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Catalase-Modulated Heterogeneous Fenton Reaction for Selective Cancer Cell Eradication: SnFe2O4 Nanocrystals as an Effective Reagent for Treating Lung Cancer Cells Kuan-Ting Lee, Yu-Jen Lu, Fwu-Long Mi, Thierry Burnouf, YiTing Wei, Shao-Chieh Chiu, Er-Yuan Chuang, and Shih-Yuan Lu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b13529 • Publication Date (Web): 22 Dec 2016 Downloaded from http://pubs.acs.org on December 25, 2016

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ACS Applied Materials & Interfaces

Catalase-Modulated Heterogeneous Fenton Reaction for Selective Cancer Cell Eradication: SnFe2O4 Nanocrystals as an Effective Reagent for Treating Lung Cancer Cells Kuan-Ting Lee 1,¶, Yu-Jen Lu 2,¶, Fwu-Long Mi 3,4,5, Thierry Burnouf 6,7, Yi-Ting Wei 1, Shao-Chieh Chiu 8, Er-Yuan Chuang 6,7* and Shih-Yuan Lu 9 1

Plastics Industry Development Center, Technology Research Development Department, Taichung 40768, Taiwan (ROC)

2

Department of Neurosurgery, Chang Gung Memorial Hospital, Tao-Yuan 33302, Taiwan (ROC)

3

Department of Biochemistry and Molecular Cell Biology, School of Medicine, Taipei Medical University, Taipei 11031, Taiwan (ROC)

4

Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, Taipei 11031, Taiwan (ROC)

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Graduate Institute of Nanomedicine and Medical Engineering, College of Biomedical Engineering, Taipei Medical University, Taipei 11031, Taiwan (ROC)

ACS Paragon Plus Environment

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Graduate Institute of Biomedical Materials and Tissue Engineering, College of Biomedical Engineering, Taipei Medical University, Taipei 11031, Taiwan (ROC)

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International PhD program of Biomedical Engineering and Translational Therapies, College of Biomedical Engineering, Taipei Medical University, Taipei 11031, Taiwan (ROC)

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Center for Advanced Molecular Imaging and Translation, Chang Gung Memorial Hospital TaoYuan 33302, Taiwan (ROC)

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Department of Chemical Engineering, National Tsing Hua University, Hsinchu 30013, Taiwan (ROC)


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ABSTRACT Heterogeneous Fenton reactions have been proven an effective and a promising selective cancer cell treatment method. The key working mechanism for this method to achieve the critical therapeutic selectivity however remains unclear. In this study, we proposed and demonstrated for the first time the critical role played by catalase in realizing the therapeutic selectivity for the heterogeneous Fenton reaction driven cancer cell treatment. The heterogeneous Fenton reaction, with the lattice ferric ions of the solid catalyst capable of converting H2O2 to highly reactive hydroxyl radicals, can effectively eradicate cancer cells. In this study, SnFe2O4 nanocrystals, a recently discovered outstanding heterogeneous Fenton catalyst, were applied for selective killing of lung cancer cells. The SnFe2O4 nanocrystals, internalized into the cancer cells, can effectively convert endogenous H2O2 into highly reactive hydroxyl radicals to invoke an intensive cytotoxic effect on the cancer cells. On the other hand, catalase, present at a significantly higher concentration in normal cells than in cancer cells, remarkably can impede the apoptotic cell death induced by the internalized SnFe2O4 nanocrystals. According to the results obtained from the in vitro cytotoxicity study, the relevant oxidative attacks were effectively suppressed by the presence of normal physiological levels of catalase. The SnFe2O4 nanocrystals were thus proved to effect apoptotic cancer cell death through the heterogeneous Fenton reaction and were benign to cells possessing normal physiological levels of catalase. The catalase modulation of the involved heterogeneous Fenton reaction plays the key role in achieving selective cancer cell eradication for the heterogeneous Fenton reaction driven cancer cell treatment.

KEYWORDS Catalase modulation; heterogeneous Fenton reaction; lung cancer cell; selective cancer therapy; SnFe2O4; hydrogen peroxide; hydroxyl radicals


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1. INTRODUCTION Cancer therapies are practically toxic to both healthy and cancer cells. An important focus in the development of new therapeutics is to utilize characteristic differences between healthy and cancer cells so that the therapeutic treatment can be highly selective and targeting. Tumor biology has illustrated that cancer cells display augmented intrinsic oxidative stresses. Compared with normal cells, most cancer cells have intrinsically elevated levels of H2O2.1 This oxygencontaining active chemical reacts with nucleic acids, proteins and lipids. Increased H2O2 levels have been detected in cancer cells, as compared to normal cells, due to their fast proliferation and enhanced metabolic rates.2 As previously elucidated, such high level of H2O2 has been utilized for developing novel smart therapeutic weapons against cancer cells.3 Fenton reaction is a biological reaction in which transition metal ions, most notably ferrous ions, of a solution transform H2O2 into reactive hydroxyl radicals to escalate oxidative stresses capable of killing the cancer cells. The Fenton reaction however suffers from the generation of iron containing sludge (Fe(OH)3), gradually diminishing its ability for hydroxyl radical production.4 Unlike the Fenton reaction, heterogeneous Fenton reactions produce hydroxyl radicals via redox reactions between the lattice ferric ions of a solid-state iron-containing catalyst and adsorbed H2O2 molecules without the iron-containing sludge as the side product, thereby maintaining continuous active generation of hydroxyl radicals.5 The heterogeneous Fenton reaction can effectively generate hydroxyl radicals, especially in high level H2O2 environment such as tumor cells. Antioxidant enzymes in healthy body can protect cells from damages induced by free radicals. Catalase is believed to play a key role in such cellular antioxidant defense mechanisms through limiting the accumulation of H2O2,6 which can function as a substrate for generation of toxic hydroxyl radicals catalyzed by the Fenton catalyst. Cancer cells are rapidly proliferating


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cells that possess high H2O2 levels with a low catalase level in comparison with normal cells.6 Thus, catalase at normal physiological concentrations protects healthy cells from attack of the hydroxyl radicals generated by the Fenton reaction.7-8 Whether the anticancer efficacy is dependent on the generated hydroxyl radicals in the intracellular or extracellular domain is another important issue. Nanotechnologies are making significant contributions to the development of new drug delivery strategies in cancer treatments, allowing the establishment of a platform for combined therapeutics with measurable functional outcomes. Assessment of inorganic nanoparticles for biomedical applications has advanced rapidly because of the extensive work already conducted to enhance their functional performances.9-11 Metal oxide nanoparticles are, among various inorganic materials, of considerable interests. Spinel oxides, in particular, are excellent catalysts for heterogeneous Fenton processes in pollutant degradation.12 Magnetite (Fe3O4) is a typical example of an intensively investigated catalyst for the heterogeneous Fenton process and other fields. Up to now, only a few studies were conducted to investigate the feasibility of the heterogeneous Fenton reaction driven cancer cell treatment, Fe3O4 nanoparticles for HeLa cell treatments as one typical example.13 On the other hand, photodynamic therapy, for example, using ZnO or TiO2 nanoparticles as the photosensitizer under UV light irradiation for cancer cell treatments, is practically difficult to implement because of the short and insufficient penetration depth of UV light to tissues.14 So far, the application of the recently synthesized spinel oxide, SnFe2O4, has been largely unexplored, although, interestingly, it exhibits outstanding degradation efficiencies as a magnetically recyclable heterogeneous Fenton catalyst.15 Investigating the anti-cancer efficacy of these SnFe2O4 nanocrystals and unveiling their overall potential medical use are of great interest.


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In this study, we produced the SnFe2O4 nanocrystals via a one-step carrier solvent-assisted interfacial reaction process,16 and applied them in the treatment of human lung cancer cell line H460. Specifically, the effect of the extent of nanocrystal aggregation on the treatment efficacy was studied. We hypothesized that smaller size of SnFe2O4 aggregates obtained with sonication treatment is advantageous for cellular uptake of the SnFe2O4 aggregates into cancer cells and subsequent production of the lethal hydroxyl radicals through the heterogeneous Fenton reaction. The cytotoxic effect derives from the high H2O2 concentration within cancer cells, which leads to highly concentrated hydroxyl radical environment created through the heterogeneous Fenton reaction on the surface of the SnFe2O4 aggregates.

2. RESULTS AND DISCUSSION 2.1. Characteristics of Test Samples There has been flurry of significant progress in the understanding of interactions between particle size and shape for development of more efficacious nanomaterial-based drug delivery systems. In most cases, agglomeration/aggregation between particles is due to attractive van der Waals forces. Sonication is known to help physically break up agglomerated particles in a suspension.17

In

addition,

higher

particle

concentrations

lead

to

more

severe

agglomeration/aggregation. Thus, it is necessary to control the particle concentration for the sonication treatment of the SnFe2O4 nanocrystals in saline solutions. Figure 1 shows TEM images of the SnFe2O4 aggregates obtained without and with the sonication treatment for 6 h at 37°C at three particle concentrations. Evidently, these SnFe2O4 nanocrystals aggregated into large crystal clusters in the saline solution because of the shielding of the electrostatic repulsive


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forces by the presence of counter-ions, Na+ and Cl-, provided by saline. Sonication, however, lessened the extent of aggregation, leading to much smaller size aggregates. The aggregate size depended on the particle concentration, as expected, and increased with increasing particle concentration from 17 ± 7, 294 ± 90, to 570 ± 124 nm for the particle concentrations of 1, 2, and 4 mmol/L, respectively. The average size and standard deviation were determined from 10 highresolution bright field photographical TEM images of 50 particles. These aggregate sizes are significantly smaller than 7-13 µm of the non-sonicated case. According to the previous literature, smaller particles exhibit higher cellular uptakes than larger ones, and only nanoscale particles, but not micron-sized particles, may be internalized by cells.18 Therefore, the small SnFe2O4 aggregates of 17 ± 7 nm in size acquired by sonication at the particle concentration of 1 mmol/L was selected as the sonicated group for subsequent studies.


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Figure 1. TEM images of non-sonicated SnFe2O4 nanocrystals (scale bar = 200 nm), and those sonicated at concentrations of 1, 2, and 4 mmol/L. To determine the structural, morphological, and physicochemical properties of the SnFe2O4 nanocrystals, EDX, XRD, and vibrating sample magnetometer measurements were conducted. Figure 2a shows the EDX spectrum of the SnFe2O4 nanocrystals. The atomic ratio of Sn vs. Fe is reasonably close to the theoretical value of 1:2 in SnFe2O4. The XRD pattern of the present SnFe2O4 product is presented in Figure 2b. Also included for comparison is the diffraction


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pattern of the SnFe2O4 from the previous literature.15 The two patterns are in good agreement, further confirming the composition of the present product to be SnFe2O4. The magnetization curve for the SnFe2O4 product measured by a VSM is shown in Figure 2c. As expected, the SnFe2O4 product exhibited paramagnetic behavior without magnetic hysteresis.

Figure 2. (a) EDX spectrum, (b) XRD patterns, and (c) VSM profile showing physicochemical characteristics of SnFe2O4 nanocrystals.

2.2. Detection of Hydroxyl Radicals


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The heterogeneous Fenton reaction generates hydroxyl radicals via redox reactions between the surface lattice ferric ions of the SnFe2O4 nanocrystals and adsorbed H2O2 molecules, without formation of iron-containing sludge as the side product.13 One thus expects that SnFe2O4 nanostructures of high specific surface areas should effectively generate hydroxyl radicals, especially in high H2O2 concentration environment such as tumor cells.19 HPF, an excellent fluorescent indicator with high stability against light, reacts with hydroxyl radicals at high specificity and, thus, can detect the presence and concentration of the hydroxyl radicals. As

shown

in

Figure

3,

the concentration

of

the

hydroxyl

radicals

produced

correlated positively with the concentrations of H2O2 and SnFe2O4. In addition, the sonicated SnFe2O4 nanocrystals exhibited much higher efficiency than the non-sonicated nanocrystals to generate the hydroxyl radicals. Furthermore, the possible re-aggregation of the SnFe2O4 nanocrystals during the reaction with H2O2 was examined. As shown in Figure S1, no significant increases in the SnFe2O4 aggregate size were observed after incubation with H2O2 for 12 h.


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Figure 3. Concentrations of hydroxyl radicals generated from heterogeneous Fenton reactions of SnFe2O4 nanocrystals and H2O2 at increasing H2O2 concentration and different sonication treatment conditions of SnFe2O4 nanocrystals.

2.3. Cellular Uptake of Test Formulations Functional behavior and internalization of particles in drug delivery are strongly affected by the geometrical structure of the particles. Usually, spherical particles can be internalized substantially more than asymmetrically shaped particles. Particle size also appears to influence the cellular uptake mechanism and the endocytic pathway of the particles, dictating the ultimate intracellular fate and accordingly inclusive biological effects of the particles. A research review commented that particles with a size of ≤500 nm achieve significantly higher cellular uptakes than particles of larger sizes.20 We first examined whether the SnFe2O4 aggregates, sonicated or not, were indeed taken up by the cells. The extent of uptake was observed and determined with a confocal laser scanning microscope (CLSM). Figure 4a indicated that a negligible cellular uptake was achieved by the non-sonicated SnFe2O4 nanocrystals, attributable to their asymmetric shape and significantly larger particle sizes (7-13 µm, >> 500 nm). On the contrary, the results of the differential interference contrast (DIC) and superimposed fluorescent image shown in Figure 4b suggested that after incubating with test cells for 12 h, the sonicated SnFe2O4 nanocrystals were effectively taken up by the cells and accumulated within the lysosomal compartments.


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Figure 4. CLSM observation of intracellular accumulation of test particles in H460 cells that were treated and then imaged after 12 hours. The gray, green, and blue colors represent (a) DIC, (b) LysoTracker, and (c) DAPI, respectively. The red triangle indicates SnFe2O4 nanocrystals accumulated within the lysosomal compartment of the test cells.

2.4. Immunofluorescence of Reactive Oxygen Species We next examined whether catalase can be taken up by the cells and then investigated its effects on generation of reactive oxygen species in the presence of the SnFe2O4 nanocrystals, sonicated or not. In a previous morphological study, the size of the catalase molecule was assessed to be around 60 nm from an electron micrograph.21 Biological cells can readily endocytose exogenous catalase through a cold-inhibitable mechanism. The capacity of some cell


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types to pinocytose catalase was also confirmed.22 As shown in Figure 5a, the DAPI-stained cells pre-treated with catalase were clearly observed by CLSM, resulting in much stronger fluorescent signals as compared to those of the untreated cells (Figure 5b). This confirms the successful absorption of the catalase molecule into the cells.

Figure 5. (a) Fluorescent images of red signal indicate absorption of Cy5-catalase into test cells, and (b) corresponding fluorescence intensities obtained using imageJ software (n = 6). *Statistical significance indicated by P < 0.05.


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The effects of the presence of the catalase molecules on the generation of reactive oxygen species were studied next. Fig. 6a shows that the group treated with non-sonicated SnFe2O4 nanocrystals, with or without the catalase, was incapable to effectively convert H2O2 from the cancer cells into visible hydroxyl radicals (HPF). This can be attributed to the low surface area and negligible cellular uptake of the non-sonicated SnFe2O4 nanocrystals. Although some H2O2 might be partially transformed into reactive hydroxyl radicals by non-sonicated SnFe2O4 nanocrystals extracellularly, hydroxyl radicals were not effective extracellularly because of their very short half-life (9-10 s) and short diffusion radius (2.3 nm), thus limiting their cytotoxic effects toward cancer cells.23 Additionally, it is known that one catalase molecule is able to convert millions of hydrogen peroxide molecules to water and oxygen in a second. Nevertheless, owing to their progressive increases in intracellular endocytosis together with high contact surface areas, the sonicated SnFe2O4 nanocrystals were able to intracellularly convert high concentrations of endogenous H2O2 into a large amount of hydroxyl radicals (Figure 6a). Interestingly, the production of hydroxyl radicals by the sonicated SnFe2O4 crystals was significantly inhibited by the presence of the catalase molecules, which were at a significantly higher concentration in normal cells than in cancer cells. This is because the H2O2, endogenously generated by cancer cells, was decomposed by the catalase molecule, thus limiting the extent of the heterogeneous Fenton reaction for the hydroxyl radical generation (Figure 6b). Furthermore, it is also known that normal cells have low concentrations of H2O2 and high concentrations of catalase,24 which should make the sonicated SnFe2O4 nanocrystals treatment less toxic to normal cells.


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Figure 6. (a) Fluorescent images of generated hydroxyl radicals in test cells, and (b) corresponding fluorescence intensities obtained using imageJ software (n = 6). *Statistical significance indicated by P < 0.05.

2.5. Cell Viability To be cytotoxic, hydroxyl radicals may assail the sugar-phosphate backbone of DNA by drawing hydrogen atom from the deoxyribose and the attacked DNA bases by addition of OH to the double bond of purine ring. If DNA is to be disrupted by hydroxyl radicals, the reaction has


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to occur in the close vicinity of DNA.25 As is well known, hydroxyl radicals are considerably more reactive, and thus much more toxic than either H2O2 or superoxide.26 As a result, test cells treated with non-sonicated SnFe2O4 nanocrystals exhibited high cell viability (Figure 7a), with or without catalase. However, after incubating with the test cells, the sonicated SnFe2O4 nanocrystals effected a cytotoxic action on the cancer cells (Figure 7a). Next, treatment with catalase remarkably blocked the apoptotic cell death induced by the sonicated SnFe2O4 nanocrystals. These observations first demonstrated that the heterogeneous Fenton reactions, functioning through the sonicated SnFe2O4 nanocrystals, amplify the reactive oxygen species stresses to induce apoptotic cell death. The SnFe2O4 nanocrystals however were benign to the cells of physiological levels of catalase. The corresponding quantitative cytotoxicity data were determined (Figure 7b). The group that received SnFe2O4 nanocrystals without the catalase treatment exhibited a significant (P < 0.05) decrease in cell viability in a dose-dependent manner, revealing the apparent cytotoxic effect of the SnFe2O4 nanocrystals. The cytotoxic effect of these SnFe2O4 nanocrystals toward normal lung cells was also investigated. We treated normal lung fibroblasts (HEL 299, fibroblast from normal lung tissue) with the SnFe2O4 aggregates of different sizes under the same experimental conditions as for the lung cancer cells, and monitored the cell growth for 48 h with the MTT assay, taking the untreated group as a control. As evident from Figure S2, no significant cytotoxicity was observed towards the normal lung cells. The results were aggregate size independent. Furthermore, no significant differences in cell growth between the treated and untreated groups were observed. As mentioned in the previous literature,27 kidney would be capable of eventually removing such nanocrystals from the vascular compartment mainly as the injected formulation in vivo and


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therefore, renal excretion would be an appropriate pathway for the SnFe2O4 nanocrystals or their digested products removal with minimal catabolism or breakdown from the human body to avoid the possible side effects. Consequently, the designed SnFe2O4 nanocrystals are of great potential as both therapeutic reagent and drug carrier for selective targets of H2O2-rich environment, such as cancer cell microenvironment.


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Figure 7. (a) Fluorescent images of slices co-stained with LIVE(green)/DEAD(red)® viability/cytotoxicity assay kit for test cells, and (b) corresponding quantitative results obtained using MTT assay (n = 6). *Statistical significance indicated by P < 0.05.

3. CONCLUSIONS H2O2-specific redox catalyst, SnFe2O4 nanocrystals, were successfully developed to serve as a potential new therapeutic reagent for cancer treatments. Our results clearly evidence that the sonicated SnFe2O4 nanocrystals play a decisive role in the high cellular uptake and significant enhancement of intracellular hydroxyl radical generation through the heterogeneous Fenton reaction, ultimately producing a considerable cytotoxicity effect toward cancer cells, as


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illustrated in Figure 8. These results confirm the efficacy of the proposed sonicated SnFe2O4 nanocrystals in promoting the anticancer effectiveness. A crucial contribution of this developed assay is the inhibition of the heterogeneous Fenton reaction with catalase through decomposition of H2O2. It has been acknowledged that the expression of catalase is regulated at the message, protein, and activity levels.28 The tumor cell lines used in the present study are low in their catalase activity as described previously.29 Rapidly proliferating cells such as cancer cells produce abnormally high H2O2 levels. This would increase the oxidative stress during neoplastic transformation and thus increase the therapeutic selection of cells through catalase concentration levels.21 In the physiological environment, tin (Sn) is an essential micronutrient of human body, and one of the initial elements humans discovered. Recent scientific investigation shows that tin can increase the metabolism of protein and nucleic acid, helpful to body growth and development. Shortage of tin leads to sluggish development of the body, especially in children. Tin deficiency will influence the normal development, and in serious cases can cause dwarfism.30 Iron is also known to be essential for almost all living creatures, being involved in a wide variety of metabolic processes, including oxygen transportation, deoxyribonucleic acid (DNA) synthesis, and electron passage.31 Moreover, iron deficiency is related to hypoxic conditions in cancer cells owing to low haemoglobin levels.32 These SnFe2O4 nanocrystals can be effectively delivered through inhalation administration for lung cancer therapy. They can be concentrated within the cancer cells via either the enhanced permeability and retention (EPR) effect or the technique of magneticallyguided drug targeting (MGDT) because of their advantageous properties including nano-sized scale and magnetically guidability. In the normal physiological environment, the internalized


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SnFe2O4 nanocrystals can be slowly degraded in lysosomes and eventually transformed into metal ions to be rapidly excreted via the kidney. On-going studies aim at defining the optimal conditions for applications in the experimental models of the cancer, and evaluating the in vivo antitumor efficacy, toxicology, biodistribution, and excretion rate. These studies will also address the physiological interactions between the SnFe2O4 nanocrystals and relevant organs.

Figure 8. Illustration showing internalized SnFe2O4 nanocrystals performing cytotoxic effect on cancer cells intracellularly and non-cytotoxic effect on cancer cells in presence of catalase.


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SUPPORTING INFORMATION Experimental section; comparison on extent of aggregation of SnFe2O4 nanocrystals before and after reaction; examination on cytotoxicity effect of SnFe2O4 treatment for normal lung fibroblasts.

AUTHOR INFORMATION ¶

The first two authors contributed equally.

Corresponding Author *To whom correspondence should be addressed: [email protected] (E.Y. Chuang) Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was financially supported by the Taipei Medical University, Taipei, Taiwan (ROC) under grant TMU105-AE1-B09, and by the Ministry of Science and Technology of Taiwan (ROC) under grant MOST-105-2314-B-038-089-MY2.


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Catalase-Modulated Heterogeneous Fenton ... - ACS Publications

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