Enhanced Cisplatin Chemotherapy by Iron Oxide Nanocarrier

Letter pubs.acs.org/NanoLett

Enhanced Cisplatin Chemotherapy by Iron Oxide NanocarrierMediated Generation of Highly Toxic Reactive Oxygen Species Ping’an Ma,† Haihua Xiao,*,∥ Chang Yu,†,‡ Jianhua Liu,§,# Ziyong Cheng,† Haiqin Song,† Xinyang Zhang,† Chunxia Li,† Jinqiang Wang,⊥ Zhen Gu,*,⊥ and Jun Lin*,† †

State Key Laboratory of Rare Earth Resource Utilization, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, P. R. China ∥ State Key Laboratory of Polymer Physics and Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, P. R. China ‡ University of Chinese Academy of Sciences, Beijing 100049, P. R. China § Department of Radiology, The Second Hospital of Jilin University, Changchun 130041, P. R. China # State Key Laboratory of Inorganic Synthesis and Preparative Chemistry, College of Chemistry, Jilin University, Changchun 130012, P. R. China ⊥ Joint Department of Biomedical Engineering, University of North Carolina at Chapel Hill and North Carolina State University, Raleigh, North Carolina 27695, United States S Supporting Information *

ABSTRACT: Reactive oxygen species (ROS) plays a key role in therapeutic effects as well as side effects of platinum drugs. Cisplatin mediates activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX), which triggers oxygen (O2) to superoxide radical (O2•−) and its downstream H2O2. Through the Fenton’s reaction, H2O2 could be catalyzed by Fe2+/Fe3+ to the toxic hydroxyl radicals (•OH), which cause oxidative damages to lipids, proteins, and DNA. By taking the full advantage of Fenton’s chemistry, we herein demonstrated tumor site-specific conversion of ROS generation induced by released cisplatin and Fe2+/Fe3+ from iron-oxide nanocarriers with cisplatin(IV) prodrugs for enhanced anticancer activity but minimized systemic toxicity. KEYWORDS: Drug delivery, cisplatin, iron oxide, Fenton’s reaction, reactive oxygen species

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drug in the cancer cells to maximize anticancer efficacy remains elusive. Fenton’s reaction occurs in almost all iron-related vital processes; H2O2 produced by the cell metabolism is catalyzed by Fe2+/ Fe3+ to form a toxic, highly reactive hydroxyl radical (•OH) or superoxide radical (O2•−).18,19 These radicals are ROS that causes oxidative damage to lipids, proteins, and DNA.18,19 Normal cells keep redox homeostasis, which helps to balance the oxidative damage through Fenton’s reaction to protect cells from death.19 However, excessive production of ROS could overwhelm this homeostasis, resulting in irreversible permanent damage to the cells and eventually cell apoptosis.18,19 For this reason, disturbing the homeostasis by means of exogenous therapeutics is already effective as a novel anticancer strategy;20−24 for example, motexafin gadolinium selectively

latinum drugs are one of the most important categories of chemotherapeutics.1−5 They are used in 80% of clinical regimens and made up 50% of all the anticancer drugs used in clinic.6−10 Reactive oxygen species (ROS) generation plays a critical role in therapeutic effects of platinum drugs, such as cisplatin.11,12 Specifically, in cancer cells, cisplatin can activate a family of enzymes named nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX),13 which can transport electrons across the plasma membrane. The activated NOX converts NADPH into NADP+ with release of electrons. An O2 molecule in turn accepts a donated electron to generate O2•−, which can be further dismutated by superoxide dismutase (SOD) enzyme to form H2O2 and its downstream highly toxic ROS species such as hydroxyl radical (•OH) (Figure 1A).13,14 However, the generation of H2O2 and downstream ROS species can also result in severe side effects (cardiac toxicity of doxorubicin;15 nephrotoxicity and ototoxicity of cisplatin16,17). Active tumor-targeting site-specific conversion of H2O2 to its downstream ROS species induced by presence of an anticancer © 2017 American Chemical Society

Received: October 11, 2016 Revised: January 6, 2017 Published: January 31, 2017 928

DOI: 10.1021/acs.nanolett.6b04269 Nano Lett. 2017, 17, 928−937

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Figure 1. Maximizing cisplatin efficacy by constructing self-sacrificing iron-oxide nanoparticles with cisplatin(IV) drugs FePt-NP2 for synergistic actions. (A) Cisplatin activates NOX, which catalyzes formation of superoxide and H2O2 from O2; iron catalyzes the Fenton chemistry to turn H2O2 into highly toxic •OH. (B) Construction of self-sacrificing iron oxide nanoparticles with cisplatin(IV) prodrug (FePt-NP2) circumvents the endocytosis of cisplatin into the cells. In this way, excess •OH are formed, which results in fast lipid and protein oxidation and DNA damage, as well as apoptosis via the ROS/Cyt C/caspase-3 pathway.

agents are commonly activated by light irradiation, which limits their use because of the inadequate tumor penetration of light.28 Herein, we report a sequential drug delivery strategy utilizing intracellular iron ions released from the iron-oxide nanocarriers to sensitize cisplatin, which is codelivered by the iron-oxide nanocarriers, for enhancing anticancer efficacy. Construction of various iron oxide nanoparticles (NPs) without Pt drugs by

accumulates in tumor cells and further kills the cells via inducing the intracellular ROS or superoxide formation.25 Moreover, phase III trials on this drug have been evaluated for cancer therapy.26 Another example of this strategy in use is the photodynamic therapy (PDT) agent, such as porphyrins, which can be promoted to an excited state upon irradiation with light to produce singlet oxygen, another ROS. This species rapidly attacks any organic compounds it encounters.27 However, PDT 929


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Figure 2. Iron pretreatment increases intracellular iron levels, sensitizing H2O2-inducing anticancer drugs by enhanced ROS generation. A) Intracellular iron uptake by A2780 and ACP cells after iron treatment (200 μM) for 0.5 and 3 h, respectively. B) Free iron levels in the cells after treatment with Fe-NP3 for 6 and 12 h, respectively. C) IC50 values of cisplatin, carboplatin, oxaliplatin, doxorubicin, and artesunate, with and without iron treatment. D) Hydroxyl radical generation and E) apoptosis rate of the cells. DFO (100 μM) was used to chelate the intracellular free iron ions and NAC (5 mM) was used to block the generated ROS. Data were shown as mean ± S.D (n = 3). Significance is defined as *P < 0.05, **P < 0.01.

efficacy and side effects of anticancer drugs,31−37 to our knowledge, this is the first demonstration that exploits both DNA targeting ability and ROS activation ability of the active drug cisplatin to generate H2O2 for the subsequent Fenton’s reaction to synergistically enhance therapy efficacy.38−40 To validate our assumption, in addition to platinum drugs, we initially screened a combination of iron with conventional anticancer drugs that are known to induce H2O2 production, such as doxorubicin.13,41,42 We also chose artesunate, a widely used antimalaria drug, which has an −O−O− bond that can be activated in a targeted way by the intracellular iron; artesunate is now frequently tested as an anticancer drug.43 Due to the oxygen bridge, artesunate could be considered as the reactant in the Fenton’s reaction.44 Fe2+, Fe3+, Fe2+/Fe3+ (mixture of Fe2+ and Fe3+), and blank iron nanoparticles with a polyethylene glycol (PEG) shell (Fe-NP3, Scheme S1) were screened as possible iron catalysts. All iron reagents showed insignificant toxicity toward A2780 cells (cisplatin sensitive) up to 2 mM (Figure S1). The subsequent treatment of a pair of cisplatin sensitive A2780 and cisplatin resistant ACP cells with various forms of iron resulted in a maximum Fe uptake for the cells treated with Fe-NP3 (Figure 2A). Specifically, addition of FeNP3 increased the iron levels in both cell lines by up to 153fold (A2780) and 227-fold (ACP) compared with the untreated cells (Figure 2A) by ICP-MS (inductively coupled plasma mass spectrometry). Only labile/reactive irons such as Fe2+ and Fe3+ are catalytic for Fenton’s reaction. To further confirm the presence of labile iron ions, a ferric ion probe, 3′,6′bis(diethylamino)-2-(4-oxopent-2-en-2-ylamino)spiro(isoindoline-1,9′-xanthen)-3-one,45 was used to detect the iron ions in the cells after treatment with Fe-NP3 for 6 and 12 h

coating with polyethylenimine (PEI) and polyethylene glycol (PEG) (Fe-NP1, bare iron NPs; Fe-NP2, PEI-coated NPs; FeNP3, PEG conjugate PEI coated NPs; Scheme S1) was first achieved. Later, loading cisplatin(IV) prodrugs, PEGylation and further rhodamine B (RhB) dye labeling could tune the iron oxide NPs with different functionalities (FePt-NP1, Pt loaded iron NPs; FePt-NP2, PEGylated Pt loaded iron NPs; FePtNP3, RhB labeled FePt-NP2; Scheme S1). The final cisplatin(IV) prodrugs loaded self-sacrificing iron-oxide NPs were FePtNP2 with PEG on the surface (Figure 1B). The design and delivery of FePt-NP2 is based on following considerations: (i) PEGylation of FePt-NP2 for keeping the NPs stealthy; (ii) MRI-guided delivery of the FePt-NP2 localizes Pt and iron accumulation into the tumor tissue; (iii) Pt and iron can be internalized maximally after in vivo accumulation of FePt-NP2; (iv) on the one hand, the loaded cisplatin(IV) prodrug can be reduced to give toxic cisplatin, forming Pt-DNA adducts, resulting in tumor-cell replication inhibition and eventually apoptosis. The more kinetically inert cisplatin(IV) prodrug was chosen rather than cisplatin due to its low toxicity, but it can be site-specifically reduced to give the toxic cisplatin inside cells. On the other hand, FePt-NP2 can be self-sacrificed by degradation via hydrolysis in acidic environment to release considerable reactive iron ions within the cancer cells. The released cisplatin activates NOX, which triggers O2 to produce abundant superoxide anion (O2•−). O2•− is subsequently converted into H2O2 by SOD; H2O2 can be further catalyzed by the released Fe2+/Fe3+ to form excess highly toxic •OH, resulting in fast lipid and protein oxidation and DNA damage.29,30 Though various drug delivery systems were reported for Pt based drugs and ROS are involved in the 930


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Figure 3. FePt-NP2 maximizes uptake of Pt and Fe and intracellular release of Pt and Fe, resulting in excess ROS generation, enhanced efficacy, and increased apoptosis by comparison to cells without addition of the drug. (A) Representative TEM image of FePt-NP2. FePt-NP2 can release both (B) Pt and (C) Fe in an acid-responsive manner. Minimized Pt and Fe release at neutral pH ensures (D) the maximized uptake of Pt drugs, which thereby results in (E) increased Pt-DNA adducts and (F) increased Fe uptake. Then enhanced Pt and Fe uptake triggers (G) abundant hydroxyl radical generation, with Pt and Fe working in concert to yield (H) enhanced Pt efficacy (expressed as IC50 values) and (I) increased apoptosis. Data is shown as mean ± SD (n = 3). Significance is defined as *P < 0.05, **P < 0.01.

(Figure S2). Intense red fluorescence was observed in both cell lines, indicating the presence of labile ferric ion in the cancer cells after internalizing Fe-NP3 (Figure S2). Quantification of the cells treated with Fe-NP3 via measuring the cell lysis supernatant by ICP-MS suggested significantly higher amount of iron ions than the nontreated cells (Figure 2B). We then investigated how the iron ions interacted with the anticancer drugs. The accumulated exogenous irons sensitized the anticancer drugs, as a reduction in IC50 (half maximal inhibitory concentration) values (Figure 2C, Figures S3−S4, Table S1) and increase in apoptosis (Figures S5−S7), were observed in the cells with iron treatment (no matter what form of iron was used, Fe2+, Fe3+, Fe2+/3+, or iron nanoparticles). The iron nanoparticles have the most prominent effect almost in all cases. To unveil the underlying mechanism on how the iron nanoparticles sensitized these drugs, specific hydroxyl radicals were tested by ELISA assay. As shown in Figure 2D, the cells with various iron reagents treatment alone and cisplatin alone showed a relative hydroxyl radical level of about 1.3- to 2.0-fold and 1.5-fold of the untreated cells. However, cisplatin combined with various iron reagents showed about 1.7- to 2.8-fold hydroxyl radical generation, which further validated the possibility of amplifying hydroxyl radical generation by a

mixture of iron and cisplatin. This amplification of hydroxyl radical generation could be almost completely blocked by a ROS scavenger NAC at 5 mM46 and iron chelator deferoxamine mesylate (DFO, 100 μM),47 substantiating the involvement of iron in this hydroxyl radical generation. The generation of total ROS production was also monitored in Figure S8. A fluorogenic dye, 2′,7′-dichlorofluorescein diacetate (DCFDA), which can be used to measure the activity of hydroxyl, peroxyl, and other reactive oxygen species (ROS) within the cells, was adopted.48 The greater ROS generation in iron and cisplatin cotreated cells (up to ∼2.5 to ∼5-fold of the nontreated cells) also validated the benefits of using iron to promote ROS generation. Cells treated with cisplatin plus blank Fe-NP3 had the highest apoptosis rate (up to ca. 45% on A2780; ca. 30% on ACP), among all the groups treated with either cisplatin with various iron sources, while NAC and DFO supplementation could result in less apoptosis ratios to only 28% and 35% for A2780 (Figure 2E). A general conclusion can be drawn that combining iron with anticancer drugs that can induce H2O2 production, especially iron NPs, could enhance hydroxyl radical formation, increase apoptosis, and eventually sensitize the anticancer drugs. This is a new way of using iron nanoparticles 931


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Figure 4. FePt-NP2 was endocytosed into the cancer cells, releasing iron and Pt that work in concert to overcome cisplatin resistance through the ROS/Cyt C/caspase-3 pathway. Uptake of FePt-NP2 was blocked by keeping the cells at 4 °C or pretreating them with 30 μg mL−1 CPZ and 120 mM NaN3, monitored by A) Pt and B) Fe uptake via ICP-MS and C) RhB-labeled FePt-NP3 by means of flow cytometry. D) Representative CLSM images of cells treated with FePt-NP3 at 37 °C, pretreated with 30 μg mL−1 CPZ and 120 mM NaN3 show the localization of the RhB-labeled FePtNP3 (red). Cell nucleus is stained by DAPI (blue). Bar = 10 μg. E) Internalized FePt-NP2 releases both Fe and Pt, which subsequently triggers ROS generation, depolarizes mitochondria, and induces mitochondria dysfunction, resulting in F) decreased membrane potential and G) release of Cyt C as well as the activation of the apoptotic cascade. Data were shown as mean ± S.D. (n=3). Significance is defined as * P < 0.05, ** P < 0.01.

63.5% Pt and 3.2% Fe at pH 4.5 (acidic) after 100 h, while at pH 7.4, only 20.5% Pt and almost no iron ion was released after 100 h. These releases correspond to 1.4 × 1019 (pH 7.4) and 4.1 × 1019 cisplatin molecules (pH 4.5) as well as none (pH 7.4) and 6.8 × 1019 (pH 4.5) iron ions released (Table S2). Almost no release of Fe and minimal Pt release at pH 7.4 but rapid release of both at pH 4.5 makes FePt-NP2 ideal for maximizing Pt and iron delivery into the cells by minimizing premature leakage of cisplatin and iron. The uptake of Fe and Pt by cells was also studied by ICPMS. A2780 and ACP cells treated with FePt-NP2 demonstrated a Pt uptake at around 230−260 pg of Pt (million cells)−1 at 1 and 4 h, while uptake was only about 10−40 pg of Pt (million cells)−1 for both cells treated with free cisplatin (Figure 3D) and cisplatin(IV) prodrug, which indicates a six- to 20-fold increase in uptake (Table S3). FePt-NP2 localized in the cell cytosol of A2780 and ACP cells treated with this nanoparticle (Figure S12). A further uptake study of the RhB-labeled nanoparticle FePt-NP3 by flow cytometry revealed a timedependent uptake of FePt-NP3 with almost no difference in uptake rates between cell lines (Figure S13). The increased Pt uptake resulted in five to ten times more Pt-DNA adducts than it was when both cells were treated with free cisplatin and cisplatin(IV) prodrug (Figure 3E, Table S3); cells treated with FePt-NP2 demonstrated a Fe uptake at about 2 to 5 ng of Fe (million cells)−1 from 1 to 4 h (Figure 3F). This uptake of Pt and Fe corresponds to an uptake of about 2−6 × 105 FePt-NP2 per cell at a Fe/Pt ratio of around 10 (Table S4). As the

for delivering H2O2-inducing therapeutics for synergistic actions. To further investigate the iron nanocarriers with anticancer drugs for in vitro usage, iron nanoparticles with kinetically inert and nontoxic cisplatin(IV) prodrugs, instead of cisplatin, were developed. Cisplatin(IV) prodrugs can be selectively activated by action of intracellular reducing agent such as glutathione to release toxic cisplatin in the tumor cells.49 Fe-NP2 with a poly(ethylenimine) (PEI) coating (13.2 w/w% by thermogravimetric analysis (TGA), Figure S9) was conjugated with the prodrug to make the Pt(IV)-drug-loaded nanoparticles, FePtNP1. The as-prepared FePt-NP1 was further coated with stealthy molecule of PEG to make FePt-NP2. PEG was introduced to minimize blood clearance and to extend circulation time (Scheme S1). The final FePt-NP2 was 5.7 nm in diameter shown by transmission electron microscopy (TEM; Figure 3A, Figure S10) and 252 nm shown by dynamic light scattering (DLS) with a zeta potential at +4.5 mV (Figure S11). At pH 7.4, the size of FePt-NP2 changed very little, while the size had a dramatic change to 173 nm at pH 4.5 for only 1 day, and they further decrease to 139 nm after 5 days. The reduction in size was possibly due to the release of Pt and PEG coating as well as the hydrolysis of the iron nanocarriers (Figure S11). Moreover, the Pt loading was 4.2 w/w% (ICP-MS), which suggests a high loading of cisplatin at 6.5 w/w%, or 270 cisplatin molecules per nanoparticle. FePt-NP2 was supposed to release both Pt and iron to take effect within the cells via acid hydrolysis.50,51 As shown in Figure 3B,C, FePt-NP2 releases 932


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Figure 5. In vivo MRI-guided delivery of FePt-NP2. MRI-guided localization of FePt-NP2, 0 (preinjection), 5, and 30 min post i.v. injection with FePt-NP2 at a Pt dose of 4 mg Pt kg−1 and a Fe dose of 37.6 mg Fe kg−1 body weight on a liver cancer mice model, with A) and without B) magnetic field. The circled region in green is the tumor and the pink region is liver. After NP administration, the tumor and liver became darker, showing that more particles were accumulated in the liver and tumor for both groups. Addition of FePt-NP2 resulted in maximized Pt and Fe accumulation (Figure S21) (C) increased ROS generation (Figure S22) and (D) enhanced tumor inhibition with limited body-weight change (Figure S25). Data is shown as mean ± SD (n = 3). Significance is defined as *P < 0.05, **P < 0.01.

6.1 μM in A2780 and ACP cells, respectively, while the values for cisplatin were 5.9 and 18.3 μM (Figure 3H, Figure S15, Table S5). FePt-NP2 had better anticancer activity and overcame cisplatin drug resistance by lowering the cisplatin resistance fold (IC50 of cisplatin on A2780/IC50 of cisplatin on ACP) from 3.0 to 0.87. FePt-NP2 also had an increased apoptosis rate at 31.8%, seen against only 16.4% for cisplatin on ACP cells. Similarly, the apoptosis rate could be reduced by use of NAC or DFO to 24.3% and 25.6%, respectively, which further confirmed the involvement of ROS in the mechanism of action of FePt-NP2 (Figure 3I, Figure S16). This drug delivery strategy demonstrates action via two pathways against cisplatin resistance. The first way in which FePt-NP2 can overcome cisplatin resistance might be the unique internalization pathway of FePt-NP2, which circumvents the biological barrier to cisplatin. A transmembrane protein, copper transporter protein (CTR1), imports copper (i) into yeast and mammalian cells, which also regulates

theoretical Fe/Pt ratio in the original intact nanoparticles is 9.41, one can envision that FePt-NP2 was almost intact with limited premature leakage of Pt and iron before cell internalization. The internalized cisplatin(IV) prodrugs together with the nanocarriers can induce H2O2 generation; these species would subsequently be catalyzed to form ROS by the released iron ions. As shown in Figure 3G, FePt-NP2 treatment showed around 15.7-fold hydroxyl radical generation compared with that in the untreated A2780 cells, while it was about 4.5- and 3.6-fold with DFO (100 μM) and ROS inhibitor NAC (5 mM) treatment, respectively. This behavior was also confirmed in ACP cells, showing the involvement of the iron from FePt-NP2 in regulating hydroxyl radical generation. Increased hydroxyl radical generation ultimately resulted in enhanced anticancer activity. Further monitoring the total ROS generation by DCFDA assay also proved the involvement of iron for ROS generating (Figure S14). FePt-NP2 had IC50 values of 6.8 and 933


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over, the Bcl-2 family proteins are involved in the cell apoptosis through the mitochondrial pathway. Therefore, Western blot protein expression of Bcl-2 (antiapoptotic protein), Bax (proapoptotic), cleaved caspase-3/9, and Cyt C in the cells treated with cisplatin and FePt-NP2, with phosphate-buffered saline (PBS) as a control, was performed and quantified by using ImageJ software (Figure 4F,G). Expression of Bax, cleaved caspase-3/9, and Cyt C for the cells was in the order PBS < cisplatin < FePt-NP2. However, Bcl-2 expression was in the reverse order. These results showed that FePt-NP2 induced apoptosis through ROS/Cyt C/caspase-3/9. To translate this dual action in vivo, FePt-NP2 was i.v. injected to mice with magnetic field for maximizing localization of nanocarriers and Pt drugs on a H22 liver cancer model. As FePt-NP2 core can work as perfect T2 MRI contrast agents (Figures S18, S19), noninvasively visualization of the localization of FePt-NP2 in the tumor site could be possible via MRI guided drug delivery.56 As such, the mice were injected with FePt-NP2 intravenously at a dose of 37.6 mg Fe kg−1 and 4 mg Pt kg−1 body weight (Fe/Pt ratio = 9.41, Table S4) with and without magnetic field (Figure 5A,B). MR images were taken 5 and 30 min postinjection. The tumor site, especially the liver, for the mice in the absence of magnetic field became darker from 5 to 30 min. However, with magnetic field, the tumor site and liver were dark even at 5 min. After 30 min, darkness was extremely evident in the tumor site. These results show that FePt-NP2 accumulated more from 5 to 30 min, for mice with magnetic field. Even greater accumulation in the tumor was observed in the presence of the magnetic field (Figure 5B, Figure S20). To further quantify the drug and nanoparticle accumulation, the levels of Pt and Fe in blood, tissues, and organs with and without magnetic field were evaluated by using ICP-MS.57,58 As shown in Figure S21, 15.7% and 18.2% of the injected Pt and Fe accumulated in the tumors with magnetic field, while the corresponding values were 1.5% and 1.96% for Pt and Fe without magnetic field, indicating an enhancement of 10.5- and 9.3-fold with magnetic field, respectively. The above biodistribution of Fe and Pt are similar to Fe/Pt ratios in the tumor with and without magnetic field, respectively (Table S6). Considering that the original injected FePt-NP2 had a Fe/Pt ratio of 9.41 (Table S4), the nanoparticle composition changed very little (+15.8% Fe/Pt ratio variation, Table S6) in the tumor with the magnetic field, hence nanoparticles remained intact during circulation with minimum premature release of Pt drugs. Accumulation of more FePt-NPs in the tumor subsequently resulted in greater ROS generation in vivo. FePt-NP2 produced an ROS level of 36.4%, while this level was only 9% for cisplatin and 24% for blank Fe-NP3. Meanwhile, if the ROS scavenger NAC was used in the mice this effect was inhibited by 62%, which demonstrated the involvement of ROS in this process (Figure 5C, Figure S22). The antitumor efficacy of FePt-NP2 was studied in an H22 cancer model. In vivo evaluation of blank Fe-NP3 showed no significant toxicity was found, from the body hematoxylin and eosin (H&E) staining of the organs collected and blood analysis of mice treated with Fe-NP3 (Figures S23, S24). The limited toxicity of Fe-NP3 could be advantageous for the in vivo use of FePt-NP2 for cisplatin(IV) loading and delivery. As shown in Figure 5D, on the 14th day, mice injected with cisplatin at 2 mg Pt (kg body weight)−1 had relative tumor growth of around 23-fold, while the blank Fe-NP3 and PBS groups showed rapid tumor growth, reaching a relative tumor

platinum drug uptake. Previous study showed that cisplatin resistance comes from the inability to accumulate drugs into the resistant cells due to the lack of CTR1.52 Here, only 40% of the mRNA level of the nonresistant CTR1 in A2780 cells was found for ACP cells (Figure S17). Treatment of the cells with cisplatin further degraded the CTR1 protein by 20% and 10% for A2780 and ACP cells, respectively, which made the subsequent internalization of cisplatin into the cells highly challenging (Figure S17). Unlike cisplatin, nanoparticles are known to be internalized via endocytosis. As endocytosis is an energy-dependent process it can be blocked by low temperature and the endocytosis inhibitor chlorpromazine (CPZ) as well as by the energy-depleting agent NaN3.53,54 As shown in Figure 4A,B, compared to the cells at 37 °C, A2780 cells treated with FePt-NP2 displayed only 37.5, 48.7, and 69.2% of Pt and 45.7, 62.2, and 60.0% of Fe uptake at 4 °C, with CPZ at 37 °C, or NaN3 at 37 °C, respectively (ICP-MS data). Moreover, the reduced uptake of the RhB-labeled iron nanoparticles, FePtNP3, was confirmed by flow cytometry and confocal laser scanning microscopy (CLSM) (Figure 4C,D) under these conditions. This reduction in uptake validated the unique internalization pathway of FePt-NP2 via endocytosis, which partially contributes to the reversal of the cell’s resistance to cisplatin. The second part of the dual action of FePt-NP2 is the increased production of ROS, which could be of great importance in overcoming resistance. As both a target and source of ROS, mitochondria plays a key role in the activation of cell apoptosis.34 In the mitochondria, a redox-active iron pool exists. Increase in the iron pool can be associated with oxidative damage to the cells and the change of mitochondrial permeability which is calcium-dependent.55 Tight regulation of mitochondrial Fe levels is crucial as excessive redox-active iron ions could result in highly toxic hydroxyl radical generation. The internalized iron nanoparticles could release iron by acid metabolism, which increases the iron levels in the mitochondria and thereby depolarizes them and induces mitochondrial dysfunction. This could further result in decreased membrane potential (MMP) and cytochrome C (Cyt C) release as well as activation of the apoptotic cascade. The MMP can be measured by a lipophilic fluorescent cationic dye, JC-1(5,5′,6,6′tetrachloro-1,1′,3,3′-tetraethylbenzimidazolyl-carbocyanine iodide). After JC-1 selectively enters the cellular mitochondria, it reversibly changes color from green to red as MMP increased. Healthy cells normally have high MMP(ΔψM) where JC-1 could spontaneously forms J aggregates which shows intense red fluorescence (590 ± 17.5 nm). However, JC-1 will remain as monomeric form in apoptotic or unhealthy cells with low ΔψM which emits green fluorescence (530 ± 15 nm). Therefore, the ratio of red to green fluorescence could be used to indicate the polarization of the mitochondrial membranes; higher ratio of red to green fluorescence means greater polarization. As shown in Figure 4E, the red emitting mitochondria localized in the upper region of the plot suggested no mitochondrial damage (control cells). Cells treated with cisplatin showed weak downward shift. However, after FePt-NP2 treatment, ACP cells significantly shifted downward, indicating a loss of MMP and mitochondrial damage. The change in the cytofluorimetric pattern suggested that FePt-NP2 treatment triggered cell death through mitochondria-mediated apoptosis pathway. Increased cellular ROS is known to contribute to induce apoptosis and cell death34 through caspase activation. More934


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Nano Letters volume of about 50-fold. In vivo, FePt-NP2 had a slightly higher tumor inhibition effect than cisplatin, with relative tumor growth of 18- and 20-fold, respectively. However, with the additional magnetic field, the tumor growth was greatly inhibited to only about 5-fold of relative tumor volume on the 14th day. The ROS scavenger NAC countered this growth inhibition effect of FePt-NP2 to some extent, with a final tumor growth of around 15-fold. These results demonstrated the participation of ROS in this process. Finally, almost no body weight losses were observed for all groups (Figure S25). Various current anticancer drugs, including platinum agents, doxorubicin, 5-FU in clinical use can induce H2O2 and its downstream ROS generation, which plays a key role in either their mechanism of action for efficacy or causing serious systemic toxicity. The ROS generation mechanism may be different for these ROS inducing anticancers. Specifically, in cancer cells, cisplatin can mediate activation of NOX,6−10 a family of enzymes that transport electrons across the plasma membrane. The activated NOX converts NADPH into NADP+ with release of electrons. An O2 molecule in turn accepts a donated electron to generate O2•−, which can be further dismutated by superoxide dismutase (SOD) enzyme to form H2O2 and its downstream ROS. The conversion of H2O2 to its downstream highly toxic ROS relies on Fenton’s chemistry where H2O2 is catalyzed by Fe2+/ Fe3+ to form a toxic, highly reactive hydroxyl radical ( •OH) or superoxide radical (O2•−).13−15 These radicals are ROS that causes oxidative damage to lipids, proteins, and DNA,13−15 resulting in irreversible permanent damage to the cells and eventually cell apoptosis. PDT through targeted delivery system was extensively developed for cancer therapy.59 So far, there is no report to actively manipulate the ROS generation induced by a certain anticancer drug. We became interested in developing a nanoparticle platform that delivers ROS inducing anticancer drugs. This platform can convert the H2O2 and its downstream ROS species produced by the anticancer drugs site-specifically for maximizing anticancer efficacy but minimizing toxicity, the key of modern chemotherapy. Starting at characteristic ROS inducing anticancer drugs, i.e., platinum agents including cisplatin, oxaliplatin, carboplatin, doxorubicin, and artesunate, we investigated the potential sensitizing effect of various iron supplements for them. Platinum drugs and doxorubicin can induce production of H2O2. Meanwhile, artesunate has an −O−O− bond that can be activated in a targeted way by the intracellular iron, which could be considered as the reactant in the Fenton’s reaction. By supplementing the cells with various iron reagents at the same dose including Fe2+, Fe3+, Fe2+/Fe3+ (mixture of Fe2+ and Fe3+), and blank iron NPs (Fe-NP3), we found that iron NPs demonstrated the most uptake of iron into the cells. Combined with the anticancer drugs, iron NPs induced the highest ROS generation, resulting in reduced IC50 values and increased apoptosis, validating iron NPs as the best sensitizing agent. We then constructed an iron oxide NP platform loaded with cisplatin(IV) prodrugs(FePt-NP2). The advantages of using this sequential drug delivery system can be multifold. First, 100% use of the carrier system and drugs can be achieved via the Fenton chemistry, i.e., cisplatin induces H2O2 production, while the released iron ions trigger the H2O2 conversion to highly toxic hydroxyl radicals. Hence, minimum amounts of excipients were codelivered to cancer cells. Second, due to the nature of iron oxide NPs, MRI-guided delivery of the FePt-NP2

can localize Pt and iron into the tumor tissue. Hence Pt and iron can be internalized maximally after in vivo accumulation of FePt-NP2. Third, the loaded cisplatin(IV) prodrug can be reduced to give toxic cisplatin, forming Pt−DNA adducts, resulting in tumor-cell replication inhibition and eventually apoptosis. Moreover, FePt-NP2 can be self-sacrificed by degradation in acidic environment to release considerable reactive iron ions within the cancer cells. The released cisplatin activates NOX, which triggers O2 to produce abundant superoxide anion (O2•−). O2•− is subsequently converted into H2O2 by SOD; this reaction can be further catalyzed by the released Fe2+/Fe3+ to form excess free hydroxyl radicals •OH, resulting in fast lipid and protein oxidation and DNA damage.15,16 In vitro, we found here FePt-NP2 released both Pt and iron in acidic conditions but with minimum release at pH 7.4. FePt-NP2 demonstrated increased uptake and Pt-DNA adducts, enhanced ROS generation, and reduced IC50 values as well as overcoming cisplatin resistance. Moreover, mechanistic study showed that FePt-NP2 depolarizes the mitochondria and induces mitochondrial dysfunction, which results in decreased membrane potential (MMP) and release of cytochrome C (Cyt C) as well as activation of the apoptotic cascade through the ROS/Cyt C/caspase-3 pathway. In vivo, mice treated with FePt-NP2 showed a Pt and Fe accumulation enhancement of 10.5- and 9.3-fold, respectively, with magnetic field compared with that without magnetic field. This enhanced accumulation of FePt-NP2 resulted in enhanced ROS generation in the tumor site and anticancer efficacy. In summary, we have shown here a programmed strategy of delivering an ROS-inducing anticancer drug cisplatin via its cisplatin(IV) prodrug form by use of iron oxide nanocarriers that can preferentially increase the Pt and Fe accumulation in the tumor site via magnetic-field mediated-localization and monitoring by MRI-guided delivery. The nanocarriers thus can enhance Pt and Fe internalization in the cancer cells after NP accumulation in the tumor. Thereafter, the loaded cisplatin(IV) prodrug can be rapidly reduced to toxic cisplatin that subsequently formed Pt-DNA adducts and also activated NOXs, which triggered a cascade reaction to form H2O2. Iron NPs degraded and were metabolized in the cancer cells, releasing excess labile iron ions that catalyze H2O2 decomposition into highly toxic ROS within cancer cells; this results in fast oxidation and deterioration of cellular membranes. The produced, abundant ROS work in a concerted way with cisplatin and showed enhanced antitumor efficacy in vivo. Ease of preparation, synergistic efficacy, and theranostic function render this new delivery method a promising anticancer strategy with clinical importance.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.nanolett.6b04269. Full experimental details, Figures S1−S23, and Tables S1−6 (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (J.L.). *E-mail: [email protected] (Z.G.). *E-mail: [email protected] (H.X.). 935


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(24) Qian, C.; Yu, J.; Chen, Y.; Hu, Q.; Xiao, X.; Sun, W.; Wang, C.; Feng, P.; Shen, Q. D.; Gu, Z. Adv. Mater. 2016, 28, 3313−3320. (25) Evens, A. M.; Lecane, P.; Magda, D.; Prachand, S.; Singhal, S.; Nelson, J.; Miller, R. A.; Gartenhaus, R. B.; Gordon, L. I. Blood 2005, 105, 1265−1273. (26) Meyers, C. A.; Smith, J. A.; Bezjak, A.; Mehta, M. P.; Liebmann, J.; Illidge, T.; Kunkler, I.; Caudrelier, J. M.; Eisenberg, P. D.; Meerwaldt, J.; Siemers, R.; Carrie, C.; Gaspar, L. E.; Curran, W.; Phan, S. C.; Miller, R. A.; Renschler, M. F. J. Clin. Oncol. 2004, 22, 157−165. (27) Ethirajan, M.; Chen, Y.; Joshi, P.; Pandey, R. K. Chem. Soc. Rev. 2011, 40, 340−362. (28) Dolmans, D. E. J. G. J.; Fukumura, D.; Jain, R. K. Nat. Rev. Cancer 2003, 3, 380−387. (29) Sharma, P.; Jha, A. B.; Dubey, R. S.; Pessarakli, M. J. Bot. 2012, 2012, 1−26. (30) Ayene, I. S.; Koch, C. J.; Krisch, R. E. Int. J. Radiat. Biol. 2007, 83, 195−210. (31) Min, Y.; Mao, C. Q.; Chen, S.; Ma, G.; Wang, J.; Liu, Y. Angew. Chem., Int. Ed. 2012, 51, 6742−6747. (32) Wang, X.; Wang, X.; Guo, Z. Acc. Chem. Res. 2015, 48, 2622− 2631. (33) Wang, X.; Guo, Z. Chem. Soc. Rev. 2013, 42, 202−224. (34) Circu, M. L.; Aw, T. Y. Free Radical Biol. Med. 2010, 48, 749− 762. (35) Min, Y. Z.; Li, J. M.; Liu, F.; Yeow, E. K. L.; Xing, B. G. Angew. Chem., Int. Ed. 2014, 53, 1012−1016. (36) Xiao, H. H.; Song, H. Q.; Yang, Q.; Cai, H. D.; Qi, R. G.; Yan, L. S.; Liu, S.; Zheng, Y. H.; Huang, Y. B.; Liu, T. J.; Jing, X. B. Biomaterials 2012, 33, 6507−6519. (37) Xu, X. Y.; Xie, K.; Zhang, X. Q.; Pridgen, E. M.; Lippard, S. J.; Langer, R.; Walker, G. C.; Farokhzad, O. C. Proc. Natl. Acad. Sci. U. S. A. 2013, 110, 18638−18643. (38) Wu, H.; Yin, J. J.; Wamer, W. G.; Zeng, M.; Lo, Y. M. J. Food Drug Anal. 2014, 22, 86−94. (39) Foy, S. P.; Labhasetwar, V. Biomaterials 2011, 32, 9155−9158. (40) Zhang, C.; Bu, W.; Ni, D.; Zhang, S.; Li, Q.; Yao, Z.; Zhang, J.; Yao, H.; Wang, Z.; Shi, J. Angew. Chem., Int. Ed. 2016, 55, 2101−2106. (41) Menna, P.; Recalcati, S.; Cairo, G.; Minotti, G. Cardiovasc. Toxicol. 2007, 7, 80−85. (42) Simůnek, T.; Stérba, M.; Popelová, O.; Adamcová, M.; Hrdina, R.; Gersl, V. Pharmacol. Rep. 2009, 61, 154−171. (43) Ho, W. E.; Peh, H. Y.; Chan, T. K.; Wong, W. S. F. Pharmacol. Ther. 2014, 142, 126−139. (44) Krishna, S.; Uhlemann, A. C.; Haynes, R. K. Drug Resist. Updates 2004, 7, 233−244. (45) Zhang, L. F.; Zhao, J. L.; Zeng, X.; Mu, L.; Jiang, X. K.; Deng, M.; Zhang, J. X.; Wei, G. Sens. Actuators, B 2011, 160, 662−669. (46) Zhang, W. J.; Wei, H.; Frei, B. Exp. Biol. Med. 2010, 235, 633− 641. (47) Eruslanov, E.; Kusmartsev, S. Methods Mol. Biol. 2010, 594, 57− 72. (48) Halasi, M.; Wang, M.; Chavan, T. S.; Gaponenko, V.; Hay, N.; Gartel, A. L. Biochem. J. 2013, 454, 201−208. (49) Xiao, H. H.; Qi, R. G.; Liu, S.; Hu, X. L.; Duan, T. C.; Zheng, Y. H.; Huang, Y. B.; Jing, X. B. Biomaterials 2011, 32, 7732−7739. (50) Feazell, R. P.; Nakayama-Ratchford, N.; Dai, H.; Lippard, S. J. J. Am. Chem. Soc. 2007, 129, 8438−8439. (51) Zhang, C.; Bu, W.; Ni, D.; Zhang, S.; Li, Q.; Yao, Z.; Zhang, J.; Yao, H.; Wang, Z.; Shi, J. Angew. Chem., Int. Ed. 2016, 55, 2101−2106. (52) Kalayda, G. V.; Wagner, C. H.; Jaehde, U. J. Inorg. Biochem. 2012, 116, 1−10. (53) Ma, P. A.; Xiao, H. H.; Li, X. X.; Li, C. X.; Dai, Y. L.; Cheng, Z. Y.; Jing, X. B.; Lin, J. Adv. Mater. 2013, 25, 4898−4905. (54) Vercauteren, D.; Vandenbroucke, R. E.; Jones, A. T.; Rejman, J.; Demeester, J.; De Smedt, S. C.; Sanders, N. N.; Braeckmans, K. Mol. Ther. 2010, 18, 561−569. (55) Urrutia, P. J.; Mena, N. P.; Núñez, M. T. Front. Pharmacol. 2014, 5, 38.

Zhen Gu: 0000-0003-2947-4456 Jun Lin: 0000-0001-9572-2134 Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This project is financially supported by the National Natural Science Foundation of China (NSFC 51672269, 51628201, 51372241, 51332008), Science and Technology Development Planning Project of Jilin Province (20140413037GH), and the National Basic Research Program of China (2014CB643803).

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REFERENCES

(1) Wilson, J. J.; Lippard, S. J. Chem. Rev. 2014, 114, 4470−4495. (2) Wang, X. Y.; Wang, X. H.; Guo, Z. J. Acc. Chem. Res. 2015, 48, 2622−2631. (3) Butler, J. S.; Sadler, P. J. Curr. Opin. Chem. Biol. 2013, 17, 175− 188. (4) Klein, A. V.; Hambley, T. W. Chem. Rev. 2009, 109, 4911−4920. (5) Rabik, C. A.; Dolan, M. E. Cancer Treat. Rev. 2007, 33, 9−23. (6) Ma, P. A.; Xiao, H. H.; Li, C. X.; Dai, Y. L.; Cheng, Z. Y.; Hou, Z. Y.; Lin, J. Mater. Today 2015, 18, 554−564. (7) Jakupec, M. A.; Galanski, M.; Arion, V. B.; Hartinger, C. G.; Keppler, B. K. Dalton Trans. 2008, 2, 183−194. (8) Kelland, L. Nat. Rev. Cancer 2007, 7, 573−584. (9) Oberoi, H. S.; Nukolova, N. V.; Kabanov, A. V.; Bronich, T. K. Adv. Drug Delivery Rev. 2013, 65, 1667−1685. (10) Johnstone, T. C.; Suntharalingam, K.; Lippard, S. J. Chem. Rev. 2016, 116, 3436−3486. (11) Marullo, R.; Werner, E.; Degtyareva, N.; Moore, B.; Altavilla, G.; Ramalingam, S. S.; Doetsch, P. W. PLoS One 2013, 8, e81162. (12) Kim, S. J.; Ho Hur, J.; Park, C.; Kim, H. J.; Oh, G. S.; Lee, J. N.; Yoo, S. J.; Choe, S. K.; So, H. S.; Lim, D. J.; Moon, S. K.; Park, R. Exp. Mol. Med. 2015, 47, e142. (13) Kim, H. J.; Lee, J. H.; Kim, S. J.; Oh, G. S.; Moon, H. D.; Kwon, K. B.; Park, C.; Park, B. H.; Lee, H. K.; Chung, S. Y.; Park, R.; So, H. S. J. Neurosci. 2010, 30, 3933−3946. (14) Itoh, T.; Terazawa, R.; Kojima, K.; Nakane, K.; Deguchi, T.; Ando, M.; Tsukamasa, Y.; Ito, M.; Nozawa, Y. Free Radical Res. 2011, 45, 1033−1039. (15) Steinherz, L. J.; Steinherz, P. G.; Tan, C. T.; Heller, G.; Murphy, M. L. JAMA, J. Am. Med. Assoc. 1991, 266, 1672−1677. (16) Rybak, L. P.; Mukherjea, D.; Jajoo, S.; Ramkumar, V. Tohoku J. Exp. Med. 2009, 219, 177−186. (17) Miller, R. P.; Tadagavadi, R. K.; Ramesh, G.; Reeves, W. B. Toxins 2010, 2, 2490−2518. (18) Bystrom, L. M.; Guzman, M. L.; Rivella, S. Antioxid. Redox Signaling 2014, 20, 1917−1924. (19) Liou, G. Y.; Storz, P. Free Radical Res. 2010, 44, 479−496. (20) Shen, J.; Zhao, L.; Han, G. Adv. Drug Delivery Rev. 2013, 65, 744−755. (21) Punjabi, A.; Wu, X.; Tokatli-Apollon, A.; El-Rifai, M.; Lee, H.; Zhang, Y.; Wang, C.; Liu, Z.; Chan, E. M.; Duan, C.; Han, G. ACS Nano 2014, 8, 10621−10630. (22) Huang, P.; Lin, J.; Wang, S.; Zhou, Z.; Li, Z.; Wang, Z.; Zhang, C.; Yue, X.; Niu, G.; Yang, M.; Cui, D.; Chen, X. Biomaterials 2013, 34, 4643−4654. (23) Ai, X.; Ho, C. J. H.; Aw, J.; Attia, A. B. E.; Mu, J.; Wang, Y.; Wang, X.; Liu, Y. X.; Chen, H.; Gao, M.; Chen, X.; Yeow, E. K. L.; Liu, G.; Olivo, M.; Xing, B. Nat. Commun. 2016, 7, 10432. 936


Letter

Nano Letters (56) Mikhail, A. S.; Partanen, A.; Yarmolenko, P.; Venkatesan, A. M.; Wood, B. J. Magn. Reson. Imaging Clin. N. Am. 2015, 23, 643−655. (57) Ho, D.; Sun, X.; Sun, S. Acc. Chem. Res. 2011, 44, 875−882. (58) Sridhar, S.; Campbell, R.; Nagesha, D.; Gultepe, E. Cancer Res. 2011, 71, 380. (59) Huang, L.; Li, Z. J.; Zhao, Y.; Zhang, Y. W.; Wu, S.; Zhao, J. Z.; Han, G. J. Am. Chem. Soc. 2016, 138, 14586−14591.

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Enhanced Cisplatin Chemotherapy by Iron Oxide Nanocarrier

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