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• Worldwide statistical reports indicate that hepatic carcinoma, one of highest mortal malignancy, is the third leading cause of cancer mortality^[1]. Although traditional treatment is feasible, it always accompa-nied with side effect, the systematic toxicity of drugs, undesirable treatment efficiency and substantial recurrence and mortality^[2].

  Photodynamic therapy (PDT), which based on the photochemical reactions of photosensitizer (PS)^[3-4], is an effective, noninvasive, nontoxic therapeutics and has attracted wide attention and is applied to studies on cancer therapy^[5]. The mechanism of PDT is that, the activated PS transfers its excited-state energy to nearby oxygen molecular to generate reactive oxygen species (ROS), such as OH·, H₂O₂ and HO₂·, which could induce oxidative damage to the structure of various biological molecules through a series of peroxidative reactions and thus result in the death of cancer cells^[6-11].

  Classical photosensitizers are mainly from porphyrins and their derivatives^[12]. However, their inherent limitations including unstable properties, high toxicity, and cumbersome synthesis procedure hinder their application of PDT. To overcome these weaknesses, titanium dioxide (TiO₂), absorbing ultraviolet (UV) light to catalyze the generation of ROS^[13-14], can be used as PS. It has the advantages of non-toxic, stability, easy synthesis. Unfortunately, TiO₂ still has some drawbacks in the clinical use, such as insufficient selectivity, poor solubility and low efficiency resulting from the deficiency of cell-specific accumulation of TiO₂ on cancer cells. Hence, an efficient TiO₂ modification for higher selectivity and killing efficiency is needed^[15-16].

  Aiming at the disadvantages of TiO₂, numerous platforms for targeting delivery have been studied, of which, magnetic nanoparticles have attracted extensive interest because of their target ability steered by an external magnetic field^[17-18]. Therefore, a rational design to modify TiO₂ with magnetic nanoparticles is fundamentally developed.

  Magnetic Fe₃O₄ nanoparticles (NPs), as promising material because of alluring properties of chemical stability, magnetic properties and biocompatibility^[19-20], have myriads of potential applications such as magnetic resonance imaging, catalysis, and drug delivery^[21-27]. Moreover, due to the small particle size of magnetic NPs (10~100 nm), they can penetrate blood capillaries and reach the depths of target tumor tissue avoiding phagocytosis of reticuloendothelial system^[28]. Thus magnetic NPs have a longer blood circulation and interaction time than common drugs. Therefore, magnetic Fe₃O₄-TiO₂ NPs were supposed to possess various advantages including targeting action, high selectivity and efficiency, low side effect^[29-30] in anticancer activities.

  In addition, a synergistic anti-cancer effect could be obtained by the combination of magnetic NPs with PSs as drug or drug delivery system. The double materials translated into double functionality: generation of ROS under excitation light source and heat production under alternating magnetic field stimulation, coupling PDT to magnetic hyperthermia (MHT)^[31]. Therefore, magnetic Fe₃O₄-TiO₂ NPs as PS have a wide prospect in MHT and PDT for cancer.

  The photokilling effect of magnetic targeted Fe₃O₄-TiO₂ NPs on hepatoma carcinoma cells (HepG2) in different external magnetic fields was investigated under different light sources. The core-shell structure Fe₃O₄-TiO₂ NPs applied in this study were reported in our previous study^[32]. In this paper, the influence of different light sources, irradiation time, magnetic field strength and the concentration of Fe₃O₄-TiO₂ NPs on the photocatalytic killing effect were investigated using HepG2 cells as a model. The changes of cell cycle, apoptosis rate and MMP of HepG2 cells treated by photocatalytic Fe₃O₄-TiO₂ NPs in external magnetic field were detected by FCM, and the mechanism was explored through the mode of action between photo excited Fe₃O₄-TiO₂ NPs and HepG2 cells. The results will provide a new idea for cancer therapy and theoretical basis for further clinical application.

  1   Experimental

  1.1   Instruments

  Digital pH-meter (pH S-3, Shanghai Leici Device Works, China). CO₂ incubator (Asheville NC Company, USA). Ultraclean workbench (GB-Ⅱ, Beijing New Technology Application Research Institute). Low-speed centrifuge (LDZ5-2, Beijing Medical Centrifuge Plant). Electric steam pressure sterilizers (LDZX-40, Shanghai Shenan Medical Instrument Factory). Intensity meter (VLX-3W, SP Company, USA). High pressure mercury lamp (GGZ, Shanghai Yaming Lighting Company). LED light (LX-1, Shenzhen Green Xing Lighting Technology Company). Flow cytometry instrument (FACS-Calibur, BD company, USA). Microplate reader (Bio-Rad Novapathtm, USA). Inverted microscope (Olympus Company, Japan). NdFeb Magnet (N10, N27, N40, China).

  1.2   Materials

  Fe₃O₄-TiO₂ NPs were prepared and characterized early by research group^[32], which displayed good dispersion, high photocatalytic activity, and excellent magnetic responsivity. The average particle size was 50 nm and the photoresponsive range of TiO₂ was extended to 444 nm.

  Human hepatoma carcinoma cell (Shanghai Institute of Cell Biology, Chinese Academy of Sciences, Shanghai, China). Dulbecco's modified Eagles medium (DMEM, Neuronbc Laboratories Company). 10%(V/V) fetal bovine serum (FBS, Yuanhengjinma Bio-technology Development Company, Beijing). 1% (V/V) Penicillin-streptomycin solution (100×, Beijing Solarbio Science & Technology Company). Trypsin-EDTA (0.25%, Beijing Solarbio Science & Technology Company). Dimethyl sulfoxide (DMSO, Beijing Solarbio Science & Technology Company). Thiazolyl blue (MTT, Beijing Solarbio Science & Technology Company). Rhodamine 123 kit (Rh-123, Keygen Biotech, Nanjing, China). Propidium Iodide (PI, Sigma, USA). Fetal bovine serum (FBS, Gibco, USA). Milli-Q (Millipore, Bedford, MA, USA).

  1.3   Cell culture and treatment

  HepG2 cells were maintained in a 37℃, 5% carbon dioxide environment in Dulbecco's modified Eagle's medium supplemented with 10% (V/V) fetal bovine serum (FBS) and 1% (V/V) Penicillin-Streptomycin.

  Fe₃O₄-TiO₂ NPs and TiO₂ were suspended by fresh medium to different concentrations of 100, 200, 300, 400 and 500 μg·mL^-1. When HepG2 cells approached the exponential growth phase, cells were transferred and cultured in a 24-well plate and allowed to grow for 24 h at 37 ℃ in a 5% CO₂ incubator. Then the medium was replaced with 1 mL of fresh culture medium containing sonicated Fe₃O₄-TiO₂ NPs and TiO₂ respectively. With different external magnetic strength, samples were irradiated by high pressure mercury lamp (dominant wavelength is 365 nm) or light emitting diode (LED, dominant wavelength is 420~425 nm) at room temperature. A band-pass filter was applied to obtain a light wavelength between 348~372 nm. The light intensity at the liquid surface was measured by a VLX-3W radiometer-photometer. The schematic diagram of Fe₃O₄-TiO₂ NPs photocatalytic killing HepG2 cells was shown in Fig. 1. After treatment, HepG2 cells were incubated for 24 h to measure cell viability and 4 h to detect cell cycle and MMP.

  Figure 1.  Schematic diagram of magnetic Fe₃O₄-TiO₂ photocatalytic killing process and mechanism

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  1.4   Experimental grouping

  In our study, the experiment was divided into six groups:

  Group Ⅰ: negative control group, routinely cultured HepG2 cells without any treatment.

  Group Ⅱ: positive control group, HepG2 cells cultured in different concentrations of TiO₂ were similarly treated with non-irradiation control group, irradiation experimental group and irradiation experimental group under magnetic field.

  Group Ⅲ: non-irradiation control group, HepG2 cells were treated with different concentrations of Fe₃O₄-TiO₂ NPs in absence of light.

  Group Ⅳ: irradiation control group, HepG2 cells were respectively irradiated by high pressure mercury lamp or LED lamp for 20, 30 and 40 minutes without any nanoparticles.

  Group Ⅴ: irradiation experimental group, HepG2 cells cultured in different concentrations of Fe₃O₄-TiO₂ NPs were similarly treated with irradiation control group.

  Group Ⅵ: irradiation experimental group under magnetic field, HepG2 cells were treated with different concentrations of Fe₃O₄-TiO₂ NPs and irradiated by high pressure mercury lamp or LED lamp under magnetic field.

  1.5   Cell viability analysis

  To assess cell viability, the modified MTT assay protocol was used^[33]. MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide) was dissolved in phosphate buffer solution (PBS, pH 7.4) at 5 mg·mL^-1 and filtered to be sterilized. 40 μL of stock MTT solution was added to each well and incubated at 37 ℃ for 4 h. Culture medium was removed and 600 μL of DMSO was added to each well and mixed thoroughly to dissolve the dark blue formazan crystals. After a few minutes to ensure that all crystals were dissolved at room temperature, the solution were extracted from each well and placed in the corresponding wells of 24-well plates. Optical density (OD) values were measured on a microplate reader at wavelength of 492 nm, taking the solution without MTT as control.

  1.6   Cell cycle analysis

  Cell cycle was assessed by flow cytometric analysis of ethanol-fixed cells stained with DNA-binding dye PI. Flow cytometry analysis was performed on a flow cytometer with Cell Quest Pro software. The light-scatter channels were set on linear gains and the fluorescence channels on a logarithmic scale. A minimum of 1×10⁴ cells were analyzed in each condition with threshold setting adjusted so that the cell debris was excluded from the data acquisition. The grain density of cells was assessed by side scatter (SSC)^[34] and the results would be presented with the DNA histograms.

  1.7   Mitochondrial membrane potential analysis

  Mitochondrial depolarization was determined using cell apoptosis rhodamine123 (Rh123) detection kit, a lipophilic cation susceptible to the changes in MMP. It has a property of entering mitochondria with membrane polarization and emitting flavo-green fluorescence with exciting of 488 nm excitation. If the mitochondrial potential is disturbed, the dye entering mitochondria will reduce, and the flavo-green fluorescence will drop^[35]. The cells were stained with Rh123 as described by the manufacturer, and the flavo-green fluorescence was detected by FCM. The strength of fluorescence corresponds to the value of MMP.

  1.8   Statistical analysis

  Experiments were repeatedly conducted for three times. Values were reported as mean ±SD. Statistical comparisons were made by t-test to detect significant differences using SPSS 17.0 software. P < 0.05 was considered to be statistically significant.

  2   Results and discussion

  2.1   Viability of HepG2 cells

  2.2   Mechanism of photocatalytic killing

  2.1.2   Influence of irradiation time

  The effect of different ultraviolet and visible light irradiation time on the relative survival rate of HepG2 cells is presented in Fig. 3. With the extension of illumination time, the relative survival rate of HepG2 cells in each group markedly decreased (^*P < 0.05). The relative survival rate of HepG2 cells in TiO₂ group was greater than Fe₃O₄-TiO₂ group at different time points.

  Figure 3.  Effect of different ultraviolet (A) and visible light (B) irradiation time on relative survival rate of HepG2 cells

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  Compared with visible light, for control and TiO₂ groups, ultraviolet light had a more significant effect on photokilling efficiency, which was consistent with the fact that ultraviolet light is necessary for TiO₂ photokilling cancer cells. However, in Fe₃O₄-TiO₂ group, no significant difference was found in the relative survival rate of HepG2 cells between visible and ultraviolet light. It is suggested that Fe₃O₄ modified TiO₂ made its light response range expanding from ultraviolet to visible region^[32].

  Taken together, Fe₃O₄-TiO₂ killed HepG2 cells in a time-dependent manner. Fe₃O₄-TiO₂ had many advantages over TiO₂ on killing cancer cells, especially high photokilling efficiency under visible light^[32].

  2.1.1   Biosafety assessment

  Biosafety assessment was vital to evaluate the potential application of Fe₃O₄-TiO₂ NPs in clinics. Here, modified MTT assay was performed to detect the toxicity of Fe₃O₄-TiO₂ NPs (Fig. 2). The survival rate of HepG2 cells gradually decreased as the concentration of TiO₂ and Fe₃O₄-TiO₂ increasing, and the cytotoxicity of Fe₃O₄-TiO₂ was slightly stronger than TiO₂ without irradiation. When their concentration was greater than 400 μg·mL^-1, a significant difference (^**P < 0.05) existed, which was different from literature that nano-TiO₂ was non-toxic to animals^[36-37]. The possible reason is that at a high concentration and after long time, the tiny particle size of TiO₂ and Fe₃O₄-TiO₂ NPs interacting with HepG2 cells might damage the cell membrane or enter the HepG2 cells to cause necrosis^[38-39].

  Figure 2.  Effect of different TiO2 and Fe₃O₄-TiO₂ NPs concentrations on relative survival rate of HepG2 cells without irradiation

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  However, when the concentrations of TiO₂ and Fe₃O₄-TiO₂ suspension were in the range of 0~300 μg·mL^-1, there is no significant difference (^**P > 0.05). Since it is widely accepted that the drugs used for the photocatalytic killing cancer cells should be non-toxic without irradiation, and thus TiO₂ and Fe₃O₄-TiO₂ NPs can be used as drugs for photocatalytic killing HepG2 cells.

  2.1.4   Morphology

  The cell morphology of Group Ⅰ, Ⅳ, Ⅴ and Ⅵ were observed by inverted microscope. The results are shown in Fig. 5. Normal HepG2 cells of Group Ⅰ adherently grew and the outlines were clear with the form of spindle or polygons. The microscopic structure could be observed, and individual cell shrank into round shape during recession phase (Fig. 5a). It was observed that HepG2 cells of Group Ⅳ, Ⅴ and Ⅵ tended to exhibit different morphological changes. Under the visible light irradiation, there was no significant change in cellular morphology (Fig. 5b). In contrast, cells irradiated by ultraviolet light markedly decreased, and parts of cells adherently grew, with others contracting into round shape and floated in the culture medium (Fig. 5c). Cellular numbers of Group Ⅴ were all reduced. Cellular debris was generated after ultraviolet light irradiation (Fig. 5e). Under visible light irradiation, cells shrank and the cell membrane became rough, indicating that cell apoptosis might occur (Fig. 5d). Compared with Group Ⅴ, the cellular number of Group Ⅵ had similar trend. Cells shrank into round shape and floated in the culture medium after ultraviolet light irradiation (Fig. 5f). After visible light irradiation, cells shrank and pseudopodia protruded, indicating that the killing effect of Fe₃O₄-TiO₂ NPs under magnetic field might have two forms, including necrocytosis and cell apoptosis (Fig. 5g).

  Figure 5.  Morphological changes of HepG2 cells treated by different manners (×400)

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  2.1.3   Influence of magnetic field strength and concentration

  The effect of magnetic field strength and concentration of Fe₃O₄-TiO₂ on the photocatalytic killing efficiency are shown in Fig. 4. With magnetic field strength ranging from 0 to 1.5 T, the relative survival rate of HepG2 cells in each group decreased in varying degrees, and Fe₃O₄-TiO₂ group varied most and its killing efficiency is the strongest (^**P > 0.05). It is suggested that magnetic Fe₃O₄-TiO₂ under magnetic field could be targeted to kill cancer cells, thereby possessing high killing efficiency. To be specific, the killing effect of Fe₃O₄-TiO₂ on HepG2 cells was markedly enhanced with the increased magnetic field strength in the range of 0~1.0 T. It has been reported that the photokilling effect of TiO₂ NPs could be prominently improved when they entered cancer cells, especially the nucleus^[40-41]. It is presumed that under external magnetic field, more nanoparticles would deposit onto the bottom of the culture plate, get in touch with the adherent growth HepG2 cells and enter the interior of HepG2 cells to kill cells. However, the trend of curve decreased slightly in the range of 1.0~1.5 T, which suggested that almost all of nanoparticles had deposited onto the bottom and the killing effect had reached its maximum limits.

  Figure 4.  Effect of Fe₃O₄-TiO₂ NPs excited by ultraviolet (A) and visible light (B) on relative survival rate of HepG2 cells under external magnetic field

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  The killing efficiency of Fe₃O₄-TiO₂ increased with the increasing of the concentration of Fe₃O₄-TiO₂. Therefore, the killing effect was speculated to be concentration-dependent. And when the concentration of suspension was 300 μg·mL^-1, the view that killing efficiency of Fe₃O₄-TiO₂ was much greater than TiO₂ has been further confirmed.

  From Fig. 4A and Fig. 4B, ultraviolet irradiation had more significant effect than visible light on the relative survival rate of HepG2 cells in control and TiO₂ group, which can be speculated that visible light played a tiny role. However, in both cases, Fe₃O₄-TiO₂ had showed strong killing effect. This further proved that its light response range was broadened to visible region (444 nm), lest tissues burned and mutations exposed to ultraviolet light.

  The results suggested that killing effect of Fe₃O₄-TiO₂ NPs on HepG2 cells could be enhanced under external magnetic field. Thus, the good magnetic responsivity of nanoparticles might be expected to be a drug for targeted therapy of cancer. It could reach the targeted tissue guided by external magnetic field, reduce damage to normal cells and enhance the killing effect.

  2.2.2   MMP analysis

  Mitochondrion is one of vital cell organs in the process of transmitting apoptosis signs, and depola-rization of MMP is the earliest event in the cascade reaction of apoptosis process^[49]. Therefore, study on the changes of MMP plays an important role in clarifying mechanism caused by photocatalytic Fe₃O₄-TiO₂ NPs. The changes on MMP of HepG2 cells caused by photocatalytic Fe₃O₄-TiO₂ NPs in external magnetic field was presented in Fig. 7.

  Figure 7.  Effect of photocatalytic Fe₃O₄-TiO₂ NPs on MMP of HepG2 cells in external magnetic field

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  Rhodamine (Rh123) was used as a fluorescent probe for assessing the damage to mitochondria. Compared with other groups, Fe₃O₄-TiO₂ group apparently increased the fluorescence of Rh123 on HepG2 cells (Fig. 7A). This phenomenon indicated a loss of mitochondrial function.

  To find whether Fe₃O₄-TiO₂ NPs can induce mitochondrial depolarization in HepG2 cells, fluore-scence intensity was quantified (Fig. 7B). Compared with cell control group, the MMP of HepG2 cells in visible light control group had no obvious change (^**P > 0.05), but a marked decrease was found (^*P < 0.05) in ultraviolet light control group, TiO₂ group and Fe₃O₄-TiO₂ group. Fe₃O₄-TiO₂ NPs excited either by ultraviolet or visible light showed no obvious difference on the depolarization of MMP (^#P > 0.05), while more significant impact than that of TiO₂ was observed in both cases. The results further indicated that the mechanism of photocatalytic killing HepG2 cells was similar for two excitation conditions.

  Mitochondria contributed to the production of ROS that is necessary for redox-dependent cellular processes. Mitochondria were carried out aerobic respiration. Excited Fe₃O₄-TiO₂ produced a large amount of superoxide in cells. Surrounded by a high concentration of ROS, mitochondrial were attacked and its functional activity was restrained by cumulative ROS. As a result, cells were approaching to apoptosis because of lack of energy, and consequently HepG2 cell viability inevitably dropped.

  Besides, to maintain mitochondrial function, normal MMP is necessary for a prerequisite in the maintenance of mitochondrial oxidative phosphorylation and the synthesis of adenosine triphosphate (ATP)^[50]. The depolarization of MMP can cause a series of biochemical changes both inside and outside on mitochondrial membrane, including release of cytochrome C with a dual function of regulating the energy metabolism and apoptosis, altering cell membrane permeability, stopping the synthesis of ATP and releasing apoptosis inducing factor (AIF). Consequently, due to disruption of the cell membrane, the cascade reaction of apoptosis process will be induced and the cells will enter a nonreversible apoptosis process^[51].

  Mitochondrion is the control center of apoptosis^[52]. In the apoptosis of mitochondrial regulation, reduction of MMP is an important indicator of the permeability and structural damage to mitochondrial membrane. Accordingly, in our experimental conditions, the photocatalytic Fe₃O₄-TiO₂ NPs might kill HepG2 cells via mitochondrial pathway to induce apoptosis.

  2.2.3   Mode of action

  Apoptosis of HepG2 cells might be induced by photocatalytic Fe₃O₄-TiO₂ NPs in two ways. One way is that ROS produced by photocatalytic Fe₃O₄-TiO₂ NPs causes imbalance in intracellular redox state and membrane damages of HepG2 cells, so the perme-ability of the mitochondrial membrane changes and its MMP reduces, inducing HepG2 cell apoptosis^[36]. Ano-ther way is that Fe₃O₄-TiO₂ NPs enter intracellular of HepG2 cells by endocytosis or passive diffusion, dire-ctly damage mitochondria and other intracellular com-ponents, and induce apoptosis through ROS or photo-generated hole-electron pair upon their surface^[38-40].

  SSC parameters (Fig. 8) obtained by FCM can be used to judge whether Fe₃O₄-TiO₂ enters HepG2 cells. The abscissa and ordinate represented FFC and SSC parameters of HepG2 cells, respectively. The quadrants were divided by solid line frame to distinguish between living and dead cells, where red and black dots stood for living and dead cells (or cell debris), respectively. Most of living cells located below quadrant in negative control group, and SSC parameters were small. While cell populations in Fe₃O₄-TiO₂ and TiO₂ groups shifted up and the SSC parameters increased, especially those in Fe₃O₄-TiO₂ group. The result indicated that more Fe₃O₄-TiO₂ NPs entered HepG2 cells than TiO₂. It allowed conclusion that photocatalytic killing effect of Fe₃O₄-TiO₂ NPs was stronger than TiO₂ in external magnetic field.

  Figure 8.  Effect of photocatalytic Fe₃O₄-TiO₂ NPs on SSC parameters of HepG2 cells under external magnetic field

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  According to the above experimental results, photocatalytic Fe₃O₄-TiO₂ NPs could induce apoptosis of HepG2 cells through the second way. Photocatalytic Fe₃O₄-TiO₂ NPs induced apoptosis through directly oxidative damage to mitochondria and other intracellular components.

  2.2.1   Cell cycle analysis

  In external magnetic fields, the effect of Fe₃O₄-TiO₂ NPs excited by ultraviolet and visible light on the cell cycle of HepG2 cells was examined by propidium iodide (PI) staining with FCM. The DNA histograms and the percentage of cell population in each phase are presented in Fig. 6.

  Figure 6.  Effect of photocatalysis Fe₃O₄-TiO₂ NPs on cell cycle of HepG2 cells under external magnetic field

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  Cell cycle analysis indicated decreased S phase and increased G₀/G₁, G₂/M phase proportion, as well as apparent blockage of G₀/G₁ phase in response to TiO₂ and Fe₃O₄-TiO₂ NPs treatment (^*P < 0.05), regar-dless of the irradiation conditions. The retardative effect and cell apoptosis rate of Fe₃O₄-TiO₂ NPs were greater than TiO₂. In the group of Fe₃O₄-TiO₂, there were no significant differences in cell cycle arrest and apoptosis rate between ultraviolet and visible light (^**P > 0.05), indicating that the mechanism might be similar in both cases.

  Many studies have reported that the most characteristic effect of radiation (such as UV, X-ray and γ-ray, etc.) on eukaryotic cell cycle is arrested in G₂/M phase^[42-43]. Moreover, our previous study on the effect of photocatalytic nano-TiO₂ on killing gastric cancer cells, demonstrated that the cell cycle was arrested in G₂/M phase after ultraviolet irradiation alone for 20 min^[44]. Therefore, it is supposed that the arrest of cell cycle in G₂/M phase was caused by ultraviolet irradiation. The increase of cell numbers in G₀/G₁ phase and the decrease in S phase were caused by TiO₂ and Fe₃O₄-TiO₂ NPs. TiO₂ and Fe₃O₄-TiO₂ NPs driven by visible light didn't cause the arrest in G₂/M phase, which also confirmed this hypothesis.

  The arrest in G₀/G₁ phase is closely related to cellular proliferation and apoptosis. In G₁ phase, mRNA, tRNA, ribosome and protein are mainly synthesized, which can provide necessary precursors for DNA synthesis in S phase^[45]. Photocatalytic Fe₃O₄-TiO₂ NPs could arrest HepG2 cells in G₀/G₁ phase and inhibit the transition to S phase. The reactive oxygen species (ROS)^[46] generated by photocatalytic Fe₃O₄-TiO₂ NPs might cause the direct damage to RNA and other substances synthesized in G₁ phase. Meanwhile, the decrease of cell numbers in S phase indicated that the reduction or inhibition of DNA synthesis in S phase might be a way of Fe₃O₄-TiO₂ NPs killing HepG2 cells.

  It is reported that different cell cycle regulatory factors, proliferating cell nuclear antigen and p53 promote the transition of different cell cycle stages. Cyclin E-Cdk2 complex is the key regulatory factor in the cycle transition from G₁ to S phase, whose activity is affected by related gene and protein^[47]. In our experiment, HepG2 cells were arrested in G₀/G₁ phase by photocatalytic Fe₃O₄-TiO₂ NPs, so it is speculated that the complex signals might be integrated and delivered by these genes and proteins, which would determine the next development of HepG2 cells such as entering into mitosis process or dormant G0 phase, causing programmed cell death or conducting self-repair^[47-48]. The reason for cell cycle arrested in G₀/G₁ can be further pursued by investigating the changes of related genes and proteins.

  3   Conclusions

  Photocatalytic activities of magnetic Fe₃O₄-TiO₂ NPs were investigated in HepG2 cells. Compared with TiO₂, the core-shell structure Fe₃O₄-TiO₂ NPs have magnetic targeted function, extend light response range to visible region and enhance photokilling efficiency. Moreover, the mechanism of photocatalytic Fe₃O₄-TiO₂ NPs killing cancer cells was explored. Entering intracellular of HepG2, Fe₃O₄-TiO₂ NPs stimulated the generation of ROS or photogenerated hole-electron pair, which directly damage mitoch-ondria and other intracellular components. Fe₃O₄-TiO₂ induced apoptosis, mediated by arresting cell cycle in G₀/G₁ phase, reducing mitochondrial membrane potential, and mitochondrial depolarization. It can be concluded that Fe₃O₄-TiO₂ magnetic NPs are of great value as effective drug in targeting photodynamic therapy. The potential of magnetic Fe₃O₄-TiO₂ in MHT and PDT for cancer could also be expected.

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  Figure 1  Schematic diagram of magnetic Fe₃O₄-TiO₂ photocatalytic killing process and mechanism

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  Figure 2  Effect of different TiO2 and Fe₃O₄-TiO₂ NPs concentrations on relative survival rate of HepG2 cells without irradiation

  Data were represented as mean ±SD (n=3); Remark: compared with the control group, ^*P > 0.05, ^**P < 0.05

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  Figure 3  Effect of different ultraviolet (A) and visible light (B) irradiation time on relative survival rate of HepG2 cells

  Data were represented as mean ±SD (n=3); Remark: compared with the irradiation time of 0 min corresponding to each group, ^*P < 0.05, ^**P > 0.05

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  Figure 4  Effect of Fe₃O₄-TiO₂ NPs excited by ultraviolet (A) and visible light (B) on relative survival rate of HepG2 cells under external magnetic field

  (a) Control of cell; (b) Control of visible light irradiation; (c) Fe₃O₄-TiO₂ NPs (100 μg·mL^-1); (d) Fe₃O₄-TiO₂ NPs (200 μg·mL^-1); (e) Fe₃O₄-TiO₂ NPs (300 μg·mL^-1); (f) TiO₂ (300 μg·mL^-1); Data were represented as mean ±SD (n=3);Remark: compared with the magnetic field strength of 1.0 T corresponding to each group, ^*P < 0.05, ^**P > 0.05

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  Figure 5  Morphological changes of HepG2 cells treated by different manners (×400)

  (a) Normal HepG2 cells; (b) Visible light irradiation; (c) Ultraviolet irradiation; (d) Fe₃O₄-TiO₂ NPs irradiated 30 min by visible light; (e) Fe₃O₄-TiO₂ NPs irradiated 30 min by ultraviolet; (f) Fe₃O₄-TiO₂ NPs irradiated 30 min by ultraviolet under external magnetic field; (g) Fe₃O₄-TiO₂ NPs irradiated 30 min by visible light under external magnetic field; Scale bar=100 μm

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  Figure 6  Effect of photocatalysis Fe₃O₄-TiO₂ NPs on cell cycle of HepG2 cells under external magnetic field

  (a) Control of cell; (b) TiO₂ excited by ultraviolet; (c) Fe₃O₄-TiO₂ NPs excited by ultraviolet; (d) TiO₂ excited by visible light; (e) Fe₃O₄-TiO₂ NPs excited by visible light; Data were represented as mean ± SD (n=3); Remark: compared with the control of cell, ^*P < 0.05, ^**P > 0.05

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  Figure 7  Effect of photocatalytic Fe₃O₄-TiO₂ NPs on MMP of HepG2 cells in external magnetic field

  (a) Control of cell; (b) Control of visible light; (c) TiO₂ excited by visible light; (d) Fe₃O₄-TiO₂ NPs excited by visible light; (e) Control of ultraviolet; (f) TiO₂ excited by ultraviolet; (g) Fe₃O₄-TiO₂ NPs excited by ultraviolet; Data were represented as mean ±SD (n=3); Remark: compared with the control of cell, ^*P < 0.05, ^**P > 0.05; Comparison of the two Fe₃O₄-TiO₂ NPs groups under irradiation of different light source, ^#P > 0.05

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  Figure 8  Effect of photocatalytic Fe₃O₄-TiO₂ NPs on SSC parameters of HepG2 cells under external magnetic field

  (a) Control of cell; (b) TiO₂ excited by visible light; (c) Fe₃O₄-TiO₂ NPs excited by visible light

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