Cellular Organelle-Dependent Cytotoxicity of Iron Oxide Nanoparticles

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Cellular Organelle-Dependent Cytotoxicity of Iron Oxide Nanoparticles and Its Implications for Cancer Diagnosis and Treatment: A Mechanistic Investigation Chieh-Cheng Huang, Zi-Xian Liao, Hsiang-Ming Lu, WenYu Pan, Wei-Lin Wan, Chun-Chieh Chen, and Hsing-Wen Sung Chem. Mater., Just Accepted Manuscript • DOI: 10.1021/acs.chemmater.6b03905 • Publication Date (Web): 26 Nov 2016 Downloaded from http://pubs.acs.org on November 29, 2016

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Chemistry of Materials

Cellular Organelle-Dependent Cytotoxicity of Iron Oxide Nanoparticles and Its Implications for Cancer Diagnosis and Treatment: A Mechanistic Investigation Chieh-Cheng Huang,† Zi-Xian Liao,† Hsiang-Ming Lu,† Wen-Yu Pan,† Wei-Lin Wan,† ChunChieh Chen†,‡ and Hsing-Wen Sung*,† †

Department of Chemical Engineering and Institute of Biomedical Engineering, National Tsing Hua University, ‡ Hsinchu 30013, Taiwan (ROC). Department of Orthopaedic Surgery, Chang Gung Memorial Hospital at Linkou, Chang Gung University, Taoyuan 33305, Taiwan (ROC)

ABSTRACT: Iron oxide nanoparticles (IONPs) have been widely used in the diagnosis and treatment of cancer; however, analysis of the relevant literature yields contradictory results concerning their toxicity. In this work, a bubble-generating liposomal system that can be thermally triggered to liberate its loaded IONPs instantly and precisely in defined cellular organelles is utilized to elucidate the mechanism that is responsible for the contradictory observations concerning IONP toxicity. As-prepared liposomes are internalized by test cells via endocytosis, and these internalized particles follow the endocytotic pathway from the endosomes to the lysosomes. The degradation of IONPs and the consequent release of iron ions depend strongly on the pH of the environment in the cellular organelles from which they are liberated, to which they are exposed, during their intracellular transportation. Higher IONP toxicity is associated with stronger in situ degradation with the release of more iron ions, and the consequent generation of more reactive oxygen species (ROS) within cells. When the amount of ROS formed exceeds that can be scavenged by the intracellular antioxidant systems, the cells experience oxidative stress, which is responsible for the observed cellular organelle-dependent toxicity profiles. Understanding the mechanism that underlies the toxicity of IONPs is critical for designing IONP nanosystems that have a wide range of clinical applications.

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(•OH), a highly reactive ROS. The toxicity of IONPs is thus directly related to their release of iron ions upon degra1,12,14,19,20 dation in acidic organelles. We hypothesize that the toxicity of IONPs that are internalized into cells by endocytosis depends on the pH of the environment to which the particles are exposed during their endocytotic transportation. Very little is currently known about the toxicity of IONPs upon exposure to various acidic organelles. Understanding the IONP toxicity that is induced in different acidic organelles is essential to the design of IONP nanosystems with a wide range of clinical applications. In a recent study, the authors presented a thermoresponsive liposomal system that contained ammonium bicar21-23 At bonate (ABC, NH4HCO3) for localized drug delivery. an elevated temperature (42 °C), the decomposition of ABC generates CO2 bubbles that creates permeable defects in the lipid bilayer of the liposomes, which rapidly and locally release their loaded therapeutic agent at high dose. To test the hypothesis that is proposed herein, this bubble-generating liposomal system with a unique mechanism for localized controlled release is utilized to liberate loaded IONPs precisely in defined acidic organelles during intracellular trafficking.

INTRODUCTION Iron oxide nanoparticles (IONPs) have been used as contrast agents in magnetic resonance imaging (MRI) for cancer di1-4 1,5,6 agnosis, as carriers of drugs/genes, and as localized heat 4,7 generators in the hyperthermia cancer treatment. Also, they have been frequently used to label cells for in vitro sepa8,9 10,11 ration and sorting and in vivo tracking magnetically. Although IONPs are generally considered to be biocompatible, the literature presents conflicting results concerning 12-15 their toxicity. The most commonly proposed explanation of IONP toxicity involves the generation of reactive oxygen species (ROS), which causes lipid peroxidation, disrupting the phospholipid-bilayer membrane, resulting in cell 1,14,16 death. Owing to their nanoscale size, IONPs are easily internal10,17,18 ized by cells via endocytosis. Upon internalization, IONPs are degraded by hydrolysis into iron ions within acidic organelles such as endosomes or lysosomes. The free iron ions are then transported across the organelle membranes through the divalent metal transporter-1 (DMT1) into cytosol, where they undergo the Fenton reaction with the mitochondrial hydrogen peroxide (H2O2) to form hydroxyl radicals

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Figure 1. Schematic illustrations showing the structure of thermoresponsive bubble-generating liposomal system and its process of spatially precise, controlled intracellular liberation of IONPs in specific cellular organelles in various endocytotic stages. The degradation of IONPs, release of iron ions, and subsequent reactive oxygen species (ROS) generation within cells are indicated. IONPs: iron oxide nanoparticles; ABC: ammonium bicarbonate; DMT1: divalent metal transporter-1. Figure 1 schematically depicts the structure of the thermoresponsive bubble-generating liposomal system and its controlled intracellular liberation of IONPs in defined cellular organelles in a spatially precise fashion. Following cellular uptake by endocytosis, the liposomes that contain ABC and IONPs are thermally triggered at 42 °C, in various stages of the process of intracellular transportation, to liberate instantly their loaded IONPs inside defined acidic organelles. Next, these IONPs are degraded to iron ions by hydrolysis in the low-pH environment. Their subsequent intracellular generation of ROS and the corresponding IONP toxicity are then determined. In this work, the hyperthermic temperature was limited to ≤ 42 °C to prevent damage to the test cells. Reportedly, cells typically undergo apoptosis or mitotic 10,24 death when tissues are heated to > 45 °C.

In Vitro Degradation of IONPs in Environments with Various pH values. Following cell internalization, IONPs + can be degraded via hydrolysis into iron ions (Fe3O4 + 8 H 2+ 3+ 10,25 The in vitro → Fe + 2 Fe + 4 H2O) in acidic organelles. degradation of IONPs and their consequent release of iron ions were studied by incubating the test particles in phosphate-buffered saline (PBS; 100 μg/mL) for various intervals at 37 °C at pH 7.2, 6.0, and 5.0, which are the pH values of the environments in cytosol, endosomes, and lysosomes, 21,26 respectively. The mean particle sizes of the test IONPs before and after degradation were measured by dynamic light scattering (DLS; Zetasizer 3000HS, Malvern Instruments Ltd., Worcestershire, UK). Their volumes were calcu3 lated as: volume = 4/3 × (πR ), where R is the radius of the test IONPs. To quantify the iron ions that were released into PBS upon degradation, the test solution was treated with Perl’s reagent (1% potassium ferrocyanide/2% hydrochloric acid, 50:50 by vol/vol) for 15 min. Ferrocyanide can chelate 3+ 3+ ferric ions (Fe ) to form ferric ferrocyanide, 4 Fe + 3 4− 27 [Fe(CN)6] → Fe4[Fe(CN)6]3, which is blue (Prussian blue). The absorbance of Prussian blue at 570 nm was then measured using a spectrophotometer (SpectraMax M5; Molecular Devices, Sunnyvale, CA, USA). Preparation of Test Liposomes. Liposome colloidal suspensions were prepared by dissolving HSPC, cholesterol, and DOTMA in a 60:40:5 molar ratio in chloroform. The organic solvent was removed with a rotavapor to produce a thin lipid film on the glass vial. The lipid film (10 mg) was then hydrated using an aqueous solution with ABC (2.7 M) and IONPs (100 μg/mL) with sonication at room tempera-

EXPERIMENTAL SECTIONS Materials. Hydrogenated soy phosphatidylcholine (HSPC) and 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA) were purchased from Avanti Polar Lipids (Alabaster, AL, USA), while cholesterol, ABC and 3,3´dioctadecyloxacarbocyanine perchlorate (DiO) were acquired from Sigma-Aldrich (St. Louis, MO, USA). IONPs that were functionalized with Cy3-labeled dextran with a diameter of around 30 nm were obtained from MagQu (New Taipei City, Taiwan). The HT1080 human fibrosarcoma cell line was obtained from the Bioresource Collection and Research Center, Food Industry Research and Development Institute, Hsinchu, Taiwan. All other used chemicals and reagents were of analytical grade.


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Chemistry of Materials assay, the cells were treated with 5 mg/mL MTT for 4 h at 37 °C. The metabolized MTT was dissolved in dimethyl sulfoxide, and its absorbance was measured at 570 nm with a spec28 trophotometer. To gain insight into the mechanism of IONP-induced cell death, the liposome-treated cells were further stained using an Alexa Fluor 488 annexin V/Dead Cell Apoptosis Kit (Life Technologies). Alexa Fluor 488 annexin V was utilized to detect the translocation of phosphatidylserine (PS) from the inner to the outer leaflet of the plasma membrane, and propidium iodide (PI), which is a nucleic acid stain, was used to 29 identify dead cells. After the HT1080 cells had been treated with the test liposomes and incubated at 37 °C for various periods, they were cultivated at 42 °C for 3 min and then incubated at 37 °C for another 2 h. The cells were then trypsinized, stained with Alexa Fluor 488 annexin V and PI at room temperature for 30 min, and analyzed using a flow cytometer. Intracellular Release of Iron Ions and Generation of ROS. Once the IONPs that were encapsulated in the endocytosed liposomes were liberated in acidic organelles, they could act as intracellular iron donors to produce free iron 2+ ions. Free iron in the form of ferrous ions (Fe ) can undergo 2+ 3+ − a Fenton reaction (Fe + H2O2 → Fe + •OH + OH ) with mitochondrial H2O2 to generate •OH radicals and ferric ions 3+ (Fe ). To determine the amount of ferric ions that were formed intracellularly, the HT1080 cells were treated with the test liposomes using the same procedure as was used in the cytotoxicity study. Next, the cells were fixed with 4% paraformaldehyde for 10 min and incubated with Perl’s reagent for 15 min to produce the Prussian blue stain, which was photographed under a phase-contrast microscope (Eclipse TE200, Nikon, Tokyo, Japan) or analyzed using a spectrophotometer at a wavelength of 570 nm. The intracellular ROS levels were obtained by treating the test cells with CellROX® Green Reagent (Life Technologies), following the manufacturer’s instructions. The treated cells were fixed in 4% paraformaldehyde and then observed under a CLSM for the qualitative determination of ROS levels; for the quantitative investigation, the cells were trypsinized, fixed with 4% paraformaldehyde, and then analyzed using a flow cytometer. Lipid Peroxidation. ROS that is formed intracellularly by IONPs can oxidize membrane lipids, yielding to break28 down products such as malondialdehyde (MDA) . Lipid peroxidation in the liposome-treated cells was qualitatively detected using an Image-iT® Lipid Peroxidation Kit (Life Technologies), which contained a fluorescence probe C11BODIPY 581/591. The MDA level in the treated cells was quantified using the Lipid Peroxidation (MDA) Assay (Abcam), according to the manufacturer’s directions. Statistical Analysis. All data are expressed as mean ± standard deviation. The one-tailed Student t test was conducted to assess differences between pairs of groups. A p value of less than 0.05 was considered to indicate statistical significance.

ture, before being sequentially extruded. The free ABC was removed by dialyzing against 10 wt% sucrose solution with 5 mM NaCl. Finally, the liposomes were passed through a G-25 column (GE Healthcare, Buckinghashire, UK) to remove the unencapsulated IONPs. Characterization of Test Liposomes. The mean particle size and zeta potential of the as-prepared liposomes were evaluated by DLS, and their morphology was examined by transmission electron microscopy (TEM, JEOL 2010 F, Tokyo, Japan). The encapsulation efficiency and content of IONPs in liposomes were obtained using fluorescence measurements following their destruction with Triton X-100. The encapsulation efficiency and content were calculated using the follow21 ing equations. Encapsulation efficiency (%) =

mass of IONPs in liposomes ×100% mass of IONPs in initial solution

Encapsulation content =

mass of IONPs in liposomes mass of lipids

The thermoresponsive characteristics of the liposomes were explored by observing the formation of CO2 bubbles in a test tube that contained prewarmed PBS at 37 °C (body temperature) and 42 °C (hyperthermic temperature). The test tubes were immersed in a water-filled tank, and the formation of CO2 bubbles was observed using an ultrasound imaging system (Z-one, Zonare; Mountain View, CA, USA). The release profiles of IONPs were obtained by incubating the test liposomes in PBS at 37 °C or 42 °C. The PBS solutions were then passed through a G-25 column to separate out the released IONPs, and their fluorescent intensity was monitored using a fluorescence spectrometer. Cellular Uptake and Intracellular Trafficking of Test Liposomes. HT1080 cells were maintained in DMEM medium that was supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin at 37 °C in a humidified incubation chamber with 5% CO2. To analyze their uptake by cells, the liposomes that were labeled with DiO (and contained 100 μg/mL IONPs) were incubated with HT1080 cells at a density 5 of 2 × 10 cells/well for predetermined periods. The cells were then trypsinized, fixed with 4% paraformaldehyde (SigmaAldrich), and analyzed using a flow cytometer (Cell Lab Quanta SC, Beckman Coulter, Inc., Fullerton, CA, USA). To monitor their intracellular trafficking following endocytosis, test liposomes were incubated with HT1080 cells for the indicated intervals before they were fixed using 4% paraformaldehyde. The fixed cells were stained with antibodies (Abcam, Cambridge, MA, USA) against endosomes (antiEEA1) and lysosomes (anti-LAMP2), counterstained with 4,6diamidino-2-phenylindole (DAPI; Sigma-Aldrich), and then observed using a confocal laser scanning microscope (CLSM; LSM 780, Carl Zeiss, Jena, Germany). Cytotoxicity of IONPs. To evaluate the cytotoxicity of IONPs that had been liberated in various endocytotic stages, HT1080 cells were treated with test liposomes for 0, 10, 15, 30, 45, or 60 min, and then incubated with prewarmed media at 37 or 42 °C for 3 min. Following incubation at 37 °C for a further 2 h, the viability of the treated cells was qualitatively determined using a live/dead assay based on calcein-AM and ethidium homodimer (LIVE/DEAD Viability/Cytotoxicity Kit; Life Technologies, Carlsbad, CA, USA) and quantitatively evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) assay. In the live/dead assay, test samples were observed using a CLSM. In the MTT

RESULTS AND DISCUSSION Owing to their combined advantages in the diagnosis and treatment of cancer, the use of IONPs has been of ongoing interest in the biomedical field. However, reports yield con12-15 tradictory findings concerning the toxicity of IONPs. The disparate results concerning IONP toxicity that have been



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of IONPs from the test liposomes depended strongly on the environmental temperature; this dependence is characteristic of a thermoresponsive carrier. As displayed in Figure 3c, the extent of release of IONPs from the liposomes at 37 °C was relatively low (ca. 15% of the initial content). In contrast, upon heating to 42 °C, many of the encapsulated IONPs (ca. 70%) were released within just 30 s. The above experimental results suggest that following endocytosis, these liposomes may be thermally triggered to produce CO2 bubbles during endocytotic transportation, structurally perturbing their lipid membranes, causing the prompt liberation of IONPs within defined intracellular spatial ranges (specific acidic organelles). To analyze their uptake by cells, the IONPs-loaded liposomes that were labeled with DiO were incubated with HT1080 cells for predetermined periods. According to the results shown in Figure 3d that were evaluated using a flow cytometer, test liposomes entered the cells rapidly. During incubation, positively charged liposomes can be adsorbed onto a negatively charged cell surface by a nonspecific ionic interaction; the adsorbed liposomes are then internalized through endocytosis. These empirical data reveal that the liposomes that were prepared in this work can be internalized by cells by endocytosis and thus may serve as efficient intracellular carriers of IONPs. Intracellular Transport of Internalized Liposomes. The intracellular transport of internalized liposomes was tracked by observing, using a CLSM, the potential colocalization of Cy3-labeled IONPs that were encapsulated in the test liposomes and cellular organelles. According to

presented in earlier investigations probably follow from variations among experimental design, which inhibits evaluation of the degradation of IONPs in cells and comparisons of their intracellular ROS generation. This work offers a general explanation for the intracellular IONP toxicity that may account for the contradictory reports, based on results obtained using a bubble-generating liposomal system that can be thermally triggered to liberate loaded IONPs in defined cellular organelles in a spatially precise manner (Figure 1). With reference to IONP toxicity that is observed in particular cellular organelles, the designs of IONPs for various biomedical applications are discussed. pH-Dependent Degradation of IONPs. In acidic envi1,30 ronments, IONPs, which mostly comprise Fe3O4, can be 2+ 3+ 10,31 degraded by hydrolysis into free iron ions (Fe /Fe ). These free ions interact with H2O2 that is generated by the mitochondria to produce highly reactive •OH radicals (Fenton reaction), resulting in oxidative stress, which can cause cell death. In this work, the kinetic release of iron ions from degraded IONPs was evaluated in vitro at the pH values in intracellular organelles. According to Figure 2a, the degradation (or volume reduction) of IONPs depended on the environmental pH. A concurrent pH-dependent release of ferric ions into the test media over time was demonstrated: it was minimal at pH 7.2 (cytosol pH) and maximal at pH 5.0 (lysosomal pH) (Figure 2b, p < 0.05). These analytical data suggest that when exposed to intracellular acidic organelles, IONPs may be degraded by hydrolysis with a pH-dependent release of iron ions, which may modulate their cytotoxicity.

Figure 2. Effects of environmental pH on (a) degradation (volume reduction) of IONPs and (b) consequent release of † ‡ iron ions (n = 6). *p < 0.05, **p < 0.001 vs. pH 7.2; p < 0.05, p < 0.001 vs. pH 6.0. Characteristics of Test Liposomes. The TEM images in Figure 3a clearly reveal that IONPs were successfully encapsulated inside the liposomes. The encapsulation efficiency and content of IONPs in the as-prepared liposomes were 78.6 ± 6.4% and 6.3 ± 0.5 μg/mg, respectively (n = 6 batches). DLS measurements indicate that the size and surface potential of the liposomes were 193.3 ± 10.2 nm and 29.5 ± 2.0 mV, respectively. The temperature responsiveness of the liposomes that were suspended in PBS was evaluated by measuring their ability to generate CO2 bubbles, which were observed macroscopically using an ultrasound imaging system, as well as the extent of the consequent liberation of their encapsulated IONPs. Whereas very few bubbles were formed in the sample of liposomes at 37 °C, many were produced when the sample was heated to 42 °C (Figure 3b). Accordingly, the liberation

Figure 3. Characteristics of as-prepared liposomes: (a) representative TEM micrographs, and (b) ultrasound images; (c) release profiles of IONPs (n = 6) when suspended in PBS at 37 or 42 °C (heat triggering). (d) Cellular uptake by HT1080 cells following their incubation with DiO-labeled liposomes for prespecified durations (n = 6).


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Figure 4. CLSM images of intracellular transport of IONPs in HT1080 cells that were incubated with test liposomes for predetermined periods. Lower panels magnify white boxes in corresponding upper panels. The white arrow highlights the event of IONP-endosome/lysosome co-localization, while the white arrowhead indicates the IONPs that were observed in cytoplasm alone. homodimer stained the nuclei of dead cells with red fluores32 cence. Similar to the untreated control cells, the cells that had been treated at 37 °C with liposomes without IONPs, exhibited no apparent cytotoxicity (p > 0.05; Figure 5a), indicating that the cationic liposomes did not cause significant cell death. Additionally, most of the cells in the group that received liposomes that contained no IONPs and incubated at 42 °C were viable compared to the untreated control cells, suggesting that the liposomes that generated only CO2 bubbles (Figure 3b) did not result in significant cell death. In the group of cells that were treated with the liposomes that contained IONPs but did not undergo thermal activation (at 37 °C), minimal toxicity was observed throughout the study. Conversely, when these cells were activated thermally at 42 °C, cell viability initially declined substantially to an extent that depended on incubation time, reaching a minimum at 30 min. At this time, the endocytosed liposomes were transported to the organelles of the lysosomes (Figure 4); after they were heated, their encapsulated IONPs were rapidly liberated locally (Figure 3c). Heat triggering at > 45 min following incubation significantly increased cell viability (Figure 5a and 5b, p < 0.05). Notably, during this interval, relatively many IONPs were freed into the cytosol as a result of the lysosomal fusion (Figure 4) before heat triggering. To elucidate the IONP-induced cell toxicity, the treated cells were further stained with annexin V and PI and then analyzed using a flow cytometer. Annexin V binds to the exposed PS on the outer plasma membranes, and so can be used to detect changes in the surfaces of cells that occur early during apoptosis. PI, a nonspecific DNA intercalating agent, is excluded by the plasma membranes of living cells, and so can be exploited to distinguish necrotic cells from apoptotic and living cells by supravital staining without prior 29 permeabilization. Therefore, flow-cytometric analysis of the stained cells can differentiate cells into four groups which are – – + – viable (annexin V /PI ), early apoptosis (annexin V /PI ), late + + – + apoptosis (annexin V /PI ), and necrotic (annexin V /PI ) cells.

Figure 4, ten to 15 minutes following incubation, the IONPs (green) exhibited high co-localization with early endosomes (stained orange with the anti-EEA1 antibody) of the cells, suggesting that the internalized liposomes were mostly localized in early endosomes at this instant. Notably, at 15 min, a few green dots of IONPs were observed in the cytoplasm alone (as indicated by the white arrowhead), implying that these IONPs might have been liberated from the internalized liposomes by the fusion of their membranes with those of early endosomes. When cationic lipids from liposomes come into close contact with anionic phospholipids from endosomes, membrane destabilization and lipid mixing are induced, and may be responsible for liberating the liposomal 25,26 contents into the cytosol (endosomal escape). In 30 min, the early endosomes had matured into late endosomes/lysosomes (stained purple with the anti-LAMP2 antibody). At that time, most of the green IONP dots overlapped the endocytotic vesicles. However, the number of free IONPs in the cytoplasm increased significantly after 30 min, probably because of the fusion between the membranes of liposomes and those of lysosomes, liberating IONPs. By 45 to 60 min of incubation, an even greater number of free IONPs had appeared in the cytosol. Once the fusion process had been initiated, lipid mixing was autocatalytic, because membrane destabilization caused additional anionic phospholipids to appear at the site of the interaction, promoting the adhesion of cationic liposomes to the lysosomal membrane, leading to the fusion of their membranes (lysosomal fu26 sion). IONP Toxicity in Distinct Cellular Organelles. To evaluate the toxicity of IONPs, HT1080 cells were incubated with the test liposomes for prespecified intervals and then thermally activated at 42 °C to liberate their encapsulated IONPs into defined cellular organelles. The viability of the cells was qualitatively determined using a live/dead assay and quantitatively evaluated using the MTT assay. In the live/dead assay, calcein-AM was enzymatically converted into green fluorescent calcein in live cells, while ethidium



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Figure 5. Cytotoxicity of IONPs that were liberated intracellularly in various endocytotic stages. (a) Viability of HT1080 cells that were incubated with test liposomes with or without IONPs for predetermined durations at 37 or 42 °C (heat triggering), as de# ## termined by the MTT assay. *p < 0.05, **p < 0.001 vs. liposomes without IONPs at 37 °C; p < 0.05, p < 0.001 vs. liposomes with† ‡ out IONPs at 42 °C; p < 0.05, p < 0.001 vs. liposomes with IONPs at 37 °C. (b) Representative live/dead staining images and (c) corresponding results of flow-cytometric analysis of HT1080 cells that had been incubated with test liposomes with IONPs for predetermined intervals and then heat-treated at 42 °C. Figure 5c displays representative results of the flowcytometric analysis and dot plots of the regional percentage of annexin V as a function of PI for the cells that had been treated with the test liposomes for indicated incubation periods and then subjected to thermal activation to liberate their encapsulated IONPs. A larger percentage (> 85%) of these cells were apoptotic than of the cells in the control group, and only a very small fraction of cell deaths were caused by necrosis, revealing that IONP toxicity causes apoptotic but not necrotic cell death. Early apoptotic cells are known to be able to be rescued from the apoptotic program and prolifer33 ate if the apoptotic stimulus is removed. Time-dependent increments in the late apoptotic population were detected when the cells were thermally activated following their 10, 15, or 30 min of incubation with the test liposomes. Notably, when the incubation was prolonged to 45 or 60 min, the proportion of cells in late-stage apoptosis fell dramatically. During cellular transport, the liberated IONPs experience a continuous decrease of pH -- from 6.3–5.5 in the early endosomes to pH 5.5–4.5 in the late endosomes/lysosomes, 2+ 3+ triggering their release of iron ions (Fe /Fe ). To elucidate the mechanism of their cellular organelle-dependent toxicity, the amounts of iron ions that were released from IONPs in various stages of the endocytotic process were studied.

Figure 6. (a) Representative photomicrographs of treated cells with Prussian blue staining in different endocytotic stages, showing intracellularly generated ferric ions; (b) corresponding quantitative results (n = 6). *p < 0.05 vs. untreat† ed control cells; p < 0.05 vs. cells that had been incubated with test liposomes with IONPs for 15 min and then heat§ treated at 42 °C; p < 0.05 vs. cells that had been incubated with test liposomes with IONPs for 45 min and then heattreated at 42 °C.


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Figure 7. (a) CLSM images, showing levels of ROS generated within treated cells in different endocytotic stages. (b) Percentage of ROS-positive cells (n = 6) and (c) corresponding fluorescence intensity, as determined by flow cytometry. Intracellular Release of Iron Ions. The amount of fer3+ ric ions (Fe ) that were generated intracellularly was determined after the treated cells had been processed for Prussian blue staining. According to Figure 6a and 6b, the untreated control cells (t = 0) exhibited a normal round morphology and a minimal intracellular intensity of Prussian blue. After the cells were incubated with the test liposomes for the specified intervals and then thermally activated, the cells appeared to be disrupted and shrunken, and the intensity of the Prussian blue stain, representing the amount of ferric ions that were formed within the cells, was higher (p < 0.05). These effects became increasingly obviously as the incubation time increased, and peaked at an incubation interval of 30 min, which was in the endocytotic stage of the lysosomes (Figure 4). As the incubation was further prolonged to > 45 min (by which time some IONPs had already been directly freed from the internalized liposomes into the cytoplasm), the intensity of Prussian blue within the cells declined (p < 0.05), possibly because significantly fewer iron ions were produced in the neutral cytosol pH environment (pH 7.0–7.4, Figure 2a and 2b). The above experimental results indicate that the degradation of IONPs and their consequent release of iron ions depend strongly on the locations of the cellular organelles in which they are liberated; specifically, exposure of the liberated IONPs to more acidic cellular organelles causes the intracellular release of more iron ions. 3+ In cellular organelles, ferric iron ions (Fe ) must be re2+ duced to ferrous ions (Fe ) by the metalloreductase STEAP3 before they can be transported into the cytosol by the membrane DMT1. These ferrous iron ions can then undergo the Fenton reaction with the mitochondrial hydrogen peroxide (H2O2) to form highly reactive ROS, such as hydroxyl radicals (•OH). When the amount of ROS formed exceeds that can be scavenged by the intracellular antioxidant systems, the cells experience oxidative stress, which is responsible for the observed intracellular toxicity profiles.

Intracellular Formation of ROS and Its Subsequent Lipid Peroxidation. The ferrous iron-induced intracellular formation of ROS was identified in cultures using the 34 CellROX Green reagent, which is a DNA fluorogenic probe. In an environment with an elevated concentration of intracellular ROS, CellROX Green reagent can be oxidized and bind to DNA. Its green fluorescence signal is therefore pri34 marily localized in the nucleus and mitochondria and can be qualitatively investigated using a CLSM (Figure 7a). The proportion of ROS-positive cells (Figure 7b) and their green fluorescence intensity (Figure 7c) were determined by flow cytometry. Consistent with the pH-dependent release of iron ions, described above, the internalized test liposomes that contained IONPs induced maximal ROS levels within the test cells following an incubation interval of 30 min and subsequent thermal activation (when IONPs were liberated in lysosomes, as presented in Figure 4). The excess intracellularly generated ROS reacted with membrane lipids, potentially inducing deleterious lipid peroxidation and producing MDA as an end-product, leading 28,35 eventually to cell death. The levels of lipid peroxidation in the treated cells as a result of exposure of IONPs to the acidic organelles, were revealed using a fluorescence probe (ImageiT® Lipid Peroxidation Kit), which shifted from red to green 36 upon oxidation in live cells. The level of MDA, a biomarker of lipid peroxidation, was determined by a colorimetric assay (Lipid Peroxidation MDA Assay). Figure 8a indicates that the levels of lipid peroxidation were low in the untreated control cells, but significantly elevated in the liposome-treated cells, whose cellular lipid peroxidation levels peaked 30 min after incubation. Similar results concerning the intracellular levels of MDA were observed (Figure 8b; p < 0.05). These experimental results suggest that the elevated intracellular ROS levels that were induced by the iron ions that were intracellularly released from the degraded IONPs caused oxidative damage to the lipid-bilayer membranes, and eventually cell death.



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Figure 8. (a) CLSM images of levels of lipid peroxidation within treated cells in various endocytotic stages; (b) intracellular lev† els of MDA (n = 6). **p < 0.001 vs. untreated control cells; p < 0.05 vs. cells that had been incubated with test liposomes with § IONPs for 15 min and then heat-treated at 42 °C; p < 0.05 vs. cells that had been incubated with test liposomes with IONPs for 45 min and then heat-treated at 42 °C. MDA: malondialdehyde. cellular toxicity; this phenomenon may favor their use in cancer treatment.

A General Explanation of Intracellular IONP Toxicity and Its Implications for Biomedical Applications. The above experimental results reveal that the toxicity of IONPs toward cells depends strongly on the cellular organelles to which they are exposed during intracellular transportation. Higher IONP toxicity arises from stronger in situ degradation and the consequently greater intracellular release of iron ions, which are closely related to the local pH environment to which the IONPs are exposed and cause greater oxidative damage by the ROS mechanism. The toxicity of IONPs is minimal in early endosomes, peaks in late endosomes/lysosomes, and then decays as the transportation proceeds further (Figure 1). Although the IONPs that are liberated in early endosomes (at 10 and 15 min following incubation) may ultimately encounter the low pH environment in late endosomes/lysosomes, the iron ions that are released from the IONPs and transported into the cytosol through the membrane DMT1 promote the synthesis of a large amount of iron2,40 storage protein ferritin (Figure 1). The synthesized ferritin may then prevent the accumulation of excess iron ions (known as iron overload) in the cytosol when the IONPs are exposed to a highly acidic organelle. Conversely, activation of liposomes in late endosomes/lysosomes (at 30 min after incubation) leads to the direct exposure of IONPs to low pH, resulting in their relatively rapid hydrolysis and, thus, the accumulation of very many iron ions in the cytosol. The resultant cytoplasmic iron overload induces the formation of 1-3,12 ROS mediated by the Fenton reaction . The mechanisms of the toxicity of IONPs in defined cellular organelles must be considered in the development of their potential biomedical applications. To use IONPs in MRI diagnosis, magnetic cell separation/sorting, or cell tracking, the early endosomal escape of IONPs is necessary to reduce their intracellular toxicity before their extensive degradation by hydrolysis during endocytotic transportation. Materials, 37,38 39 such as fusogenic peptides or polyethyleneimine, that favor endosomal escape can be integrated into the IONP coating. Additionally, the fusion of the membranes of cationic liposomes and the endocytotic vesicles, which can promote the liberation of liposomal contents (such as IONPs, as demonstrated in this work) into the cytoplasm, can also reduce the intracellular toxicity of IONPs. However, when the IONPs are located in lysosomes, the locally low pH causes massive dissolution of iron oxide as iron ions, increasing

CONCLUSION The model that is proposed herein demonstrates that the local environment in cellular organelles, in which the pHdependent degradation of IONPs and the release of iron ions occur, critically affects the amount of intracellularly generated ROS, which causes lipid peroxidation and eventual cell mortality. The contradictory results in the literature concerning IONP toxicity may therefore be strongly related to the environment to which the IONPs are exposed during their intracellular transport. For applications in cancer diagnosis and cell separation/sorting and tracking, the early endosomal escape of IONPs is crucial to preventing toxicity toward target cells. Conversely, the direct exposure of IONPs in lysosomes can significantly elevate their intracellular toxicity, potentially improving their effectiveness in cancer treatment.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]

Author Contributions C.C.H. and Z.X.L. contributed equally to this work.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This study was supported by grants from the Ministry of Science and Technology (MOST 103-2221-E-007-022-MY3 and MOST 104-2314-B-007-001-MY3), Taiwan.

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Cellular Organelle-Dependent Cytotoxicity of Iron Oxide Nanoparticles

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