Nano-Fenton Reactors as a New Class of Oxidative Stress

Carbon Convergence Materials Research Center, Korea Institute of Science and .... fibroblast cell line) were obtained from Korean Cell line Bank (Kore...
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Nano-Fenton Reactors as a New Class of Oxidative Stress Amplifying Anticancer Therapeutic Agents Byeongsu Kwon,† Eunji Han,† Wonseok Yang,† Wooram Cho,† Wooyoung Yoo,† Junyeon Hwang,§ Byoung-Mog Kwon,∥ and Dongwon Lee*,†,‡ †

Department of BIN Convergence Technology, Chonbuk National University, Jeonju, Chonbuk 561-756, Republic of Korea Department of Polymer-Nano Science and Technology, Chonbuk National University, Jeonju, Chonbuk 561-756, Republic of Korea § Carbon Convergence Materials Research Center, Korea Institute of Science and Technology, Wanju, Chonbuk 565-905, Republic of Korea ∥ Laboratory of Biology and Genomics, Korea Research Institute of Bioscience and Biotechnology, Daejeon, 305-806, Republic of Korea ‡

S Supporting Information *

ABSTRACT: Cancer cells, compared to normal cells, are under oxidative stress associated with an elevated level of reactive oxygen species (ROS) and are more vulnerable to oxidative stress induced by ROS generating agents. Thus, manipulation of the ROS level provides a logical approach to kill cancer cells preferentially, without significant toxicity to normal cells, and great efforts have been dedicated to the development of strategies to induce cytotoxic oxidative stress for cancer treatment. Fenton reaction is an important biological reaction in which irons convert hydrogen peroxide (H2O2) to highly toxic hydroxyl radicals that escalate ROS stress. Here, we report Fenton reaction-performing polymer (PolyCAFe) micelles as a new class of ROSmanipulating anticancer therapeutic agents. Amphiphilic PolyCAFe incorporates H2O2-generating benzoyloxycinnamaldehyde and iron-containing compounds in its backbone and self-assembles to form micelles that serve as Nano-Fenton reactors to generate cytotoxic hydroxyl radicals, killing cancer cells preferentially. When intravenously injected, PolyCAFe micelles could accumulate in tumors preferentially to remarkably suppress tumor growth, without toxicity to normal tissues. This study demonstrates the tremendous translatable potential of Nano-Fenton reactors as a new class of anticancer drugs. KEYWORDS: cancer, Fenton reaction, hydroxyl radical, oxidative stress, polymeric micelles

1. INTRODUCTION

ROS-mediated signaling, which is required for the increased rate of growth.6,7 Nearly all nonsurgical anticancer treatments including chemotherapy, radiotherapy, and photodynamic therapy share a common mechanism, in which overproduction of ROS disrupts redox homeostasis and causes apoptotic cell death.8−11 Recently, induction of oxidative stress specifically in cancer cells has been emerging as a promising strategy for cancer treatment.6,12−16 In this regard, great efforts have been devoted to the development of logical strategies to induce ROS stress to kill cancer cells preferentially.17−20

Reactive oxygen species (ROS) are a group of small, highly reactive oxygen-derived species including superoxide, H2O2, and hydroxyl radicals, which play essential roles in living organisms.1 Despite their fundamental roles such as a second messenger in cell signaling, overproduction of ROS causes oxidative damages to biological molecules, leading to various physiological disorders such as inflammation, cancer, and neurodegenerative diseases.2,3 It has been well-known that cancer cells produce a large amount of ROS to drive cell proliferation and other events required for tumor development.4,5 Ironically, cancer cells are highly susceptible to oxidative damages induced by exogenous ROS generating agents because they function with a heightened basal level of © XXXX American Chemical Society

Received: December 22, 2015 Accepted: February 18, 2016

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DOI: 10.1021/acsami.5b12523 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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Figure 1. A schematic diagram of Nano-Fenton reactors as novel anticancer therapeutic agents. Dual acid-sensitive amphiphilic PolyCAFe is selfassembled to formulate micelles, which serve as a nanoplatform of Fenton reactions to produce toxic hydroxyl radicals, leading to apoptotic cell death.

cancer cell destruction. In this regard, we developed NanoFenton reactors as a novel anticancer therapeutic agent, which release BCA in acidic environments to generate H2O2 that is rapidly converted into hydroxyl radicals by ferrocene, leading to apoptotic cell death (Figure 1). Here, we report the translational potential of PolyCAFe micelles as a new ROS amplifying anticancer agent using cell culture models and tumor xenograft mouse models.

Approximately 70% of anticancer therapeutic agents are derived from natural compounds.21 One of the natural compounds gaining attention in medicine is cinnamaldehyde, a major component of cinnamon, which has been widely used as a flavoring agent in food and beverage.22 Cinnamaldehyde contains α,β-unsaturated carbonyl Michael pharmacophore and has been reported to exert antiproliferative activities and cause apoptotic cell death through the generation of ROS including H2O2, ROS-mediated mitochondrial permeability transition and caspase activation.23−26 BCA (benzoyloxycinnamaldehyde) is derived from cinnamaldehyde and has higher anticancer activity than cinnamaldehyde.24 However, its clinical applications are hampered by the low bioavailability and less therapeutic activity than commercial anticancer drugs.25,27 Therefore, delivery of BCA to cancer cells with other therapeutic agents in synergic combination is a logical strategy to maximize the anticancer efficacy of BCA. One of reasonable approaches to amplify ROS stress in anticancer therapy involves Fenton reaction, in which H2O2 is converted into highly toxic hydroxyl radical catalyzed by Fe2+/ Fe3+ ions.28,29 Hydroxyl radical is known to oxidize biological molecules to induce apoptotic cell death.30 In addition, a number of studies have reported that hydroxyl radicals generated via Fenton reactions are responsible for the toxicity of magnetic iron oxide nanoparticles, MRI imaging agents.31,32 It is therefore critical to develop strategies to allow Fenton reaction specifically in cancer cells to minimize toxicity to normal cells. Ferrocene is an iron-containing organometallic compound and undergoes oxidation to produce Fe2+ in acidic aqueous solutions, which catalyzes the conversion of H2O2 into hydroxyl radicals.29,30 It was therefore reasoned that codelivery of H2O2-generating BCA and ferrocene to cancer cells would yield hydroxyl radicals via Fenton reactions to enhance oxidative stress and provide a logical strategy to target selective

2. MATERIALS AND METHODS 2.1. Materials. BCA was obtained from Korea Research Institute of Bioscience and Biotechnology. 2-Hydroxyl ethyl acrylate, hydroquinone, p-toluenesulfonic acid, monomethoxy poly(ethylene glycol) (PEG, MW 2000 Da), benzene, terephthalic acid, and trimethylamine were purchased from Sigma-Aldrich (St. Louis, MO). Aminoferrocene was obtained from TCI (Tokyo, Japan). All materials were used as received without purification. Dichloromethane (Samchun, Korea) was used after drying with calcium hydride. SW620 (colon cancer cell line), DU145 (prostate cancer cell line), HEK 293 (human embryonic kidney cell line), and NIH3T3 cells (mouse fibroblast cell line) were obtained from Korean Cell line Bank (Korea). 2.2. Synthesis of BCA-Containing Diacrylate 1,(3-(2Benzoyloxy)phenylprop-2-ene-1,1-diyl) Bis(oxy)bis(ethnane2,1-diyl) diacrylate. 2-Hydroxyl ethyl acrylate (59.46 mmol) and hydroquinone (7.93 mmol) were added in dry benzene at 50 °C, to which were added BCA (19.82 mmol) and p-toluenesulfonic acid (0.1 mmol). The mixture was heat up to 92 °C and the reaction was allowed for 12 h. The reaction mixture was cooled down to room temperature and quenched by the addition of trimethylamine. The product was obtained after solvent evaporation using a rotary evaporator (Eyela, Japan). The compound, 1 was collected from column chromatography using a mixture of hexane/ethyl acetate (8/2) and vacuum-dried. 1H NMR was utilized to confirm the chemical structure of 1. 2.3. Synthesis of PolyCAFe. 4.4′-Trimethylene-dipiperidine (0.45 g, 2.14 mmol), aminoferrocene (0.048 g, 0.24 mmol), methoxy PEG monoacrylate (0.49 g, 0.24 mmol), and 1 (1 g, 2.14 mmol) were B

DOI: 10.1021/acsami.5b12523 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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0.1% Formic acid in acetonitrile): 0 → 1 min, 5% B; 1 → 10 min, 5% B; 10 → 14 min, 100% B, 14 → 20 min, 5% B) at a flow rate of 0.5 mL/min. Multiple reaction monitoring (MRM) was conducted by m/z 148 → 118.9 for 2′-hydroxycinnamaldehyde. Ions were generated in negative ionization mode using electrospray ionization interface. The fragmentor potential was set to 90 V and the collision energy (CID) was set to 15. 2.9. Cytotoxicity of PolyCAFe Micelles. 3-(4,5-Dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay was used to determine the cytotoxicity of PolyCAFe micelles. SW620, DU145, HEK 293 or NIH3T3 cells (3 × 105 cells/well in a 24 well plate) were cultured for 24 h to reach ∼90% confluency. Cells were treated with various formulations for 24 h. One hundred microliter of MTT solution was added to each well and cells were incubated for 2 h. Each well was treated with 900 μL of dimethyl sulfoxide for 10 min to dissolve the resulting formazan crystals. The absorbance of each well was recorded using a microplate reader (Biotek Instruments, Winooski, VT) at 570 nm and the cell viability was determined by comparing with the absorbance of untreated control cells. 2.10. Flow Cytometry for Apoptosis Assay. SW620 cells (5 × 105) cultured in a 12 well culture plate with 90% confluency were treated with free BCA, ferrocene, BCA along with ferrocene or PolyCAFe micelles for 24 h. Cells were washed with fresh medium twice and resuspended in 1× binding buffer at a concentration of 1 × 105 cells/mL. The cell suspension (100 μL) was transferred to a 5 mL culture tube for analysis. For ROS detection, cells were treated with DCFH-DA (2′,7′-dichlorofluorescein-diacetate). For apoptosis assay, cells were treated with Annexin V-FITC and propidium iodide, followed by gentle mixing. The cells were incubated for 15 min at room temperature in the dark and added with 400 μL of 1× binding buffer. The stained cells were analyzed by flow cytometry (FACS caliber, Becton Dickinson, San Jose, CA). A total of 1 × 104 events were counted for analysis. 2.11. DNA Fragmentation. SW 620 cells (2 × 106/well) cells were treated with various concentrations of BCA, ferrocene, combination of BCA with ferrocene or PolyCAFe micelles for 12 h and then washed with fresh PBS twice. Chromosomal DNA was extracted from the apoptotic cells using Quick Apoptotic DNA Ladder Detection Kit (Life Technologies, Frederick, MD). In brief, cells were lysed with TE buffer by pipetting up and down several times. The crude lysates were treated with Enzyme Solution until they became clear and mixed with ammonium acetate and ethanol. The mixtures were vortexed at −20 °C for 10 min to allow DNA to precipitate. The mixtures were centrifuged at 10 000×g to collect precipitated DNA. After careful removal of supernatant, DNA pellets were washed with 70% cold ethanol and centrifuged at 10 000×g for 10 min. The supernatants were discarded and DNA was air-dried at room temperature. DNA was then suspended with DNA Suspension Buffer by pipetting up and down several times. DNA was separated on agarose gel (1.8%), stained with ethidium bromide and photographed under UV light. 2.12. Mouse Model of Tumor Xenografts. SW620 cells (2.5 × 106 cells) were injected into the flank of nude mice (4 weeks old, Orient Bio, Seoul, Korea) to establish a subcutaneous tumor. Mice were intravenously injected through a tail vein with one of the following: BCA (0.6 mg/kg), ferrocene, (0.15 mg/kg), and BCA along with ferrocene or PolyCAFe micelles (3 mg/kg). Mice were treated every 3 days for 21 days. The tumor volume and body weight of mice were recorded every 3 days. Tumor volume was determined using the following formula: (width2 × length)/2. For the biodistribution studies, mice were intravenously injected with IR820-labeled PolyCAFe micelles (6 mg/kg), and the fluorescence images were made using a fluorescence imaging system (FOBI, Neoscience, Korea). For LC−MS/MS analysis, tumors were frozen in liquid nitrogen and ground into a fine power using a pestle and mortar. Tumor lysates were analyzed using the same procedure for cell lysates. Animal experiments were conducted in accordance with the national guidelines and approved by the Institution Animal Ethical Committee (CBU2014−00024).

completely dissolved in dry dichloromethane. The solution was heated to 35 °C to initiate the polymerization reaction. After 96 h of polymerization reaction, the mixture was added into cold hexane to terminate the reaction. Wine colored solid polymer was obtained from vacuum drying. Its chemical structure was confirmed by 1H NMR. 1H NMR in CDCl3 on a 400 MHz spectrometer: δ 3.77 (m, 4H), 3.89 (m, 4H), 5.23 (d, 2H), 5.84 (d, 2H), 6.16 (m, 4H), 6.43 (t, 1H), 7.24−7.7 (m, 8H), and 8.2 (m, 2H). Its molecular weight was measured using gel permeation chromatography (Polymer Laboratories/PL-GPC 110). The ratio of blocks in PolyCAFe was confirmed by Energy Dispersive X-ray Spectroscopy (EDS, SUPRA 40VP, Carl Zeiss, Germany). 2.4. Synthesis of IR820-Conjugated PolyCAFe. 4.4′-Trimethylene-dipiperidine (0.35 g, 1.66 mmol), aminoferrocene (0.05 g, 0.25 mmol), tyramine (0.065g, 0.48 mmol), methoxy PEG monoacrylate (0.49g, 0.24 mmol), and 1 (1 g, 2.14 mmol) were completely dissolved in dry dichloromethane. Polymerization was allowed at 35 °C to for 4 days and terminated by precipitation in cold hexane. After identifcation with 1H NMR, the obtained polymer was dissolved in dimethylformamide, to which IR-820 (Sigma-Aldrich, St. Louis, MO) was added. Conjugation was allowed for 24 h at room temperature in dark. IR820-conjugated PolyCAFe was obtained from multiple precipitation in cold hexane and filtration. FT-IR (Spectrum GX, Perkin Elemer) was used to confirm the conjugation of IR820 to the OH groups of tyramine in the bacbone of PolyCAFe. 2.5. Preparation and Characterization of PolyCAFe Micelles. PolyCAFe (1 mg) was dissolved in 100 μL of acetone. The solution was added into 10 mL of phosphate buffer saline (PBS, pH 7.4). After complete evaporation of acetone using a rotary evaporator, polyCAFe micelles (100 μg/mL) were obtained. The hydrodynamic size of polyCAFe micelles at a concentration above CMC (critical micelle concentration) were measured using a particle size analyzer (90Plus, Brookhaven Instrument Corp., Holtsville, NY) with 2 min of data acquisition time. The micelles negatively stained with a tungstic acid solution were observed using a transmission electron microscope (BioTEM, HITACHI Corp., Japan). 2.6. Determination of CMC of PolyCAFe Micelles. The CMC of PolyCAFe micelles was determined using a fluorospectrometer (FP6500, JASCO Corp., Japan) and pyrene as a fluorescence probe. Various concentrations of PolyCAFe micelles were prepared in PBS containing the same concentration of pyrene (2.5 μM). The fluorescence emission spectra of pyrene-loaded PolyCAFe micelles were recorded with excitation wavelength at 334 nm. The intensity ratio of pyrene at 373 and 384 nm was plotted against the PolyCAFe concentrations to determine the CMC. 2.7. Release Kinetics of BCA from PolyCAFe Micelles. A membrane dialysis tube (Cutoff MW 1000, Sigma-Aldrich, St. Louis, MO) was loaded with 1 mL of PolyCAFe micelles (1 mg/mL). The tube was immersed in 50 mL of buffer solution (pH 7.4 or 5.4) and was mechanically stirred at 37 °C. At appropriate time intervals, 1 mL of solution was removed and replaced with the same volume of fresh buffer solution. The amount of BCA released from the micelles was determined using high performance liquid chromatography (HPLC, Futex, Korea) at 294 nm by comparing with BCA standard solutions. 2.8. Liquid Chromatography−Mass Spectroscopy (LC−MS/ MS) of Cell Lysates. SW620 cells were seeded in a 10 cm culture dish containing 10 mL of medium and allowed to attach for 24 h. Cells were treated with 100 μM of BCA or 100 μg/mL of PolyCAFe micelles. After 24 h of incubation, cells were washed with new medium, and cell pellets were added with 100 μL of methanol. The mixture was vortexed and then additional 900 μL of methanol was added. The contents were thoroughly mixed by vortexing and high molecular weight materials were removed by sequential centrifugation (10 000×g) for 10 min. The supernatant was immediately stored at −80 °C until analysis. Upon analysis, 5 μL was injected and the peak for 2′-hydroxycinnamaldehyde was analyzed using a LC−MS/MS spectrometer (6410 Triple Quad LC/MS/MS, Agilent Technologies, Willington, DE) equipped with a column (Synergi 4 μ Hydro RP 80A, 150 × 2.00 mm). 2′-Hydroxycinnamaldehyde was eluted with a gradient of water and acetonitrile (A = 0.1% Formic acid in water; B = C

DOI: 10.1021/acsami.5b12523 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces Scheme 1. Synthetic and Degradation Routes of PolyCAFe

Figure 2. Characterization of pH-sensitive PolyCAFe micelles. (A) A representative dynamic light scattering of PolyCAFe micelles. The inset is a representative transmission electron micrograph. (B) 1H NMR spectrum of PolyCAFe micelles formulated in D2O. (C) Determination of CMC of PolyCAFe micelles based on the index of micelle hydrophobicity. (D) Change of a mean diameter of PolyCAFe micelles in the presence of 10% serum as a function of time. HCl was added to reduce the pH to 6.0. Values are mean ± s.d. (n = 3). (E) The effects of pH on the stability of PolyCAFe micelles. PolyCAFe micelles were prepared at different pH values from 5.0 to 8.0 and the fluorescence intensity was measured immediately after micelle formation. (F) Release kinetics of BCA from PolyCAFe micelles at different pH values. Values are mean ± s.d. (n = 4). 2.13. Statistical Analysis. Values were expressed as mean ± s.d. Comparison between groups was conducted with one-way ANOVA using GraphPad Prism 5.0 (San Diego, CA). A difference of p < 0.05 was considered statistically significant.

molecularly engineered to possess both BCA and ferrocene in its hydrophobic backbone and have hydrophilic PEG, allowing micelle formation by self-assembly in aqueous solutions. The hydrophobic backbone of PolyCAFe has amino groups which could undergo acid-triggered hydrophobic−hydrophilic transitsion. In addition, BCA was incorporated in the backbone of PolyCAFe through an acetal linkage that is rapidly cleaved at acidic pH.33,34 We therefore hypothesized that the micellar

3. RESULTS 3.1. Synthesis and Characterization of PolyCAFe Micelles. Nano-Fenton reactors are formulated from a dual acid-sensitive biodegradable polymer (PolyCAFe) that was D

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Figure 3. Amplification of intracellular ROS stress by PolyCAFe micelles. (A) Generation of intracellular ROS in SW620 cells determined by flow cytometric analysis. (B) Fluorescence intensity of terephthalic acid in cells treated with PolyCAFe micelles. Data are a representative of three independent experiments.

mL. As shown in Scheme 1, PolyCAFe was designed to have two acid-sensitive moieties, an acetal linkage and a protonable amine group and undergo pH-dependent phase transition behaviors. We therefore investigated the pH-responsiveness of PolyCAFe micelles over a wide range of pH. PolyCAFe micelles were stable at physiological pH in the presence of serum for 24 h, but the reduction of pH induced rapid dimicellization (Figure 2D). The rapid pH-dependent disruption of PolyCAFe micelles is because of tertiary amino groups (pKb = 6.5) in the hydrophobic backbone that are protonated at acidic pH and become hydrophilic, leading to rapid dissociation of micelles.25,36 pH-dependent dissociation of PolyCAFe micelles was also evidenced by the gradual decrease of I384/I373 (Figure 2E). BCA was incorporated into the backbone of PolyCAFe via acid-cleavable acetal linkages.25,37 1H NMR was employed to examine the pH-dependent release of BCA from PolyCAFe micelles. As shown in Figure S5, during hydrolytic degradation of PolyCAFe at pH 5.4, the signal intensity at 5.2 ppm corresponding to acetal protons decreased gradually. However, the intensity of the aldehyde proton at ∼9.6 ppm increased with time. PolyCAFe showed pH-dependent BCA releases, with >40% release within 24 h at pH 5.4, but ∼15% release within 72 h at pH 7.4 (Figure 2F). These observations demonstrate that PolyCAFe micelles undergo dual acidresponsive changes, rapid micelle dissociation, and acidtriggered BCA release. 3.2. Induction of ROS Stress in Cells. In proof-of-concept studies using cell cultures, we first investigated whether PolyCAFe micelles deliver BCA to cancer cells. Human colon cancer SW620 cells were treated with PolyCAFe micelles for 12 h and lysed for LC−MS/MS analysis. The generation of BCA from PolyCAFe micelles in cells was confirmed by the detection of 2-hydroxycinnamaldehyde, a putative metabolite of BCA in a body (Figure S6).27 The results demonstrate that PolyCAFe micelles are readily taken up by cells via probably endocytosis and release BCA. The ability of PolyCAFe micelles to amplify ROS stress in cancer cells was investigated using DCFH-DA which can be converted to fluorescent DCF (2′,7′-dichlorofluorescein) by ROS (Figure 3A).38 Ferrocene alone (12.5 μM) marginally increased the ROS stress due to Fenton reactions with endogenous H2O2 in cancer cells. BCA (100 μM) also induced moderate ROS stress as previously reported.26,39 Co-delivery of BCA and ferrocene induced significantly higher DCF

Nano-Fenton reactors readily dissociate in acidic environments, leading to concomitant release of BCA and ferrocene.35 For the synthesis of PolyCAFe, we first synthesized BCAcontaining diacrylate 1, 3-(2-(benzoyloxy)phenyl)prop-2-ene1,1-diyl)bis(oxy))bis(ethane-2,1-diyl)diacrylate from the reaction of 2-hydroxyethyl acrylate and BCA. BCA was covalently incorporated in 1 via an acid-cleavable acetal linkage, confirmed by 1H NMR (Figure S1 of the Supporting Information). Methoxy PEG monoacrylate was synthesized using monomethoxy PEG with acryloyl chloride. PolyCAFe was synthesized from the reaction of BCA-containing diacrylate 1, aminoferrocene, trimethylene dipiperidine, and PEG monoacrylate at a molar ratio of 0.9:0.1:0.9:0.1 (Scheme 1). The random block copolymer PolyCAFe was obtained as winecolored solid after polymerization at 35 °C for 4 days. 1 H NMR was utilized to confirm the chemical structure of PolyCAFe. Acetal protons appear at 5.2 ppm and ethylene protons between ester and amine groups appear at 2.8 and 3.6 ppm (Figure S2). PolyCAFe has the same composition ratio as the feeding ratio, determined by the ratio of Fe and N atoms in the EDS spectrum (Figure S3). These observations demonstrate that PolyCAFe was successfully synthesized which covalently incorporates BCA in its backbone via acetal linkages. Its weight-average molecular weight was determined to be ∼8000 Da by gel permeation chromatography (Figure S4). Amphiphilic PolyCAFe self-assembled to form micelles with a mean diameter of ∼160 nm determined by dynamic light scattering, which is further confirmed by the transmission electron micrograph (Figure 2A). 1H NMR was also utilized to confirm the formation and structure of PolyCAFe micelles. Figure 2B shows the 1H NMR spectrum of PolyCAFe micelles formulated in D2O. One strong signal at ∼3.7 ppm corresponds to ethylene protons of PEG. However, other protons in the polymer backbone are not observed, indicating that BCA-based hydrophobic segments form the core of micelles and hydrophilic PEG is on the surface of micelles as a corona. The CMC of PolyCAFe micelles was determined using pyrene as a hydrophobic fluorescent probe. We monitored the fluorescence emission intensity of pyrene-loaded PolyCAFe micelles at 384 and 373 nm, and their ratio was measured as an index of micelle hydrophobicity. Figure 2C shows the index of micelle hydrophobicity (I384/I373) as a function of PolyCAFe concentrations, indicating that PolyCAFe forms thermodynamically stable micelles at concentrations higher than ∼15 μg/ E

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Figure 4. In vitro anticancer activity of Fenton reaction-performing PolyCAFe micelles. Cytotoxicity of PolyCAFe micelles to SW620 cells (A), DU145 cells (B), and HEK293 cells (C). *p < 0.05, ***p < 0.001. Values are mean ± s.d. (n = 4). (D) Nucleosomal DNA fragmentation in SW620 cells induced by PolyCAFe micelles. Data are a representative of three independent experiments.

fluorescence, indicating that BCA synergized with ferrocene to escalate ROS stress due to Fenton reactions. PolyCAFe micelles caused remarkable elevation of ROS stress in a concentration dependent manner, which is attributed to BCA-induced H2O2 and subsequent hydroxyl radical production via Fenton reactions. The concentrations of ferrocene (12.5 μM) and BCA (100 μM) are almost equivalent to those released from 100 μg/mL of PolyCAFe micelles. PolyCAFe micelles-mediated ROS stress was significantly inhibited by the pretreatment with H2O2-scavenging catalase, because H2O2 generated by PolyCAFe was decomposed by catalase, and the lower extent of Fenton reactions was allowed to generate hydroxyl radicals. The cellular generation of hydroxyl radicals by PolyCAFe micelles was further substantiated by the increased fluorescence intensity of terephthalic acid, which captures hydroxyl radicals to emit unique fluorescence (430 nm) (Figure 3B).40 PolyCAFe micelles induced the significantly higher fluorescence intensity of terephthalic acid than cotreatment of ferrocene and BCA. In addition, the fluorescence intensity was significantly reduced by hydroxyl radical-scavenging thiourea. These observations demonstrate that PolyCAFe micelles induce the generation of hydroxyl radicals via Fenton reactions between BCA-mediated H2O2 and ferrocene. 3.3. Anticancer Activity of PolyCAFe Micelles in Vitro. Cytotoxicity of PolyCAFe micelles was evaluated using cancer cells (colon cancer cell line SW620 and prostate cancer cell line DU145) and normal cells (human embryonic kidney cell line HEK 293 and mouse fibroblast NIH3T3 cell). BCA (100 μM) significant killed SW620 and DU145 cells. Ferrocene alone (12.5 μM) showed negligible effects on cell viability. However,

the combination of BCA and ferrocene exerted remarkably higher cytotoxicity than BCA and ferrocene. PolyCAFe micelles induced dose-dependent cytotoxicity, with more than 70% cell death at a dose of 100 μg/mL. PolyCAFe micelles-mediated cytotoxicity was significantly inhibited in the presence of catalase, indicating that H2O2 was decomposed by catalase and less extent of Fenton reaction was allowed to generate toxic hydroxyl radicals. In good accordance with Figure 3A, these observations indicate that cytotoxicity of PolyCAFe micelles is mainly attributed to oxidative stress and H2O2 is a primary reactive oxygen intermediate of their synergistic anticancer activity (Figure 4A,B). Interestingly, PolyCAFe micelles exhibited no or minimal toxicity to HEK293 cells and NIH3T3 cells (Figure 4C, Figure S7). Cancer cell-specific toxicity of oxidative stress amplifying PolyCAFe micelles can be explained by the rationales that normal cells have sufficient antioxidants and cancer cells are more susceptible to ROS insults due to altered redox balance.14,15,41 We studied the effects of PolyCAFe micelles on DNA fragmentation which is a hallmark of apoptosis.42 As shown in Figure 4D, after 12 h of treatment, BCA alone and ferrocene alone induced minimal DNA fragmentation. BCA synergized with ferrocene to escalate ROS stress, inducing more DNA fragmentation. PolyCAFe micelles induced considerable nucleosomal DNA fragmentation as clear DNA ladders of multiples of 180 bp, characteristics of apoptotic DNA fragmentation.43 However, PolyCAFe-induced DNA fragmentation was remarkably suppressed by catalase. The results of DNA fragmentation were further supported by the flow cytometric assay using Annexin V-FITC as an apoptosis marker and propidium iodide as a cell viability marker. As shown in F

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Figure 5. Flow cytometric analysis of SW620 cells using Annexin V-FITC and propidium iodide. Cells were treated with various formulations for 24 h. A total of 1.0 × 104 cells stained were subjected to flow cytometric analysis to determine the distribution of cells. The data shown are representative of three independent experiments.

Figure 6. PolyCAFe micelles as therapeutic anticancer agents. (A) Gross images of tumor bearing mice treated with various formulations. Arrows indicate tumors, and the bottom panel is the images of tumors removed. (B) Changes in tumor volumes during the treatment. Arrows indicate the date of treatment. ***p < 0.001 relative to BCA + Ferrocene (n = 4). (C) Changes in the body weight of tumor bearing mice during the treatment. Arrows indicate the date of treatment.

Figure 5, treatment with BCA alone and ferrocene alone induced slight apoptosis. Co-treatment of BCA and ferrocene showed a greater population of apoptotic cells, demonstrating that BCA synergized with ferrocene for inducing apoptotic cell death. However, PolyCAFe micelles induced more apoptotic cell death than BCA, ferrocene and their combination, in a dose dependent manner. More than 80% of cells were in the late stage of apoptosis by the treatment of 100 μg/mL of PolyCAFe micelles. Pretreatment with catalase remarkably blocked the apoptotic cell death induced by PolyCAFe micelles. These observations demonstrate that Fenton reaction-performing PolyCAFe micelles amplify the ROS stress to induce apoptotic cell death. 3.4. Anticancer Activity of PolyCAFe Micelles in Vivo. Anticancer therapeutic activity in vivo of PolyCAFe micelles

was evaluated using a mouse model of tumor xenografts. Human colon cancer SW620 cells were inoculated in the flank of nude mice and mice were treated with various formulations through tail vein injection after tumors reached about ∼5 mm3. Untreated mice showed gradual and continuous tumor growth (Figure 6A, B). Treatment with ferrocene alone or BCA alone showed moderate inhibitory effects on tumor growth. Cotreatment of ferrocene and BCA showed negligible synergistic anticancer effects, despite their synergistic anticancer activities in vitro. Insufficient anticancer effects of BCA and cotreatment with ferrocene can be explained by their short half-life and lack of ability to target tumors. However, remarkable reduction in tumor volume was observed with PolyCAFe micelles which generate toxic hydroxyl radicals via Fenton reactions, without apparent changes in body weight (Figure 6C). G

DOI: 10.1021/acsami.5b12523 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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Figure 7. H&E staining of tumors treated with various formulations (×400).

Figure 8. TUNEL staining of tumors treated with various formulations (×200).

Figure 9. Fluorescence imaging of tumors with IR820-labeled PolyCAFe micelles. (A) Chemical structure of polyCAFe labeled with IR820. (B) Representative fluorescent images of tumor-bearing mice treated with IR820-labeled PolyCAFe micelles. (C) Fluorescence images of organs from tumor-bearing mice treated with IR820-labeled PolyCAFe micelles.

Histological examination was performed to further confirm the therapeutic efficacy of PolyCAFe micelles. Anticancer

effects of PolyCAFe micelles were evidenced by a number of dead cells without nuclei and disruption of cell membranes H

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concurrently H2O2-generating agents and ferrocene into the site of tumors to achieve maximal therapeutic activity through Fenton reactions, while minimizing side effects to normal cells. In this context, we molecularly engineered PolyCAFe as a platform for Nano-Fenton reactors, which incorporates both H2O2-generating BCA and ferrocene in a backbone of biodegradable poly(β-amino ester). Poly(β-amino ester) was employed as a scaffold because of its excellent chemical tunability, biocompatibility, and ease of synthesis.33,46,47 In addition, PolyCAFe was designed as a dual acid-sensitive polymer to afford both BCA and ferrocene in the acidic tumor site. BCA synergized with ferrocene to generate highly toxic hydroxyl radicals and escalate ROS stress, leading to significant elimination of cancer cells in vitro (Figures 3 and 4). As expected, PolyCAFe micelles could serve as a Nano-Fenton reactor, which releases BCA and ferrocene in cells to generate hydroxyl radicals through Fenton reactions. In cell culture models, the combination of BCA and ferrocene exerted the significantly higher cytotoxicity than BCA alone and ferrocene alone. However, cotreatment of BCA with ferrocene exhibited no synergic therapeutic anticancer activity in tumor-bearing mice. The doses of BCA (0.6 mg/kg) and ferrocene (0.15 mg/ kg) are almost equivalent to those released from PolyCAFe micelles (3 mg/kg) which displayed significant inhibitory effects on tumor growth. It can be explained by the rationale that there is very little chance that BCA and ferrocene concurrently accumulate in tumors due to their hydrophobicity and lack of targeting ability. These observations clearly suggest the necessity for the development of strategies to deliver H2O2generating agents and irons concurrently into the tumor site to achieve Fenton reaction. As a Nano-Fenton reactor, PolyCAFe micelles exerted significant anticancer activity in tumor-bearing mice (Figure 6). The results of bioimaging and LC−MS/MS analysis suggest that the remarkable therapeutic anticancer effects of PolyCAFe micelle can be explained by their preferential accumulation in tumors resulting from the hydrophilic surface and small size (∼160 nm) (Figure 9, Figure S11). Previously, Huang et al. reported that combination of iron oxide nanoparticles with ROS-generating β-lapachone effectively and specifically kill cancer cells by generating hydroxyl radicals through Fenton reaction.30 However, this twocomponent anticancer system has a drawback due to the slow release of iron ions from the nanoparticles. Cells were pretreated with iron oxide nanoparticles for 48 h to allow the sufficient release of iron ions before exposure to β-lapachone, which could limit their clinical applications. In contrast, PolyCAFe micelles are a single component system, in which both H2O2-generating agents and irons are incorporated in the same backbone of polymers, which provides an advantage over the two-component anticancer system. Moreover, although in vitro anticancer efficacy of iron oxide nanoparticles in combination with β-lapachone was well established, their therapeutic anticancer efficacy in vivo was not investigated. In the present work, we demonstrated that Fenton reactionperforming PolyCAFe micelles could induce apoptotic cancer cell death and suppress the tumor growth in the study of mouse tumor xenograft models. To our best knowledge, this is the first study reporting the well-established anticancer activity of Fenton reaction-performing polymeric systems in the study of both cell culture and mouse models. However, safety and toxicity of PolyCAFe micelles will need to be thoroughly

(Figure 7). In order to verify whether PolyCAFe micelles induce apoptotic cell death, TUNEL staining was also performed (Figure 8). The combination of BCA and ferrocene induced apoptotic cell death more than ferrocene alone and BCA alone. However, a number of green TUNEL-positive apoptotic cells were observed with a PolyCAFe micelles-treated group. In addition, PolyCAFe micelles showed no apparent damages to liver and heart (Figures S8−S9), indicating that PolyCAFe micelles exerted limited adverse side effects to organs. In order to demonstrate the accumulation of PolyCAFe micelles in tumors, we developed PolyCAFe labeled with fluorescent IR820 (Figure 9A; Figure S10). PolyCAFe-IR820 micelles had almost the same size and stability as unlabeled PolyCAFe micelles, indicating that conjugation with IR820 did not affect the micelle formation. After intravenous injection of PolyCAFe-IR820 micelles into tumor-bearing mice, a strong fluorescence signal was observed at tumors preferentially, which lasted for more than 48 h (Figure 9B, C), demonstrating that PolyCAFe micelles are stable enough to circulate in bloodstream and passively target tumors due to enhanced permeation and retention effects.34,44 Tumor targeting of PolyCAFe micelles was further confirmed by LC−MS/MS analysis of tumor lysates (Figure S11). 2-Hydroxycinnamaldehyde, an active metabolite of BCA, was detected in only tumors of mice treated with PolyCAFe micelles, demonstrating that PolyCAFe micelles passively target tumors due to their size and release BCA in the acidic tumor environment. These results also support the superior in vivo anticancer activities of PolyCAFe micelles over the combined use of BCA with ferrocene which lack the ability to target tumor sites.

4. DISCUSSION ROS are known to have double-edged properties that could determine the fate of cancer cells, depending mainly on their concentration and duration time.45 Cancer cells are under oxidative stress associated with the increased generation of ROS which promote proliferation and migration of cancer cells. However, excessive accumulation of ROS could induce apoptosis of cancer cells.34,42 On the basis of the elevated ROS level and altered redox balance in cancer cells, modulation of ROS stress has recently became a new therapeutic strategy in the development of anticancer drugs.16,20 Fenton reaction is an essential biological reaction in which ferrous ions convert mild oxidant H2O2 to highly deleterious hydroxyl radical which is the most reactive among ROS. There are a few studies which report the potential of anticancer agents which exploit Fenton reaction to kill cancer cells preferentially.17,29,30 In particular, paramagnetic iron oxide nanoparticles in combination with ROS-generating drug β-lapachone generate hydroxyl radicals to improve anticancer drug efficacy.30 It was reasoned that iron ions released from iron oxide nanoparticles react with H2O2 generated from β-lapachone to produce hydroxyl radicals through Fenton reaction, leading to enhanced cancer cell death. Tamoxifen-ferrocene conjugate was also developed as a cancer cell-specific prodrug that generates apoptosis-stimulating hydroxyl radicals.29 Tamoxifen was served as a carrier to deliver ferrocene, which could react with overproduced H2O2 in cancer cells, leading to elimination of cancer cells. However, as the toxicity of iron oxide nanoparticles is ascribed to mainly hydroxyl radicals generated from the Fenton reaction,31,32 it is critical to deliver I

DOI: 10.1021/acsami.5b12523 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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(8) Wang, J.; Yi, J. Cancer cell killing via ROS: To increase or decrease, that is the question. Cancer Biol. Ther. 2008, 7, 1875−1884. (9) Trachootham, D.; Zhou, Y.; Zhang, H.; Demizu, Y.; Chen, Z.; Pelicano, H.; Chiao, P. J.; Achanta, G.; Arlinghaus, R. B.; Liu, J.; Huang, P. Selective killing of oncogenically transformed cells through a ROS-mediated mechanism by β-phenylethyl isothiocyanate. Cancer Cell 2006, 10, 241−252. (10) Fruehauf, J. P.; Meyskens, F. L., Jr. Reactive oxygen species: a breath of life or death? Clin. Cancer Res. 2007, 13, 789−794. (11) Kimani, S. G.; Phillips, J. B.; Bruce, J. I.; MacRobert, A. J.; Golding, J. P. Antioxidant Inhibitors Potentiate the Cytotoxicity of Photodynamic Therapy. Photochem. Photobiol. 2012, 88, 175−187. (12) Hagen, H.; Marzenell, P.; Jentzsch, E.; Wenz, F.; Veldwijk, M. R.; Mokhir, A. Aminoferrocene-based prodrugs activated by reactive oxygen species. J. Med. Chem. 2012, 55, 924−934. (13) Watson, J. Oxidants, antioxidants and the current incurability of metastatic cancers. Open Biol. 2013, 3, 12014410.1098/rsob.120144 (14) Trachootham, D.; Alexandre, J.; Huang, P. Targeting cancer cells by ROS-mediated mechanisms: a radical therapeutic approach? Nat. Rev. Drug Discovery 2009, 8, 579−591. (15) Wang, J.; Yi, J. Cancer cell killing via ROS To increase or decrease, that is the question. Cancer Biol. Ther. 2008, 7, 1875−1884. (16) Gorrini, C.; Harris, I. S.; Mak, T. W. Modulation of oxidative stress as an anticancer strategy. Nat. Rev. Drug Discovery 2013, 12, 931−947. (17) Hagen, H.; Marzenell, P.; Jentzsch, E.; Wenz, F.; Veldwijk, M. R.; Mokhir, A. Aminoferrocene-Based Prodrugs Activated by Reactive Oxygen Species. J. Med. Chem. 2012, 55, 924−934. (18) De Raedt, T.; Walton, Z.; Yecies, J. L.; Li, D.; Chen, Y.; Malone, C. F.; Maertens, O.; Jeong, S. M.; Bronson, R. T.; Lebleu, V.; Kalluri, R.; Normant, E.; Haigis, M. C.; Manning, B. D.; Wong, K.-K.; Macleod, K. F.; Cichowski, K. Exploiting Cancer Cell Vulnerabilities to Develop a Combination Therapy for Ras-Driven Tumors. Cancer Cell 2011, 20, 400−413. (19) Trachootham, D.; Zhou, Y.; Zhang, H.; Demizu, Y.; Chen, Z.; Pelicano, H.; Chiao, P. J.; Achanta, G.; Arlinghaus, R. B.; Liu, J. S.; Huang, P. Selective killing of oncogenically transformed cells through a ROS-mediated mechanism by beta-phenylethyl isothiocyanate. Cancer Cell 2006, 10, 241−252. (20) Cairns, R. A.; Harris, I. S.; Mak, T. W. Regulation of cancer cell metabolism. Nat. Rev. Cancer 2011, 11, 85−95. (21) Liang, J.-A.; Wu, S.-L.; Lo, H.-Y.; Hsiang, C.-Y.; Ho, T.-Y. Vanillin Inhibits Matrix Metalloproteinase-9 Expression through Down-Regulation of Nuclear Factor-kappa B Signaling Pathway in Human Hepatocellular Carcinoma Cells. Mol. Pharmacol. 2009, 75, 151−157. (22) Cabello, C. M.; Bair, W. B.; Lamore, S. D.; Ley, S.; Bause, A. S.; Azimian, S.; Wondrak, G. T. The cinnamon-derived Michael acceptor cinnamic aldehyde impairs melanoma cell proliferation, invasiveness, and tumor growth. Free Radical Biol. Med. 2009, 46, 220−231. (23) Chew, E. H.; Nagle, A. A.; Zhang, Y.; Scarmagnani, S.; Palaniappan, P.; Bradshaw, T. D.; Holmgren, A.; Westwell, A. D. Cinnamaldehydes inhibit thioredoxin reductase and induce Nrf2: potential candidates for cancer therapy and chemoprevention. Free Radical Biol. Med. 2010, 48, 98−111. (24) Cabello, C. M.; Bair, W. B., 3rd; Lamore, S. D.; Ley, S.; Bause, A. S.; Azimian, S.; Wondrak, G. T. The cinnamon-derived Michael acceptor cinnamic aldehyde impairs melanoma cell proliferation, invasiveness, and tumor growth. Free Radical Biol. Med. 2009, 46, 220−231. (25) Kim, B.; Lee, E.; Kim, Y.; Park, S.; Khang, G.; Lee, D. Dual AcidResponsive Micelle-Forming Anticancer Polymers as New Anticancer Therapeutics. Adv. Funct. Mater. 2013, 23, 5091−5097. (26) Han, D. C.; Lee, M.-Y.; Shin, K. D.; Jeon, S. B.; Kim, J. M.; Son, K.-H.; Kim, H.-C.; Kim, H.-M.; Kwon, B.-M. 2′-Benzoyloxycinnamaldehyde Induces Apoptosis in Human Carcinoma via Reactive Oxygen Species. J. Biol. Chem. 2003, 279, 6911−6920. (27) Lee, K.; Kwon, B. M.; Kim, K.; Ryu, J.; Oh, S. J.; Lee, K. S.; Kwon, M. G.; Park, S. K.; Kang, J. S.; Lee, C. W.; Kim, H. M. Plasma

investigated in normal tissues and organs. In addition, further mechanistic studies are also needed to establish the therapeutic window for PolyCAFe micelles.

5. CONCLUSIONS We developed Nano-Fenton reactors as novel anticancer drugs that amplify ROS stress to induce cancer cell death preferentially. Nano-Fenton reactors were formulated from a dual acid-sensitive PolyCAFe which possesses both BCA and ferrocene in its hydrophobic backbone and has hydrophilic PEG, allowing micelle formation by self-assembly in aqueous solutions. PolyCAFe micelles rapidly dissociated at acidic pH and released BCA in an acid-triggered manner. Proof-ofconcept studies using cell cultures and mouse tumor models revealed that Nano-Fenton reactors generate hydroxyl radicals in cancer cells and remarkably suppress tumor growth without apparent toxicity to other organs. We anticipate that Fenton reaction-performing PolyCAFe micelles pave a way for the development of a new class of ROS-manipulating anticancer agents.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.5b12523. Characterization of PolyCAFe using NMR, LC−MS/MS, and histological examination (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel: +82-63-270-2344. Fax: +82-63-270-2341. E-mail: dlee@ chonbuk.ac.kr (D.L.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was supported by the grant of Korean Health Technology R&D Project (HI15C1619), Ministry of Health & Welfare, and KRIBB Research Initiative Program, Republic of Korea.

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REFERENCES

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DOI: 10.1021/acsami.5b12523 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX