Corrosion‐Activated Chemotherapeutic Function of ...download.xuebalib.com/xuebalib.com.38829.pdf · line therapy for the specific purpose of overcoming cisplatin resistance. Recent

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Corrosion-Activated Chemotherapeutic Function of Nanoparticulate Platinum as a Cisplatin Resistance-Overcoming Prodrug with Limited Autophagy InductionHsien-Jen Cheng, Te-Haw Wu, Chih-Te Chien, Hai-Wei Tu, Ting-Shan Cha, and Shu-Yi Lin*

unveiled. These seemingly contradictory effects of cellular protection and cytotoxicity are puzzling, and might involve an adverse consequence from multiple enzymatic activities of nano-Pt,[12] including superoxide dismutase (SOD), catalase, as well as peroxidase mimetics, that regulate the consump-tion and induction of reactive oxygen species (ROS). Among these enzyme-mimicking actions of nano-Pt, the SOD-like activity can convert superoxide anion (O2˙

−) into oxygen and hydrogen peroxide (H2O2) that can subsequently be con-sumed by the catalase- and peroxidase-like activities. The final and intermediate products from catalase and peroxidase mimetics are oxygen (O2) and hydroxyl radical (HO˙, a highly toxic ROS),[18] respectively. Although the peroxidase-like activity of nano-Pt differs depending on environmental pH values and the size and surface facet of nano-Pt,[15,19–21] the production of highly active ROS (i.e., HO˙) by any nano-Pt DOI: 10.1002/smll.201602374

Despite nanoparticulate platinum (nano-Pt) has been validated to be acting as a platinum-based prodrug for anticancer therapy, the key factor in controlling its cytotoxicity remains to be clarified. In this study, it is found that the corrosion susceptibility of nano-Pt can be triggered by inducing the oxidization of superficial Pt atoms, which can kill both cisplatin-sensitive/resistance cancer cells. Direct evidence in the oxidization of superficial Pt atoms is validated to observe the formation of platinum oxides by X-ray absorption spectroscopy. The cytotoxicity is originated from the dissolution of nano-Pt followed by the release of highly toxic Pt ions during the corrosion process. Additionally, the limiting autophagy induction by nano-Pt might prevent cancer cells from acquiring autophagy-related drug resistance. With such advantages, the possibility of further autophagy-related drug resistance could be substantially reduced or even eliminated in cancer cells treated with nano-Pt. Moreover, nano-Pt is demonstrated to kill cisplatin-resistant cancer cells not only by inducing apoptosis but also by inducing necrosis for pro-inflammatory/inflammatory responses. Thus, nano-Pt treatment might bring additional therapeutic benefits by regulating immunological responses in tumor microenvironment. These findings support the idea that utilizing nano-Pt for its cytotoxic effects might potentially benefit patients with cisplatin resistance in clinical chemotherapy.

Drug Resistance

Dr. H.-J. Cheng, T.-H. Wu, Dr. C.-T. Chien, Dr. S.-Y. LinInstitute of Biomedical Engineering and NanomedicineNational Health Research Institutes35 Keyan Road, Zhunan 35053, TaiwanE-mail: [email protected]

H.-W. Tu, Dr. T.-S. ChaNational Synchrotron Radiation Research CenterNo. 101, Hsin-Ann Road, Hsinchu 30076, Taiwan

1. Introduction

Nanoparticulate platinum (nano-Pt) possessing a cisplatin-like chemotherapeutic function[1–6] (i.e., killer) as well as acting as an artificial antioxidant[7–17] (i.e., protector) has been

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would cause biosafety concerns.[19,22,23] In fact, experimental results at the in vitro level have demonstrated that nano-Pt that mimics peroxidase and also has SOD-like and catalase-like activities can only act as an antioxidant for cellular pro-tection.[12,17] Presumably, the cellular level of ROS in a given physiological state would be balanced by natural defense molecules, such as glutathione and methallothioneins, for homeostasis.[24,25] Thus, the significant cytotoxicity of nano-Pt should not be due to its peroxidase-like activity, meaning, again, that the key factor or factors underlying that cytotox-icity remain to be clarified.

Pioneering studies have shown that the superficial atoms of noble metal-based artificial nanoparticles, including nano-Pt, are key active sites for performing redox-type cat-alytic cycles, allowing such nanoparticles to mimic various enzyme abilities.[20,21] Following such catalytic cycles, the catalytic efficiency would be improved while the number of exposed Pt atoms would be increased, thus increasing the number of active sites. Despite the fact that the mechanism underlying the enzyme-like behavior of nano-Pt has not yet been clarified completely, the activity is possibly rel-evant to the association and dissociation of oxygen species during the ROS-harvesting process.[21,26,27] Thus, the adsorp-tion process can increase ROS decomposition and dehy-dration, resulting in the generation of atomic oxygen.[21] Owing to the fact that atomic oxygen can easily diffuse into the inner shell of nano-Pt, resulting in the growth of surface oxides via interface oxidization,[28,29] such oxidiz-ability of superficial atoms would be dramatically increased with increasing active sizes. In other words, when the size of individual nano-Pt particles is shrunk to ≈1 nm, at least 76% of the Pt atoms on the surface are exposed,[30] meaning

that the coverage of surface oxides on such particles would be higher than those of large size nano-Pt.[31–34] With such oxidization and subsequent dissolution in the acidic orga-nelles (i.e., endosomes/lysosomes) of cells, a typical corro-sion process similar to that of silver nanoparticles would be triggered.[23] We thus hypothesized that the cytotoxicity of nano-Pt might be related to the loss of chemical inert-ness rather than originating from its peroxidase-like activity. Up to now, little is known as to whether the cytotoxicity of nano-Pt might be increased by increased susceptibility to corrosion. In the present study, we attempted to manipulate the corrosion susceptibility of nano-Pt (Scheme 1, square box) to understand the correlation between such dissolution and its cytotoxic effects.

Furthermore, it is well-known that nano-Pt can be taken up by cells via an endocytosis-mediated pathway.[5] This makes nano-Pt unlike cisplatin, a well-established Pt-based prodrug commonly used as a first-line therapy,[35] which can rely almost exclusively on membrane transports to be taken up by certain types of cancer cells.[36] In general, cancer cells with these specific forms of membrane transporters are sensi-tive to cisplatin and can easily be eradicated. However, cancer cells can develop acquired drug resistance after a period of cisplatin treatment, in which some were found to express the specific transporters at lower levels that led to a substantial decrease in cisplatin uptake.[37,38] Nano-Pt, with its different way of cellular entry, might thus provide an efficient second-line therapy for the specific purpose of overcoming cisplatin resistance. Recent studies have reported that autophagy is related to the acquired cisplatin resistance of different types of cancer cells, such as ovarian, lung, and esophageal cancers.[39–42] Autophagy is a cellular process that supports

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Scheme 1. A pictorial diagram showing the cellular effects, cytotoxicity, and autophagy induction of cisplatin and nano-Pt treating on cancer cells. Hydrolysis-activated cisplatin is known to kill cancer cells by apoptosis, though it could also cause severe autophagy induction leading to acquired cisplatin resistance for cancer cells. The dashed arrows mean that further verification is needed for corrosion-activated nano-Pt.



cell survival in response to stress or starvation. During autophagy, cytoplasmic proteins and damaged organelles are initially surrounded by pre-autophagosomal structures with a double-layered membrane called phagophores. After the elongation and rounding of the membrane of phagophore, the mature autophagosome then fuses with a lysosome, called an autolysosome, and hydrolytic enzymes then digest the contents within this acidic environment.[43] Autophagy could protect cancer cells against stressful challenges of anti-cancer drugs by maintaining minimum metabolic homeostasis. Therefore, autophagy has been suggested to be one of the mechanisms initiating the acquired drug resistance of cancer cells during cisplatin treatment (Scheme 1, upper panel). In cancer therapy, the role of autophagy is still a topic of intense debate, with autophagy being thought to potentially function as either a pro-survival or pro-death mechanism to coun-teract or mediate the cytotoxic effects of anticancer drugs.[44] The autophagic responses induced in cancer cells by cisplatin could thus either protect them from apoptosis or, conversely, contribute to enhanced cell death.[45,46] Recently, the pros and cons of nanoparticle-induced autophagy in cancer therapy have been given greater attention.[47] If nano-Pt possesses sufficient susceptibility to corrosion to give it cisplatin-like toxicity for killing cancer cells, it is important and necessary to investigate whether autophagy could be induced in cancer cells during chemotherapy with nano-Pt prodrugs (Scheme 1, lower panel).

2. Results and Discussion

2.1. The Difference between As-Prepared and Aged Nano-Pt

Details on the preparation of the nano-Pt used in the present work have been reported elsewhere,[5] in which a low-genera-tion and amine-terminated dendrimer (G2NH2) acts as a cage for nano-Pt preparation. Since the sizes of nano-Pt are very small, only transmission electron microscopy (TEM) images with high magnification (Figure S1, Supporting Information) can be used to confirm the presence of nano-Pt. Specially, Figure S1B (Supporting Information) shows an eye-like structure from the nano-Pt caged by a G2NH2. Meanwhile, NMR spectra (Figure S2, Supporting Information) clearly reconfirm the presence of dendrimer from the caged nano-Pt. The average size of “naked” nano-Pt particles encap-sulated within a polyamidoamine (PAMAM) dendrimer is ≈1 nm (as shown in Figure 1A), regardless of whether fresh (denoted as as-prepared nano-Pt) or un-fresh (denoted as aged nano-Pt) preparation is used. In testing various enzyme-mimicking activities, the SOD-like catalytic ability can indeed be observed (Figure 1B) for both the as-prepared and aged nano-Pt. Comparatively, the O2˙

−-scavenging capability of the as-prepared nano-Pt is slightly superior to that of the aged nano-Pt. Presumably, the catalytic sites of aged nano-pt are occupied by oxides to decrease the O2˙

− affinity for performing a redox reaction. Meanwhile, we found that both

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Figure 1. The properties of two kinds of nano-Pt, namely, as-prepared nano-Pt and aged nano-Pt, which are labeled (1) and (2). The A) size distributions, B) peroxidase-like and SOD-like activities, C) cytotoxicity levels, and D) levels of toxic ions released by the two types of nano-Pt in neutral and acidic conditions. The measurement in (D) was performed by inductively coupled plasma mass spectrometry (ICP-MS). Ctr1 and Ctrl 2 in the photograph in (B) refer to nano-Pt samples without TMB and with HRP (horseradish peroxidase, a natural enzyme), respectively.



types of nano-Pt can consume H2O2 to cause the oxidization of a typical peroxidase substrate (TMB, 3,3′,5,5′-tetramethyl-benzidine[48]), resulting in a blue-color or a yellow-color product (photographs in Figure 1B). These results confirmed that both the as-prepared and aged nano-Pt possess perox-idase-like activity for the production of HO˙. Accordingly, the behaviors of both the as-prepared and aged nano-Pt in terms of exhibiting enzyme-mimicking activity are very sim-ilar. Theoretically, their trends in terms of causing either cell survival or cell death would thus be similar to each other. Unexpectedly, however, significant toxicity was only found for cells being treated with the aged nano-Pt, as shown in Figure 1C. In contrast, the as-prepared nano-Pt did not cause cell damage. It should be noted, however, that the aged nano-Pt itself was derived from as-prepared nano-Pt intentionally dried and then preserved for a period time in atmospheric conditions. Otherwise, endotoxin contamination of the aged nano-Pt has been ruled out as a cause of its toxicity. Owing to the fact that the superficial atoms of nano-Pt serve as pos-sible active sites for exerting catalytic functions, the signifi-cant cell death caused by the aged nano-Pt might be related to a significant difference in the intrinsic properties of “naked metal” compared with those of as-prepared nano-Pt. Thus, we hypothesized that a corrosion phenomenon similar to that undergone by silver nanoparticles (nano-Ag) might be involved in the cell death observed.[23] The corrosion differ-ences between the as-prepared and the aged nano-Pt were thus investigated and described. Since the nano-Pt can be taken up by cells via a typical endocytosis pathway,[5] the end

location of such intracellular trafficking is inside endosomes/lysosomes. Once the corrosive nano-Pt thus becomes local-ized in the acidic organelles, the toxic ions would be released, causing cell damage. Figure 1D and Figure S3 (Supporting Information) show the dissolution and release profile of the aged nano-Pt, respectively, implying the oxidization of the “naked metal” of the aged nano-Pt.

2.2. The Differences in Corrosion between the As-Prepared and the Aged Nano-Pt

Oxidizability is an important index of corrosion in metals.[29] Two powerful tools, X-ray photoelectron spectroscopy (XPS) and X-ray absorption near edge structures (XANES) have been used to indirectly and directly confirm whether the growth of surface oxides occurs on nano-Pt. Figure 2A shows the XPS spectrum of as-prepared nano-Pt, in which the binding energy of the Pt (4f7/2) component is very sensitive to the oxi-dizability of superficial atoms.[49] The main peak appearing at ≈73 eV is higher than that for large-size nano-Pt (≈71 eV), a difference which can be attributed to the size effect.[49] Illus-trating the results of an analysis of the Pt electronic states of XPS spectra of nano-Pt of different ages, Figure 2B summa-rizes the values at the binding energy of Pt (4f7/2). We found that the binding energy was slightly increased as the aging time of the nano-Pt was increased. This variation in chemical shifts strongly implied the surface oxidization of nano-Pt.[49,50] Note that the control sample (with 0 h of aging) represented

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Figure 2. The formation of nano-Pt oxides. A,B) XPS data, C) XANES data at Pt LIII-edge, and D) FT EXAFS spectra data of nano-Pt and Pt foil.



the as-prepared samples, but the production process cannot completely eliminate air. Direct evidence to confirm whether the growth of surface oxides on nano-Pt has occurred can be supplied using XANES. According to an analysis of the XANES spectra (Figure 2C), the typical peaks at ≈11567 eV originating from the Pt LIII-edge are very sensitive to the oxi-dation status of nano-Pt.[51] As expected, smaller (1 nm) nano-Pt particles can easily be oxidized compared to larger (5 nm) nano-Pt particles and bulk (Pt foil) particles. Comparatively, the intensity of the absorption signal of 1 nm aged nano-Pt is dramatically stronger than that of as-prepared nano-Pt. These results clearly indicated the existence of surface oxides.

2.3. The Formation of PtO Bonds in the Aged Nano-Pt

It is interesting to further explore how the structural differ-ences between the as-prepared and the aged nano-Pt occur; that is, whether the PtO bonds can or cannot form after particles are covered with surface oxides. Such fine struc-tures at atomic levels can be investigated using the extended X-ray absorption fine structures (EXAFS) technique, with the backscattering of photoelectrons being able to provide specific messages about the neighboring atoms of superficial Pt atoms in nano-Pt. Figure 2D shows the Fourier transfor-mations (FT) of Pt LIII-edge EXAFS signals from short-time (1 h) and long-time (8 h) aged nano-Pt and two control

samples. As can be seen, the FT amplitude had a pronounced peak near ≈2.7 Å, a value indicating the distance between two Pt atoms.[52] Comparatively, bulk-Pt (i.e., Pt foil) can be easily observed in the first nearest PtPt bond than those of three kinds of nano-Pt. These results can easily be under-stood because the backscattered photoelectrons can cause resonance with the nearest atoms in the EXAFS process.[53] More importantly, a main peak near ≈2.0 is contributed by the PtO bond,[52] which can be significantly increased when contributed by the aged 1 nm nano-Pt. These results directly elucidated that the growth of surface oxides can occur via the formation of PtO bonds when the as-prepared nano-Pt is allowed to remain in contact with air for a period time.

2.4. Nano-Pt Exhibits Cytotoxicity against A2780cis as well as A2780 Cells

Nano-Pt exhibits cytotoxicity against A2780cis as well as A2780 cells. In order to determine whether nano-Pt pos-sesses cytotoxicity against cancer cells similar to that of cis-platin, the ovarian cancer cell lines A2780 and A2780cis were selected as the targets of treatment. The A2780 cell line displays a round-shaped appearance and grows in clusters. The acquisition of cisplatin resistance in the A2780cis cell line has induced morphologic changes giving A2780cis cells a spindle-shaped appearance (Figure 3A). First, we treated

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Figure 3. A) The morphological characteristics of cisplatin-resistant A2780 ovarian cancer cells compared with their parental A2780 cells. B) Cytotoxic activity of cisplatin and nano-Pt against A2780 and A2780cis cell lines. The dashed line in (B) shows the resistance factor of cisplatin and nano-Pt, respectively.



A2780 and A2780cis cells with nano-Pt or with cisplatin to determine whether nano-Pt is able to kill cancer cells as well as cisplatin. After 24 h period of treatment, the cell viabili-ties were determined using WST-1 assays after 24 h period of treatment. The results showed that low concentrations of cisplatin could easily kill A2780 cells, but that high concen-trations of cisplatin were required to effectively kill A2780cis cells. On the other hand, different concentrations of nano-Pt could kill both A2780 and A2780cis cells at nearly the same rates (Figure 3B). The RF (resistance factor = IC50A2780cis/IC50A2780) for cisplatin was 3.7, while the RF for nano-Pt was about 0.87. These data revealed that nano-Pt exerted cytotoxicity roughly similar to that of cisplatin against A2780 cells, while also exhibiting cytotoxicity against A2780cis cells in spite of their cisplatin resistance. These results also sug-gested that the application of nano-Pt to chemotherapy could not only inhibit cancer progression but also could overcome acquired drug resistance. That is the most critical advantage of nano-Pt over cisplatin.

2.5. Nano-Pt Induces Fewer Autophagic Responses Than Cisplatin in A2780 and A2780cis Cells

Autophagy is known to be one of the survival mechanisms of cancer cells during chemotherapy and is associated with the regulation of cytotoxicity of anti-cancer drugs as well

as acquired drug resistance.[40–42] To reveal the role played by autophagy in cancer cells treated with nano-Pt, we first examined whether autophagy could be induced in both the A2780 and A2780cis cell lines by treatment with nano-Pt. The induction of autophagy in A2780 and A2780cis cells was determined by western blot analysis hybridized with anti-LC3 antibody, an autophagosome marker. The conver-sion of LC3 (from LC3-I to LC3-II), a key step during the formation of autophagosomes, is widely used to monitor autophagy,[54,55] with the levels of LC3-II expression rep-resenting the relative number of autophagosomes in the cells. After a 24 h period of treatment, cisplatin signifi-cantly induced higher LC3-II expression in both A2780 and A2780cis cells. Relatively speaking, nano-Pt caused only a slight increase in LC3-II expression (Figure 4A). To further validate the differing levels of autophagy induced by cisplatin treatment and nano-Pt treatment, we assessed the levels of autophagic flux and autophagosome accumulation. Cyto-ID, a fluorescence dye that selectively labels autophagic vacuoles, was used for monitoring the autophagic flux and measuring the accumulated autophagosomes in live cells.[56] First, the level of autophagic flux was determined by flow cytometric analysis (Figure 4B). The histogram results showed that cis-platin (green) induced higher levels of autophagic flux than nano-Pt (red), both in the A2780 (25.3% vs 12.0%) and A2780cis cells (25.0% vs 6.0%). Comparing between these two types of cells, A2780 cells can be triggered to produce

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Figure 4. Autophagic responses: A) LC3-II expression, B) autophagic flux, and C) autophagosome accumulation were induced by cisplatin and nano-Pt in A2780 and A2780cis cell lines. Hereinafter, “M” represents protein markers, “NT” means non-treated control.



severe autophagic responses relative to A2780cis cells under drug treatments, and this finding was consistent with the LC3-II expression patterns in the preceding western blotting analysis (seeing Figure 4A). In addition, the accumulation of autophagosomes was also observed by confocal microscopy (Figure 4C). The autophagosome signals (green) of untreated A2780cis cells were fewer and weaker than those of A2780 cells, with Hoechst33342 and LysoTracker being used for nuclear counterstaining (blue) and to indicate lysosome loca-tions (red), respectively. With cisplatin treatment, the accu-mulation of autophagosomes was significantly increased in both the A2780 and A2780cis cells. The yellow signals in the merged images indicated that autophagosomes fused with lysosomes to form autolysosomes. However, unlike cisplatin treatment, nano-Pt treatment only induced minor accumu-lation of autophagosomes. The minor autophagic responses induced by nano-Pt might result from two phenomena that were observed in our previous study: First, the endocytic pathway of cellular entry retained nano-Pt in endosomes until fusion with lysosomes.[5] Second, the increased level of intracellular ROS, a stress factor for autophagy induction,[43] caused by nano-Pt in cancer cells was significantly lower than that caused by cisplatin.[5,57] According to the results men-tioned above, treatment with nano-Pt might induce no drug resistance or at least less drug resistance than cisplatin treat-ment because of its minor induction of autophagic responses. This is the second advantage of nano-Pt over cisplatin.

2.6. Nano-Pt Caused a Different Pattern of Cell Death from Cisplatin in A2780cis Cells

The nano-Pt showed the similar cytotoxicity against cancer cells as cisplatin, while also exhibiting a lower ability to induce autophagic responses than cisplatin. Cisplatin has been known to kill different types of cancer cells by inducing apoptosis.[57] Therefore, we sought to determine the type of cell death that nano-Pt triggers in A2780 and A2780cis cells in order to clarify the mechanisms of its cytotoxicity. After treating A2780 and A2780cis cells with cisplatin or nano-Pt for a period of 24 h, the levels of apoptotic cell death were determined by Annexin V and PI staining and flow cytometric analysis. Annexin V and PI staining is widely used to deter-mine if cells are viable, apoptotic, or necrotic through differ-ences in phosphatidylserine transfer to outer cell membranes and plasma membrane integrity.[58] The results showed that cisplatin induced early apoptosis (Annexin V+/PI−) in A2780 cells in a dose-dependent manner, while only a minor degree of necrosis/late apoptosis (Annexin V+/PI+) was induced (Figure 5, panel I). This indicated that cisplatin killed A2780 cells by means of inducing apoptosis with a slow progres-sion. In addition, while cisplatin treatment did not trigger cell death obviously in A2780cis cells, apoptosis was still weakly detected (Figure 5, panel II). On the other hand, nano-Pt treatment induced significant necrosis/late apoptosis in both A2780 and A2780cis cells in a dose-dependent manner, whereas only minor early apoptosis was induced in A2780 cells (Figure 5, panel III and IV). That indicated that nano-Pt killed A2780 cells by means of inducing apoptosis with a

rapid progression. Alternatively, nano-Pt directly induced both apoptosis and necrosis in A2780cis cells. In our pre-vious study, the dual apoptotic and necrotic cell death caused by nano-Pt was also observed in another cisplatin-resistant cancer cell line, MDA-MB-231.[5] The results above revealed the possibility that nano-Pt could trigger a different cell death pathway in drug-resistant cancer cells from that triggered by cisplatin. Again, we also found that nano-Pt treatment indeed can change cytokine and chemokine profiles of cancer cells, the data shown in Tables S1 and S2 (Supporting Information). They also implied that inflammatory immune responses could be induced by these apoptotic cells and necrotic debris in tumor microenvironments after nano-Pt treatment.

3. Conclusion

In the present study, the chemotherapeutic function of nano-Pt can be activated by inducing the oxidization of superficial Pt atoms to kill cisplatin-sensitive as well as cisplatin-resistant cancer cells. More importantly, the activated nano-Pt only triggers minor autophagy responses in both cisplatin-sensitive and cisplatin-resistant cancer cells. Thus, the pro-survival role of autophagy in cancer cells treated with nano-Pt would be limited. With this advantage, the possibility for further devel-opment of autophagy-related drug resistance could be sub-stantially reduced or even eliminated in cancer cells treated with nano-Pt. Additionally, our nano-Pt is demonstrated to kill cisplatin-resistant cancer cells not only by inducing apop-tosis but also by inducing necrotic cell death for pro-inflam-matory and inflammatory responses. Thus, nano-Pt treatment might bring additional therapeutic benefits by regulating immunological responses in tumor microenvironment. These findings support our concept that utilizing nano-Pt as a novel second-line anticancer drug could prove useful in nanomedi-cine-related prodrug development.

4. Experimental Section

Nano-Pt Synthesis and Purification: H2PtCl6 (Acros, 200 μL, 30 μmol, 150 mm) was added to 20 mL of deionized water con-taining G2NH2 (Aldrich, 94.7 μL, 5 μmol, 20 wt% methanol solu-tion). The reaction mixture of G2NH2 and either H2PtCl6 or K2PtCl4 was incubated at room temperature overnight before being irradi-ated by microwave (CEM, Discover LabMate System, 300 W/120 °C and 30 min). After reduction, precipitated large platinum nanopar-ticles were filtered through a 0.22 μm membrane filter (Millipore, PES membrane, for platinum nanoclusters). Note that the solution should be freeze-dried and then dissolved in ≈1 mL water for fur-ther purification. To further remove extra anions (PtCl6

2−), anionic exchange chromatography (Merck, Fractogel EMD TMAE Hicap) was used to obtain the purified nano-Pt. High-resolution transmission electron microscopy (HRTEM, JEOL-2010) was then used to iden-tify the sizes of the as-prepared and aged nano-Pt particles. After that, the surface area of nano-Pt is ≈40.03 m2 g−1 by BET surface area measurements using nitrogen adsorption (Micrometric ASAP 2010 apparatus). The data are shown in Figure S4 (Supporting Information).

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Cytotoxicity Pretest after Nano-Pt Synthesis: Cells of the MDA-MB-231 line (Bioresource Collection and Research Center, Taiwan) were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum under 5% CO2 atmosphere at 37 °C. 24-well cell culture plates were inoculated with 1 × 105 cells/well and kept at 37 °C for 24 h. The culture medium was changed in the presence of various concentrations of different treatments (G2NH2 alone, carboplatin, cisplatin, and the nano-Pt) in culture medium (three replicates). The culture medium was removed and the cells were incubated with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazo-lium bromide (MTT, Sigma, MO, USA) at 37 °C for 1 h. After treat-ment, the formazan product from MTT was dissolved in DMSO and quantified using a conventional ELISA reader at 570 nm. For cali-bration, a blank test was performed in a 24-well plate under the same conditions.

XPS Measurement: XPS measurements were carried out with a system (PHI 1600) equipped with an Al/Mg dual anode (15 kV, 400 W). The nano-Pt samples were examined without Ar sput-tering, and the ultra-high vacuum (UHV) pressure used was below 5 × 10−10 Torr.

XANES and EXAFS Measurements: The XANES and EXAFS spectra were recorded at the National Synchrotron Radiation Research Center (NSRRC) in Hsinchu, Taiwan, with a beamline 01C1 operated at 1.5 GeV with a current of 360 mA. The Si (111) double crystal monochromatic was used with a resolution ΔE/E better than 1 × 10−4. The Pt LIII-edge absorption spectra were recorded by the fluorescence yield (FY) mode at room temperature using a Lytle detector for liquid samples. Standard Pt metal foil was used for energy calibration and also for comparing different electronic valence states. The EXAFS data analysis was performed

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Figure 5. Patterns of cell death induced by cisplatin and nano-Pt in A2780 and A2780cis cell lines. The abbreviations C1, C2, and C3 represent three concentrations in increasing of cisplatin and nano-Pt, respectively.


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using the ATHENA and ARTEMIS programs in the Demeter com-puter package.[59,60] For the EXAFS analysis, the backgrounds of the pre-edge and the post-edge were subtracted and normalized with respect to the edge jump step from the XAS spectra (χ(E)). The normalized (χ(E)) spectra were transformed from energy to k-space and further weighted by k3 to distinguish the contribu-tions of backscattering interferences from different coordination shells. Then, k3-weighted data in the k-space ranging from 2.85 to 12.5 Å−1 for the Pt LIII-edge were Fourier transformed to r-space data in order to separate the EXAFS contributions from different coordination shells. The phase correction was set on all spectra in the r-space.

Cell Culture: The A2780 and A2780cis human ovarian cancer cell lines were purchased from the European Collection of Authen-ticated Cell Cultures (ECACC). The cisplatin-resistant A2780cis cell line was developed by chronic exposure of the parent cisplatin-sensitive A2780 cell line to increasing concentrations of cispl-atin. Both cell lines were grown in RPMI-1640 medium (Gibco by Life Technologies) supplemented with 10% FBS and incubated at 37 °C with 5% CO2. The cisplatin-resistant A2780cis cells were fed with an extra 0.3 μg mL−1 cisplatin every 2–3 passages in order to maintain their resistance. The characteristics of A2780 and A2780cis cells were observed and imaged under bright field by inverted microscopy (IX71, Olympus).

Cytotoxicity Assay: The cytotoxicity of cisplatin and nano-Pt against A2780 and A2780cis cells was demonstrated by using WST-1 assay (Roche-CELLPRO-RO, Sigma-Aldrich). A2780 or A2780cis cells were seeded in 96-well microtiter plates in a volume of 100 μL/well medium. A cell concentration of 5 × 103 cells/well and an incubation time of 24 h were used for experimental treat-ments with cisplatin or nano-Pt at particular concentrations. After the incubation period, the culture medium was replaced with 0.1 volume of WST-1 in RPMI-1640 medium. The cells were fur-ther incubated for 1–1.5 h. Then the samples in 96-well microtiter plates were analyzed against a background control by using an ELISA reader (SpectraMax M2, Molecular Devices), and the wave-length for measuring the absorbance of the formazan product was 450 nm. The data of cell relative viabilities were normalized to con-trol groups (non-treatment). For each experimental repeat, each condition was tested with six wells of cells.

Autophagy Assay: Cellular Protein Extraction and Western Blot Analysis: A2780 or A2780cis cells were seeded in 10 cm cul-ture dishes in a volume of 10 mL medium. A cell concentration of 3 × 106 cells/dish and an incubation time of 24 h were used for experimental treatments with cisplatin or nano-Pt at particular con-centrations. After the incubation period, experimentally treated A2780 and A2780 cells were washed with phosphate-buffered saline (PBS) and scraped after they had been immersed in Prote-oJET mammalian cell lysis reagent (Fermentas Life Sciences) con-taining EDTA-free protease inhibitor cocktail (Roche-CELLPRO-RO, Sigma-Aldrich). After a 15 min reaction on ice, the lysis samples were centrifuged at 14 000 rpm and 4 °C for 15 min to collect the cellular proteins from the supernatant. The protein samples were quantified by Pierce BCA protein assay kit (Thermo scientific) and kept at −80 °C before use. Quantified protein samples were sepa-rated in Bolt 4%–12% Bis-Tris Plus gels by using Bolt min gel tank and transferred to 0.2 μm polyvinylidene difluoride membranes (PVDF, Immobilon-P, Millipore) with Bolt mini blot module (NOVEX by Life Technologies). After blocking with 5% skim milk in 0.05%

PBS-Tween 20 for 30 min, the PVDF membranes were incubated with anti-LC3 (1:3000 dilution, Novus Biologicals) and anti-β-actin antibodies (1:300 dilution, GeneTex, Inc.), respectively, at 4 °C over-night. After they had been washed with 0.05% PBS-Tween 20, the membranes were incubated with anti-rabbit IgG antibodies (1:3000 dilution, Millipore) at room temperature for 1.5 h. The membranes were then washed with 0.05% PBS-Tween 20 and soaked in western HRP substrate (Luminata Crescendo, Millipore) for 1 min. Then the membrane was exposed and imaged to reveal specific proteins by Amersham Imager 600 (GE Healthcare Life Sciences).

Fluorescence Staining and Flow Cytometric Analysis: Cyto-ID Autophagy detection kit (ENZO) was used to monitor autophagic responses in live cells. For the detection of autophagic flux, A2780 or A2780cis cells were seeded in six-well microtiter plates in a volume of 1 mL/well medium. A cell concentration of 2 × 105 cells/well and an incubation time of 24 h were used for experimental treatments with cisplatin or nano-Pt at particular concentrations. Experimentally treated A2780 and A2780 cells were washed with PBS buffer and then harvested by trypsin (0.25%, with 2.21 × 10−3 m EDTA, Corning Life Sciences) digestion and serum neutralization. The harvested cells were then stained by the fluo-rescence dye Cyto-ID Green detection reagent, according to the manufacturer’s instructions. The stained cells were analyzed using flow cytometry (FACSCalibur, BD Biosciences).

Fluorescence Staining and Confocal Microscopic Observa-tion: In observations of autophagosome accumulation, A2780 or A2780cis cells were seeded in μ-Dish35 mm, high (ibidi) dishes alternatively in a volume of 1 mL medium. A cell concentration of 2 × 105 cells/dish and an incubation time of 24 h were used for experimental treatments with cisplatin or nano-Pt at particular con-centrations. Experimentally treated A2780 and A2780 cells were washed with PBS buffer and then stained by the fluorescence dyes Cyto-ID Green detection reagent, Hoechst33342 Blue detection reagent (nuclear stain, ENZO), and LysoTracker Red DND-99 (lyso-somal stain, Molecular Probes, Invitrogen), according to the manu-facturer’s instructions. The stained cells were observed by using confocal laser microscopy (FV10i, Olympus).

Cell Apoptosis Assay: Alexa Flour 488 Annexin V/Dead cell Apoptosis kit (Molecular Probes, Invitrogen) was used to validate cell death phenotypes including apoptosis and necrosis. A2780 or A2780cis cells were seeded in six-well microtiter plates in a volume of 1 mL/well medium. A cell concentration of 2 × 105 cells/well and an incubation time of 24 h were used for experimental treatments with cisplatin or nano-Pt at particular concentrations. Experimentally treated A2780 and A2780 cells were washed with PBS buffer and then harvested by trypsin (0.25%, with 2.21 × 10−3 m EDTA, Corning Life Sciences) digestion and serum neutralization. The harvested cells were then stained by the fluo-rescence dyes annexin V-FITC and propidium iodide (PI), according to the manufacturer’s instructions. The stained cells were analyzed using flow cytometry (FACSCalibur, BD Biosciences).

Cytokine Enzyme-Linked Immunosorbent Assay: Cancer cells (3 × 105 cells/well; six-well plate) were treated with nano-Pt for 24 h. Cytokines and chemokine were detected using Luminex, the Bio-Plex multiplex system (Bio-Rad); the operation is according to the manufacturer’s instructions. The ELISA assay contains IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12(P40), IL-12(P70), IL-13, IL-17A, eotaxin, G-CSF, GM-CSF, IFN-r, KC, MCP-1, MIP-α, MIP-β, RANTES, and TNF-α.


6133© 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim www.small-journal.comsmall 2016, 12, No. 44, 6124–6133

Supporting Information

Supporting Information is available from the Wiley Online Library or from the author.

Acknowledgements

H.-J.C. and T.-H.W. contributed equally to this work. The authors thank the National Health Research Institutes of Taiwan (NHRI-BN-104-PP-30/32) and the Ministry of Science and Technology of Taiwan (MOST-103-2325-B-400-014, MOST-104-2325-B-400-004) for pro-viding financial support for this research. Special thanks go to Mr. Chia-Yeh Liu, Mr. Wei-Neng Liao (from Dr. Jen-Kun Chen), and Dr. Ching-Ping Liu for helping with the confocal imaging measurements, BET surface area measurement, and discussion, respectively.

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Received: July 18, 2016Revised: August 26, 2016Published online: September 22, 2016

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