CCH 2 NH 3 ) 3 ]Á 6H 2 O (AsMo 6 ) on lung adenocarcinoma cells (A549) was investigated by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) assay, the results indicated that the inhibitory effect was higher than that of 5-fluorouracil (5-FU) [39]. And in the study of anti-leukemia, the compound showed low inhibitory effect on normal human umbilical vein endothelial cells (HUVEC) with an IC 50 value of 889.18 μM and a significant anticancer effect on human HL-60 and U937 leukemia cells [40]. Herein, we aim to evaluate the cytotoxicity of pure POMs and nanoparticles on MCF-7 cell line. The K 2 Na[As III Mo 6 O 21 (O 2 CCH 2 NH 3 ) 3 ]Á6H 2 O doped silica nanospheres (POM@SiO 2 ) was synthesized by reverse microemulsion method and confirmed by TEM and FT-IR analysis. Furthermore, their release profile was evaluated by ICP-MS. And the morphological changes of MCF-7 cells were detected by confocal laser scanning microscope. In addition, at physiological conditions, the binding interactions of the nanoparticles with HSA were investigated by multispectroscopic techniques.

Reagents and materials
All reagents were obtained from commercial supplies and used without further purification. The K 2 Na[AsMo 6 O 21 (O 2 CCH 2 NH 3 ) 3 ]Á6H 2 O (POM), was prepared according to the previously described approach [40]. RPMI-1640 Medium and Fetal bovine serum (FBS) were purchased from Hyclone, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), phosphate buffer saline (PBS) and HSA were obtained from Sigma-Aldrich. The Propidium Iodide (PI) and Hoechst 33342 dye were obtained from Dingguo Chemicals Company. The solution of HSA was prepared by dissolving the reagent in PBS and stirred for 24 h to get a homogeneous solution. RIPA lysis buffer (Solarbio, China) and antibodies (Abcam) were prepared for the western blot analysis.

Preparation of POM@SiO 2 nanoparticles
The POM@SiO 2 nanoparticles were prepared by water-in-oil microemulsion method according to the literature with a little modification [41]. A W/O microemulsion containing 9.29 mL cyclohexane, 2.26 mL n-octanol, 2.23 mL Triton X-100 and 200 μL tetraethoxysilane (TEOS) was prepared in a breaker, in the meantime, the POM (0.024g) suspended in 1 mL H 2 O was mixed with the other microemulsion containing 9.29 mL cyclohexane, 2.26 mL n-octanol, 2.23 mL Triton X-100 and 200 μL ammonia. The two microemulsions were mixed under moderate stirring at room temperature for 24 h, after that, acetone was added to precipitate the nanoparticles. The obtained white solid was washed with ethanol after centrifuging at 8000 rpm. The POM@SiO 2 nanocomposites were dried in a desiccator under vacuum.

Characterization of POM@SiO 2 nanocomposites
The morphology of nanocomposites were analyzed by a transmission electron microscopy (TEM, JEM-1011) operating at an acceleration voltage of 100 kV and the POM-based particles were deposited on carbon-coated copper grids. The hydrodynamic diameter of nanoparticles was determined by dynamic light scattering (DLS) (Brookhave) analysis. The IR peaks of the nanoparticles were identified by fourier transform infrared spectroscopy (Perkin Elmer Spectrum RX-1) with the frequency range spanning from 2500 to 400 cm −1 . The samples were prepared in the form of potassium bromide (KBr) pellets.

Entrapment and in vitro release of POMs studies
The upload efficiency and in vitro release of POMs within the nanoparticles was determined by inductively coupled plasma mass spectrometry (ICP-MS) analysis. The in vitro release of POMs was performed in phosphate buffered saline (pH 5.5 and pH 7.4) at room temperature. The nanoparticles were incubated in 1.5 mL PBS buffer solution, at predetermined time intervals, the nanocomposites were centrifuged at 8000 rpm for 5 min, then 0.5 mL of the supernatant was taken out. After that, the pellet was redispersed and the same volume of PBS was supplemented. The supernatant was analyzed by the ICP-MS analysis. The entrapment efficiency was calculated by all the AsMo 6 released from the SiO 2 shell. It was determined by the following formulae: where Mnp is the mass of AsMo 6 in the nanocomposites, Mf is the mass of POMs used in the formulation.

Cytotoxicity studies
The breast cancer cells MCF-7 (provided by the department of the 1 st hospital, Jilin University) were cultured in RPMI 1640 medium (Hyclone) supplemented with 10% (V/V) fetal bovine serum (FBS, Hyclone) and 50 U/mL penicillin-streptomycin in 5% carbon dioxide atmosphere at 37˚C in an incubator. The medium was changed every other day and the cells were sub-cultured after attaining about 80% confluency. The antitumor activity of the nanoparticles was tested by the MTT assay. Briefly, The MCF-7 cells were seeded into a 96-well plate at a density of 1×10 4 cells/well. After incubating the cells for 24 h, the dilutions of POM@SiO 2 nanocomposites at different doses (containing real concentrations of POM 2.5-80 μg/mL at specific intervals) were added and incubated for 24 h, 48 h and 72 h. After that, the culture medium was discarded and the wells were washed with PBS twice, followed by the addition of 20 μL MTT dye (0.5 mg/mL) each well. The cells were incubated for another 4 h at 37˚C. After removing all the culture medium, 150 μL DMSO was added per well. The percentage of cell viability was measured on a microplate reader (Biotek Co., USA) at the wavelength of 490 nm. The cell inhibitory rate was calculated using the following equation:

Morphological observation
To observe the morphological changes on MCF-7 cells by the nanoparticles, cells were seeded in confocal culture dishes for 24 h at 37˚C, after that, cells were treated with POM@SiO 2 nanocomposites. The cells were then stained with Hoechst 33342/PI solution (1:1, V/V) at room temperature for 10 min in the dark. The cellular morphology was observed using the fluorescence confocal microscopy (Olympus FV1000, Japan).
Flow cytometry analysis of cell apoptosis MCF-7 cells were placed on a 12-well plate for 24 h. The nanoparticles solution with a corresponding POM concentration of 80 μg/mL was then added and incubated at 37˚C. After 24 h, the cells were harvested and then washed three times with cold PBS. After centrifugation, the supernatant was discarded, and the pellets were resuspended in Annexin-V-FITC/PI buffer and left in the dark for 15 min at room temperature. Cells were analyzed on a flow cytometer using Cell Quest Software. Only single cells were gated for fluorescence analysis.

Flow cytometry analysis of cell cycle distribution
Briefly, MCF-7 cells were seeded into a 12-well plate (1 × 10 6 cells per well) at 37˚C for 24 h. After that, they were exposed to different concentrations of the nanoparticles (corresponding POM concentrations of 80, 40 and 20 μg/mL). The cells were harvested using trypsin and collected by centrifugating at 1200 rpm, then, washed three times with cold PBS. After that, Triton X-100 (0.1%, 100 μL), DNase free RNase (200 μg/mL, 100 μL) and propidium iodide (50 μg/mL, 200 μL) were added to the cells. The cells were incubated for 30 min at room temperature in dark.

Western blot
After treatment of POM@SiO 2 nanoparticles for 24 h, total cellular proteins were prepared using RIPA lysis buffer from MCF-7 cells. The protein concentrations were established by bicinchoninic acid (BCA) assay. Equal amount of protein was separated by 12% polyacrylamide gels (SDS-PAGE) and then transferred onto nitrocellulose membranes. The membranes were blocked with 5% milk at 4˚C overnight and then incubated with specific primary antibodies. After washing with TBST (containing 0.1% Tween 20) 3 times, the membranes were incubated with the corresponding HRP-conjugated secondary antibodies in TBST at 37˚C for 1 h. The protein β-actin was used as a housekeeping control for normalization. Finally, the expression levels of proteins were visualized and analyzed using ImageJ software.

HSA binding experiments
UV-vis absorption spectra. The absorption spectra of 4 mg/mL POM@SiO 2 nanoparticles, 2.5 μM HSA and the mixture of them were measured by a UV spectrophotometer at room temperature. The absorption spectra was set from 190 to 600 nm, 10.0 mm optical path length quartz cuvette was used in this test.
Fluorescence titration experiments. The absorption titration experiments were performed by sequential addition of specified volume of POM@SiO 2 nanoparticles stock solution into HSA solution. Quartz cuvette with 10.0 mm optical path length was used. Excitation of the sample took place at a wavelength of 290 nm and the emission spectra were recorded from 300 to 450 nm. The concentration of HSA was 2.5 μM, whereas the nanocomposites concentrations increased stepwise from 0 to 0.04 μg/mL. At the same time, by fixing the excitation wavelength and emission wavelength at a Δλ value of 60 nm, the synchronous fluorescence intensity of the mixture solution (POM@SiO 2 nanoparticles and HSA) was detected. The widths of the slits were all 5.0 nm.

Statistical analysis
All statistical analyses were performed using the SPSS Version 17.0 for Windows (significance was established at P < 0.05). Data were expressed as mean ± SD (standard deviation), statistical significance was evaluated by one-way analysis of variance (ANOVA) combined with Duncan's multiple range tests. All experiments were performed in triplicate, unless otherwise indicated.

Synthesis and characterization of the POM@SiO 2 nanoparticles
The POM@SiO 2 nanoparticles were obtained by the hydrolysis of tetraethoxysilane (TEOS) method (Fig 1). The TEM image showed morphological uniform nanosized spheres (Fig 2A), which provided direct evidence of the formation. It is clearly evident from the image that nearly monodispersed POM@SiO 2 nanoparticles were obtained by this technique, the average particle diameters were about 39 nm. The POMs were encapsulated in SiO 2 with an evident core/shell structure. EDX mapping confirmed the presence of arsenic and molybdenum from POM and the presence of silica from the shell (S1 Fig). As shown in Fig 2B that the hydrodynamic diameter of POM@SiO 2 nanoparticle solution was between 30 and 52 nm by the dynamic light scattering (DLS) measurements. The image was supported by the above transmission electron microscopy (TEM) observation. Particles between 33 and 43 nm reached about 48%. These results also demonstrated the reduction of POM aggregation due to the encapsulation. The FT-IR spectra (Fig 2C) of the POM exhibited the absorption characteristic peaks at 928, 688, 538 and 426 cm -1 , which were attributed to ν(Mo-O bridge -Mo) and ν(Mo-O terminal ) of polyoxoanion as described in the previous study [39], the corresponding peaks were exhibited in the POM@SiO 2 solid line as shown in the image (860, 617, 546, 430 cm -1 ), indicating that these compounds still retain the basic structure after encapsulated in SiO 2 . The strong absorption band at 1110 cm -1 as shown in the POM@SiO 2 line was attributed to the Si-O stretching, which further demonstrated the formation of POM@SiO 2 nanoparticles.
To characterize the release of POMs, the POM@SiO 2 nanoparticles were incubated at pH values of 5.5 and 7.4 PBS solutions. The nanoparticles were centrifuged at predetermined time intervals and analyzed by ICP-MS. From these releasing profile studies (Fig 2D), the AsMo 6 reached about 34% and 42%, respectively. The release efficiency increased stepwise during the intervals and the SiO 2 resulting in the controlled release from the shells. Efficiency at pH 5.5 was a little higher than that at pH 7.4. Entrapment efficiency was calculated by all the POMs which released from the nanocomposites. The entrapment efficiency of POM within the nanoparticles was calculated to be 42.6%.

Anticancer activity studies
To determine whether the doped POMs nanoparticles could enhance their antitumour activity, experiments were performed in vitro at different concentrations by MTT assay on MCF-7 cells. As shown in Fig 3, the nanoparticles showed significant cytotoxicity to MCF-7 cells at a concentration of 40 μg/mL (calculated by the encapsulated POM) with the cell viability about 51.9%, the anti-proliferative effects were dependent on its concentration. The cell viability was down to 31.3% at 24 h, 1.7% at 48 h and still kept about 2.0% at 72 h with POM concentration of 80 μg/mL that contained in the nanoparticles. The IC 50 value of the corresponding POM concentration in the POM@SiO 2 nanoparticles was calculated to be 40 μg/mL at 24 h, 10.8 μg/ mL at 48 h, 1.7 μg/mL at 72 h. Particles showed antitumor activity in a time dependent manner in these three periods. For one reason, the cells took more particles and the drug exhibited more antitumor effect with longer time during a specific time frame, which could enhance the The cell viability was 79.9%, 69.5%, 66.5% comparing to that of 63.2%, 51.8% and 31.3% as shown in results of the nanoparticles. It can be concluded that the antitumor activity of the POM@SiO 2 nanoparticles is higher than the free POM .

Hoechst 33342/PI double staining assay
Hoechst 33342/PI double staining assay was performed to detect the necrotic cell by confocal laser scanning microscopy (Fig 4). The nuclei of normal cells can be stained into light blue (Hoechst 33342), apoptotic cells can be stained into brilliant blue and light red (Hoechst 33342 and PI) and the dead cells were brilliant red (PI). The results were presented in the CLSM images. In the control group, nuclei were all stained into blue and no red cells appeared in the image. In the presence of POM@SiO 2 nanoparticles, the cell numbers reduced dramatically and there were a large number of necrotic cell residues (stained into red by PI). Massive necrotic and apoptotic cells were found in the drug group at the corresponding POM concentration of 40 μg/mL. The number of dead cells were increased with the adding of nanoparticles. Therefore, it can be concluded that the nanoparticles inhibit this cell line growth significantly. The changes of nuclei and cells morphology become much clearer under the magnification of 60 (S4 Fig). Cells in the control groups nested very well and grew strongly at a high density (bright images of DIC channel). Comparing to the intact and polygonal shape in the control group, cells exposed to the nanocomposites showed shrink margin loss of contact with the adjacent ones and apparently the sharp decrease in amount. Hoechst 33342/PI cytological investigations elucidated the membrane instability and cytoskeleton disturb effect by the nanoparticles.

Flow cytometry analysis of cell apoptosis
To further confirm and detect the apoptosis effect on MCF-7 cells induced by the nanoparticles, Annexin V-FITC /propidium iodide (PI) double-staining technique was used. It is sensitive to detect the early apoptosis phase cells based on distinct double staining patterns: viable (Annexin V-and PI-, lower left square), early apoptotic (Annexin V+ and PI-, lower right square), late apoptotic (Annexin V+ and PI+, upper right square) and necrotic cells (Annexin V-and PI+, upper left square). The results (Fig 5) showed that the proportion of late apoptotic cells increased with the addition of the nanoparticles. The proportion of late apoptotic cells induced by the nanoparticles is nearly 34% higher than the control group (5.71% in control group, 39.47% in drug group). Taken together, the POM@SiO 2 nanoparticles induced the apoptosis of MCF-7 cells and inhibited their proliferation, which were consistent with the MTT results mentioned above.

Flow cytometric analysis for cell cycle distribution
To detect whether the growth inhibition of MCF-7 cells by nanoparticles is a result of cell cycle arrest, MCF-7 cells were seeded and exposed to different concentrations of the POM@SiO 2 nanoparticles (corresponding POM concentrations of 20, 40, 80 μg/mL) for 24 h, stained with PI and examined by FCM to analyze the changes of cell cycle. Generally, a doubling of DNA and other cellular contents are necessary for a cell replication process. Cell cycle distribution is divided into four distinct phases: G 1 phase, S phase, G 2 phase and M phase, which are defined by cell entry checkpoints. The major event of S phase is the synthesis of DNA. The cell prepares to divide during G 2 phase and division takes place during M phase. Passing these checkpoints, the cell will proceed with division. External factors such as drugs (5-fluorouracil), radiation, and reactive oxygen species result in DNA-damage-related cell death during S phase [42,43]. The abnormality of DNA prevents the progress of replication and influences the next new one. Then the DNA repair occurs during the G 2 phase, before entering the M phase for mitosis. For the cell cycle analysis, as shown in Fig 6, the results showed increase level in S phase from 26.73% in the control group to 30.41%, 33.58%, 32.81%, respectively with the corresponding reduction in G 1 and G 2 /M phase. The level of G 1 phase was 66.1%, 62.04%, 59.86% and 59.58%, respectively, which suggested the inhibition mechanism on MCF-7 cells was S phase arrest. Since DNA replication occurs during S phase, DNA damage is responsible for S phase arrest as well as the inhibition of proliferation versus the control group.

Western blot
The flow cytometry analysis indicated that the apoptosis rate increased in MCF-7 cells as the POM@SiO 2 nanoparticles concentration increased. To further confirm the effects of nanoparticles in MCF-7 cells, western blot assay was performed to investigate the molecular mechanism for the observed apoptosis. As shown in Fig 7, a remarkable upregulation of cleaved caspase 3 protein levels and a decrease of Bcl-2 protein levels were observed as the nanoparticle concentration increased. As we know Bcl-2 is an apoptosis inhibitor which could restrain cell apoptosis. The cleaved caspase 3/β-actin ratio in MCF-7 cells treated with nanoparticles (corresponding POM concentration of 80 μg/mL) was 0.616, which was significantly higher than that of the blank control. Therefore, our findings showed that nanoparticles could promote apoptosis by inhibiting the Bcl-2 protein and upregulating the cleaved caspase 3 expression in cells. HSA binding UV-vis absorption. UV-vis measurement is an easy and effective way to investigate the structural changes and complex formation between the protein and nanoparticles [44]. As shown in Fig 8, the characteristic absorption peaks of isolate HSA were 206 nm and 278 nm, meanwhile isolate POM@SiO 2 nanoparticles were found near 200 nm at the same concentration. With the addition of nanoparticles solution, the absorption peak of the protein was slightly increased, which indicated that an interaction occurred between HSA and POM@SiO 2 nanoparticles.

Fluorescence spectroscopic experiments
The fluorescence measurements can be applied to investigate the binding information of small molecule substances to protein, such as the binding mechanism, binding constants, binding mode, and the number of binding sites. Fluorescence quenching is the decrease of quantum yield from a fluorophore induced by quencher molecule, there are variety molecular interactions between them, such as excited-state reaction, energy transfer and collision quenching. That means the fluorescence changes can give information about the molecular micro-environment. And in another level, changes in emission spectra of residues is often considered in the investigation of protein folding and association reactions [5,45]. Fluorescence quenching is a well-established method to study the interactions between ligand and protein. It has been used to analyze the interactions between different POMs and HSA [46][47][48][49][50]. But all of these investigations were previously performed between plain POMs and protein. In this study, the interaction between POM uploading silica nanoparticles and HSA was performed.
HSA belongs to endogenous fluorophores with tryptophan residue at position 214 nm in the molecule. In these steady state fluorescence experiments, the concentration of HSA was kept constant, while the concentration of nanoparticles was increased stepwise. The fluorescence emission spectra of the complexes in the absence and presence of HSA were shown in Fig 9A. [51]. Fluorescence intensity of HSA decreased regularly at about 330 nm with the enhancement of nanoparticles (fluorescence quenching), which indicated that particle interacted with HSA and a complex is formed between them [45]. Furthermore, there was a red shift at the maximum emission peaks of HSA after the addition of particles, which suggested that the fluorescing residues in nonpolar hydrophobic cavities are moved to a hydrophilic environment [5].
In the case of fluorescence quenching procedures, the absorption of nanoparticles can absorb protein fluorescence and the presence of particles can scatter the input and output fluorescence, further reducing the fluorescence signal, which are termed as inner filter effect (IFE). This also results in a decreased fluorescence intensity that is not a quenching effect. In this study, the following formula was used for correcting fluorescence intensity: where F cor and F obs are the correct and observed fluorescence intensity, respectively. A ex and A em are the absorbance value at the excitation and emission wavelengths, respectively. Fluorescence intensities were corrected for inner filter and dilution effects before the following analysis [45,52]. There are two possible reasons for the complexes quenching HSA fluorescence, the static quenching and dynamic quenching. The analysis of the results was done with the help of Stern-Volmer equation [53]: where F 0 and F are the fluorescence intensity in the absence and presence of a quencher, respectively, [D] is the concentration of quencher, K q and K sv are the quenching rate constant, Stern-Volmer dynamic quenching constant, respectively, τ 0 is the average lifetime of molecule without the quencher and its value is 10 −8 s. Based on the experimental data, the Stern-Volmer plot for the quenching of HSA with POM@SiO 2 nanoparticles was given in the inset of Fig 8A. If the quenching process is the dynamic one, the dependence of F 0 /F on the quencher concentration is linear with slope equal to the value of K sv . Moreover, the maximum quenching rate constant of various quenchers is 2.0 × 10 10 Lmol -1 s -1 . The fluorescence quenching coefficient of the POM@SiO 2 is 1.3 × 10 18 Lmol -1 s -1 , which is far greater than the K q value of the dynamic quenching for various quenchers. It suggested that the quenching belonged to a static quenching rather than the dynamic one. In static quenching, small molecules bind to equivalent sites on a macromolecule independently; whereas in the dynamic interaction, the quencher binds to the fluorophore during the excited state [5]. Furthermore, static quenching often changes absorption spectrum of the luminophore molecule, while dynamic quenching is on the contrary. According to the UV-vis analyses, the absorption spectrum of HSA was changed by the POM@SiO 2 . Therefore, the fluorescence quenching of the nanoparticles was static quenching and the nanoparticles were likely to bind to HSA. These results indicated the interaction between POM@SiO 2 nanoparticles and HSA molecules, and the nanospheres can be transported in the serum albumin.
To further study the binding effect of nanoparticles to HSA, synchronous fluorescence (SFS) study was performed. The test could provide the information of tryptophan residue of the protein when the wavelength interval was established in 60 nm [33]. Fig 9B showed that the fluorescence intensities of tryptophan residue were decreased as the nanoparticle solutions added stepwise to HSA stock solution. The emission wavelength peak of tryptophan residue in the investigated concentration range was a slightly blue shift (Δλ = 60 nm). It suggested that the nanoparticles changed the conformation of the protein.

Conclusions
In summary, we report the in vitro anti-proliferative effects on MCF-7 cells of the POM@SiO 2 nanoparticles with small size around 40 nm. The nanoparticles significantly enhanced the cytotoxicity on the cells comparing to that of the pure POM. SiO 2 shells controlled the release efficiency as the drug delivery system. The higher antitumor activity could be due to their higher cellular penetration for the small size and surface modification by the shells. The Flow cytometry analysis results showed that the nanoparticles could induce apoptosis and cell cycle S phase arrest. The HSA binding experiment showed that the complex changed the conformation and quenched the fluorescence of the protein, which belonged to a static quenching. Furthermore, the antitumour effect of other cell lines and modification with target molecules on the spheres should be further investigated.