Boosting Chemodynamic Therapy by the Synergistic Effect of Co-Catalyze and Photothermal Effect Triggered by the Second Near-Infrared Light

The MoS2 nanosheets served as co-catalyst could reduce Fe3+ ions with lower Fenton reaction activity into the highly reactive Fe2+ ions, thereby boosting the production of hydroxyl radical (•OH) for high efficiency chemodynamic therapy (CDT). The photothermal effect of MoS2 nanosheets motivated by second near-infrared light could further improve the treatment effectiveness by synergetic photothermal-enhanced CDT and photothermal therapy. The MoS2 nanosheets served as co-catalyst could reduce Fe3+ ions with lower Fenton reaction activity into the highly reactive Fe2+ ions, thereby boosting the production of hydroxyl radical (•OH) for high efficiency chemodynamic therapy (CDT). The photothermal effect of MoS2 nanosheets motivated by second near-infrared light could further improve the treatment effectiveness by synergetic photothermal-enhanced CDT and photothermal therapy. In spite of the tumor microenvironments responsive cancer therapy based on Fenton reaction (i.e., chemodynamic therapy, CDT) has been attracted more attentions in recent years, the limited Fenton reaction efficiency is the important obstacle to further application in clinic. Herein, we synthesized novel FeO/MoS2 nanocomposites modified by bovine serum albumin (FeO/MoS2-BSA) with boosted Fenton reaction efficiency by the synergistic effect of co-catalyze and photothermal effect of MoS2 nanosheets triggered by the second near-infrared (NIR II) light. In the tumor microenvironments, the MoS2 nanosheets not only can accelerate the conversion of Fe3+ ions to Fe2+ ions by Mo4+ ions on their surface to improve Fenton reaction efficiency, but also endow FeO/MoS2-BSA with good photothermal performances for photothermal-enhanced CDT and photothermal therapy (PTT). Consequently, benefiting from the synergetic-enhanced CDT/PTT, the tumors are eradicated completely in vivo. This work provides innovative synergistic strategy for constructing nanocomposites for highly efficient CDT.


Introduction
Nowadays, cancer is an illness which threatens the human health owing to its high mortality rate and recurrence rate [1,2]. Several treatment strategies have been applied in clinic, such as surgery [3], radiotherapy [4], and chemotherapy [5,6], but they still suffer from limited treatment effect and significant side effects. Recently, chemodynamic therapy (CDT) based on Fenton and Fenton-like reactions (denoted as generating highly oxidative hydroxyl radicals from hydrogen peroxide by ferrous or non-ferrous ions to remove organic pollutant) has drawn more attentions because it can generate the highly toxic reactive oxygen species [hydroxyl radicals (•OH)] in the tumor lesion area, resulting in high specificity and low side effects [7][8][9][10][11][12][13]. However, the weak acid and low transformation efficiency of Fe 3+ to active Fe 2+ in the tumor microenvironments lower the Fenton reaction efficacy, which leads to unsatisfactory therapy effect of CDT [14,15]. Therefore, it is urgent to develop new strategy to improve the Fenton reaction efficiency in the tumor microenvironments.
Recently, some promising strategies have been used to improve the treatment effect of CDT. For instance, Bu and coworker take advantage of photothermal effect to enhance the Fenton reactions efficiency, and obtain the good treatment effect of combinatorial CDT/PTT [7]. Our group has successfully utilized the light, ultrasound, and near-infrared II (NIR II) light-triggered photothermal effect to assist the Fenton reaction and improve the treatment effect. In addition to these methods that depend on the external stimulus to enhance the Fenton reactions efficiency [16,17], the strategy that introduces a catalyst to accelerate the rate-controlling step of Fe 3+ /Fe 2+ conversion has been attracted much more attentions recently in the field of treating persistent pollutants [18,19]. However, the research on improving the therapeutic efficacy of CDT with this co-catalytic strategy is rarely reported till now [20,21]. If the catalyst also has good photothermal performance response to NIR II light for PTT, the improved therapeutic efficacy of CDT will be achieved through the synergistic effect.
Building from these ideas, herein, we design and synthesize novel FeO/MoS 2 nanocomposites modified by bovine serum albumin (FeO/MoS 2 -BSA) for magnetic resonance imaging guided highly efficient CDT (Scheme 1). FeO/MoS 2 -BSA exhibits higher production efficiency of •OH compared to that of FeO and MoS 2 individually, demonstrating that the co-catalysis strategy can improve the Fenton reaction efficacy. Moreover, owing to the absorption ability of MoS 2 in NIR II region (1000-1350 nm), the photothermal (PT) effect of FeO/MoS 2 -BSA triggered by NIR II light (1064 nm) has been first employed to further improve the production efficiency of •OH. As the results, not only the cancer cells could be effectively killed by synergetic enhanced CDT and photothermal therapy (PTT), but also the tumors are eliminated completely in vivo experiments, indicating their highly efficient therapeutic efficiency in vitro and in vivo. In addition, the good magnetic properties of FeO endows FeO/MoS 2 -BSA with great potential as contrast agents for MRI [28,29]. This work thus presents a synergistic strategy of NIR II light motivate photothermal effect [30][31][32][33] and co-catalysis to construct nanotheranostic agents with high potency and low side effects.

Preparation of MoS 2 Nanosheets
The large-scale MoS 2 nanosheets were prepared following to the previous literature [23]: ammonium molybdate (50 mg, 0.25 mmol) and NAC (50 mg, 0.3 mmol) were dissolved in ultrapure water (30 mL) in ice-water bath under stirring vigorously. Then, thiourea (40 mg, 0.5 mmol) was poured into the precursor solution under vigorous stirring for 30 min. The mixture was added into a 40-mL Teflon-lined stainless steel autoclave at 200 °C for 8 h. After cooling to room temperature, collected by centrifugation at 8000 r min −1 for 5 min and washed with water for three times. The small MoS 2 nanosheets were obtained large-scale nanosheets under ultraphonic effect of ultrasonic cell disruptor for 1 h, and then collect the upper suspension after centrifugation and re-dispersed in 10 mL water.

Preparation of FeO Nanoparticles
The FeO nanoparticles were synthesized by the thermal decomposition method [34]. The iron(III) acetylacetonate (500 mg), oleic acid (0.52 mL), and trioctylamine (20 mL) were sequentially added into three-necked flask and heated at 150 °C for 30 min to remove water under argon gas protection, then slowly heated to 270 °C and kept for 10 min (rise rate: 2 °C min −1 ). After cooling to room temperature, FeO nanoparticles were collected by centrifugation at 8000 rpm for 10 min, and washed with cyclohexane and ethyl alcohol mixed solution for three times. The hydrophilic FeO nanoparticles were obtained by ligand-exchange strategy as follows [35]: 5 mL of FeO nanoparticles were dispersed in hexane (∼5 mg mL −1 ), and then 5 mL of dichloromethane solution of NOBF 4 (0.01 M) was added into solution at room temperature under stirring vigorously for 10 min. After that, the hydrophilic FeO nanoparticles were obtained by centrifugation at 2000 r min −1 for 5 min and washed with toluene and hexane (1:1 by volume) for two times.

Preparation of FeO/MoS 2 -BSA Nanocomposites
The FeO nanoparticles solution (1 mL, 1000 ppm) was dropped into 10 mL of MoS 2 nanosheets with mass concentration of Mo (200 ppm), respectively, under stirring vigorously for 20 min, and then BSA solution (1 mL, 5 mg mL −1 ) was added into above mixed solution individually for 1 h. The nanocomposites were collected by centrifugation at 8000 r min −1 for 10 min and washed with water for two times.

Characterization
Powder X-ray diffraction (XRD) was tested on a Bruker D8 Focus powder X-ray diffraction with Cu Kα radiation (λ = 1.5418 Å) at 40 kV and 40 mA. Inductively coupled plasma (ICP) analyses were obtained from Varian Liberty 200 spectrophotometer to determine the contents. The UV−vis−NIR spectra were recorded on spectrometer (SHI-MADZU, UV-3600). Infrared thermal imaging camera (FLIR T420, Fluke, USA). Transmission electron microscopic (TEM) images were obtained from a TECNAI G2 high resolution transmission electron microscope, operating at 200 kV. X-ray photoelectron spectroscopy (XPS) spectra were performed on an ESCALAB-MKII 250 photoelectron spectrometer (VG Co.) with Al Kα X-ray radiation as the X-ray source for excitation. Discovery MR750w, GE, America. Bruker Avance III (9.4 T, 400 MHz) NMR spectrometer for magnetic resonance imaging. Fluorescence spectrometer (F-4800) for detection of PTA.

Detection of Extracellular •OH
Series of sample solutions were prepared by adding different samples into total reaction volume of 2 mL PBS buffer solution (pH 6.0) containing H 2 O 2 (0.5 mM) and PTA as fluorescence probe (25 mg L −1 ) for fluorescence intensity detection (EX: 315 nm, EM: 425 nm) with time. Samples are as follows: FeO/MoS 2 -BSA, MoS 2 nanosheets, FeO nanoparticles.

Photothermal Effect and Thermal Stability of FeO/ MoS 2 -BSA Nanocomposites
The

MRI imaging Property
The

Cell Culture
Human cervical adenocarcinoma epithelial cells (HeLa) were cultured with regular growth medium containing high glucose DMEM at 37 °C in a 5% CO 2 environment.
Cell culture media with different pH were adjusted by HCl.

Cytotoxicity Measurement of FeO/MoS 2 -BSA Nanocomposites
HeLa cells were seeded into 96-well plates for 24 h (37 °C, 5% CO 2 ). Then, FeO/MoS 2 -BSA nanocomposites (0, 50, 100, 200, and 400 μg mL −1 ) were added into 96-well plates for 24 h. Then, the HeLa cells were washed with PBS for 2 times. Then CCK-8 solution (100 μL) was added and maintained for 3 h. At last, the 96-well plates were putted into plate reader for analysis and the absorbance at 450 nm was recorded to measure the cell viability. Except for adding H 2 O 2 and adjusting pH value of DMEM (pH = 6.5), the other experiment is same as mentioned above for cytotoxicity measurement of FeO/MoS 2 -BSA nanocomposites in stimulated TME.

In Vitro Photothermal-assisted CDT of FeO/ MoS 2 -BSA Nanocomposites
HeLa cells were seeded in 96-well plates for 24 h. Then, FeO/ MoS 2 -BSA nanocomposites solution (0, 50, 100, 200, and 400 μg mL −1 ) were added into the HeLa cells containing H 2 O 2 (0.01 mM) for 24 h. Then, the HeLa cells were irradiated with 1064 nm laser (0.75 W cm −2 ) for 10 min. After that, the HeLa cells were washed with PBS for 2 times and incubated with CCK-8 solution (100 μL) for 3 h. At last, the 96-well plates were putted into plate reader for analysis and the absorbance at 450 nm was recorded to measure the cell viability.

Detection of Intracellular •OH by DCFH-DA
HeLa cells were seeded in six-well plate containing H 2 O 2 for 12 h. FeO/MoS 2 -BSA with mass concentration of 200 μg mL −1 were added into and incubated for 4 h. After incubating with DCFH-DA (10 μM) with or without 1064 nm laser irradiation (0.75 W cm −2 ) for 5 min and after washing with PBS for two times, the fluorescence imaging of cells were monitored by confocal microscopy.

PI/AM Co-staining
HeLa cells were seeded in six-well plate for 12 h. FeO/ MoS 2 -BSA with mass concentration of 200 μg mL −1 in DMEM containing H 2 O 2 were added and incubated for 4 h. Then, cells were irradiated with or without 1064 nm laser (0.75 W cm −2 ) for 10 min. After washing with PBS for two times and co-stained with calcein AM and propidium iodide (PI) for 30 min. After washing with PBS for two times, the cellular modality of cells was monitored by fluorescence microscopy.

Animal Experiment
All of the animal experiments were carried out under the NIH guidelines for the care and use of laboratory animals (NIH Publication No. 85-23 Rev. 1985) and approved by the Jilin University Animal Care and Use Committee. Kunming mices (20 g) were purchased from Laboratory Animal Center of Jilin University (Changchun, China).

In Vivo Therapy
The U14 cells were subcutaneously injected into the underarm of Kunming mice. Thirty mice bearing U14 tumor (≈120 mm 3 ) were randomly allocated into four groups:

TUNEL and H&E Staining
The tumor-bearing mice were killed on the third day, the tumor tissues were dissected out for hematoxylin and eosin (H&E) staining and TdT-mediated dUTP nick end labeling (TUNEL). Tumor-bearing mice were necked off on the 14th day, and the heart, liver, spleen, lung, kidney were dissected out and immersed in formalin and processed in paraffin for H&E staining.

Characterization of FeO/MoS 2 -BSA Nanocomposites
The MoS 2 nanosheets with average size of 150 nm were obtained after sonicating the solution of large MoS 2 sheets (> 1 μm) which is shown in Fig. 1a, b, and as shown in Fig.  S1a, the X-ray diffraction (XRD) pattern can be indexed well to 2H-MoS 2 phase (JCPDS No. 37-1492). The ligand-free FeO nanospheres were synthesized following the previous report [34], and the average diameter was determined to be 8 ± 3 nm by transmission electron microscopy which is (TEM) shown in Figs. 1c and S2. The high resolution TEM image in the inner picture of Fig. 1c shows that the lattice fringes with d-spacing of 0.20 nm corresponds to the (4 0 0) plane of FeO, which is consistent with the FeO (JCPDS No. 19-0629, Fig. S1b). Afterward, FeO/MoS 2 nanocomposites were obtained by electrostatic interaction, and modified by bovine serum albumin (BSA) to improve their biocompatibility (Fig. S3). The TEM image in Fig. 1d illustrates that FeO nanospheres were successfully anchored on the surface of MoS 2 nanosheets. The energy dispersive X-ray (EDX) mappings shown in Fig. 1e confirm the chemical composition of FeO/MoS 2 -BSA nanocomposites. The characteristic diffractions of FeO and MoS 2 can be indexed in XRD pattern and selected area electron diffraction (SAED) of FeO/ MoS 2 -BSA nanocomposites (Figs. S1c and S4), further demonstrating the successful synthesis of FeO/MoS 2 -BSA nanocomposites. The X-ray photoelectron spectroscopy (XPS) analysis provides more information of the composition and surface electronic states of FeO/MoS 2 -BSA nanocomposites (Fig. S5). The high-resolution Fe 2p spectrum shows two peaks at 710.5 and 724.3 eV, which were attributed to Fe 2+ . The peaks at 228, 232, and 225 eV correspond to the data reported for Mo 3d 5/2 (Mo 4+ ), Mo 3d 3/2 (Mo 4+ ), and S 2s, and two peaks at 161 and 162 eV represent the 2p 3/2 and 2p 1/2 of S 2− , revealing the existence of MoS 2 in the nanocomposites [23]. The presence of the peaks of N 1s and C 1s confirms the successful modification BSA on the surface of FeO/MoS 2 . The Fourier transform infrared spectroscopy (FT-IR) results further evidence the successful BSA functionalization (Fig. S6). The hydrodynamic diameters of FeO/MoS 2 -BSA nanocomposites in normal saline and PBS solution are 227.1 and 246.0 nm with low PDI value, respectively, indicating their good dispersity and stability in physiological environments (Fig. S7, Table S1).

Synergetic Enhanced CDT/PTT of FeO/MoS 2 -BSA in vitro
To evaluate the •OH production capacity of FeO/MoS 2 -BSA nanocomposites, p-phthalic acid (PTA) was choosed as fluorescence probe to monitor the •OH production in phosphate buffer solution (PBS) with pH value of 6.0, whose fluorescence emission at 425 nm enhanced as the increasing of the amount of •OH. As shown in Fig. 2a, the •OH production ability of FeO/MoS 2 -BSA is not only higher than that of FeO and MoS 2 separately, but also the total amount of them (FeO + MoS 2 ), indicating that the co-catalyst MoS 2 can actually improve the generation efficiency of •OH. This result maybe can ascribe to the fact that the active Mo 4+ ions on the surface of MoS 2 nanosheets can reduce Fe 3+ ions into Fe 2+ ions, accelerating the conversion of Fe 3+ to active Fe 2+ . Moreover, FeO/MoS 2 -BSA nanocomposites exhibit stronger absorption at 808 and 1064 nm than that of FeO, indicating MoS 2 nanosheets endow them with good photothermal property (Fig. S8). Then, we compared the photothermal effects of different concentrations of FeO/MoS 2 -BSA nanocomposites exposed to 808 and 1064 nm laser with maximum permissible exposure (MPE, 0.3 W cm −2 for 808 nm and 1 W cm −2 for 1064 nm), respectively. As shown in Fig. 2b, c, the temperature of FeO/MoS 2 -BSA nanocomposites (200 μg mL −1 ) could increase to 57 °C after irradiation with 1064 nm laser for 10 min. In contrast, it just reaches to 30 °C under illumination with 808 nm laser in the same conditions. In fact, even irradiation with 1064 nm laser at 0.75 W cm −2 (lower than MPE: 1 W cm −2 ), FeO/ MoS 2 -BSA nanocomposites show good photothermal performances (Fig. S9). The photothermal conversion efficiency was calculated to be 56% for 1064 nm (Figs. S10 and S11), demonstrating the outstanding photothermal performance of FeO/MoS 2 -BSA nanocomposites responsive to NIR II light. As shown in Fig. S12, there is no absorption decrease in FeO/MoS 2 -BSA after irradiation for 1 h, revealing that the FeO/MoS 2 -BSA possess good photothermal stability. Importantly, the •OH production capacity of FeO/MoS 2 -BSA nanocomposites was effectively enhanced after irradiation with 1064 nm laser for 12 min (Figs. 2d and S13), which indicates that the photothermal effect of FeO/MoS 2 -BSA nanocomposites triggered by NIR II light could not only cause cancer cells death for PTT, but also achieve highly efficient CDT.
For further application in vivo, the potential cytotoxicity of FeO/MoS 2 -BSA nanocomposites was evaluated by a standard Cell Counting Kit-8 (CCk-8) assay. As shown in Fig. 3a, after incubated with different concentrations of FeO/MoS 2 -BSA nanocomposites for 24 h, no significant cytotoxicity for HeLa cells was observed, showing their good biocompatibility (Fig. S14). However, the cell viability decreased with the concentration increase in FeO/ MoS 2 -BSA nanocomposites under stimulated tumor microenvironments (100 μM H 2 O 2 ), which could be attributed to the DNA damage of HeLa cells caused by •OH production through Fenton reaction. After irradiation with 1064 nm laser for 10 min, the cell viability is no more than 10%, showing the highly efficient antitumor efficacy of combinatorial PTT and PT-enhanced CDT. To confirm the generation of active •OH in cells, 2,7-dichlorofluorescein diacetate (DCFH-DA) was used as fluorescence probe for tracking ROS production, which produces green fluorescence triggered by •OH. As shown in Fig. 3b, compared to the control and laser groups, the weak green emission was observed after treated with FeO/MoS 2 -BSA nanocomposites, demonstrating the generation •OH in HeLa cells by Fe 2+ and overpressed H 2 O 2 through Fenton reaction. Moreover, FeO/MoS 2 -BSA+ 1064 nm laser group shows obviously enhanced green fluorescence, indicating that the photothermal effects of FeO/MoS 2 -BSA nanocomposites triggered by NIR II laser illumination can significantly improve the generation efficiency of •OH. The good treatment effect of FeO/MoS 2 -BSA nanocomposites on HeLa cells was further proved by calcein AM and propidium iodide (PI) staining. As shown in Fig. 3c, the control and laser groups show the strong green fluorescence (live cells) and ignorable red fluorescence (dead cells), but the FeO/MoS 2 -BSA nanocomposites group shows enhanced red fluorescence, indicating the destruction of HeLa cells by •OH. The stronger red fluorescence was observed after irradiation with 1064 nm laser for 10 min, revealing the good antitumor effect of FeO/ MoS 2 -BSA nanocomposites by PTT and PT-enhanced CDT. All these results show that FeO/MoS 2 -BSA nanocomposites have good potential for effective PT-enhanced CDT/PTT synergistic therapy. of control group and laser group, which could be ascribed to the good co-catalytic effect of MoS 2 and FeO for CDT. In contrast, it was found that the tumors treated with FeO/ MoS 2 -BSA nanocomposites and exposed to 1064 nm laser were thoroughly ablated on the 5th day without recurrence within 2 weeks, indicating the excellent anticancer efficacy of synergistic PT-enhanced CDT/PTT. Such result was further proved by the histological analysis of tumor tissues with hematoxylineosin (H&E) and TdT-mediated dUTP-biotin nick and labeling staining (TUNEL). As shown in Fig. 4d (Fig. S18). These results reveal that FeO/ MoS 2 -BSA nanocomposites have good potential for serving as T 2 -weighted MRI contrast agents.

Conclusions
In summary, the innovative FeO/MoS 2 -BSA nanocomposites were constructed to serve as "weapon" for highly efficient synergetic enhanced CDT/PTT of cancer. Not only the FeO nanoparticles and MoS 2 nanosheets could generate the •OH individually as Fenton reagent and nanoenzyme, but also the co-catalytic effect between Mo 4+ ions and Fe 3+ ions is able to enhance Fenton reaction efficiency for more efficiency CDT. Importantly, FeO/MoS 2 -BSA nanocomposites shown excellent photothermal properties under irradiation with 1064 nm laser, achieving synergetic PT-enhanced CDT and PTT. The outstanding treatment effect has been illustrated both in vitro and in vivo. In addition, the good magnetic property enabled FeO/MoS 2 -BSA nanocomposites apply to the promising contrast agents for T 2 -weighted MRI, supplying accurate and clear information for tumor diagnosis. This collaborative strategy based on NIR II light-motivated photothermal effect and co-catalysis may provide a new idea for constructing effective Fenton nanoagents, and boost the development of CDT in future.
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