pH-Sensitive Polymeric Nanoparticles with Gold(I) Compound

Article pubs.acs.org/molecularpharmaceutics

pH-Sensitive Polymeric Nanoparticles with Gold(I) Compound Payloads Synergistically Induce Cancer Cell Death through Modulation of Autophagy Yao-Xin Lin,†,‡ Yu-Juan Gao,† Yi Wang,†,‡ Zeng-Ying Qiao,† Gang Fan,† Sheng-Lin Qiao,†,‡ Ruo-Xin Zhang,† Lei Wang,† and Hao Wang*,† †

CAS Key Laboratory for Biological Effects of Nanomaterials and Nanosafety, National Center for Nanoscience and Technology (NCNST), No. 11 Beiyitiao, Zhongguancun, Beijing, China ‡ University of Chinese Academy of Science (UCAS), No. 19A Yuquan Road, Beijing, China S Supporting Information *

ABSTRACT: Various nanomaterials have been demonstrated as autophagy inducers owing to their endocytosis cell uptake pathway and impairment of lysosomes. pH-dependent nanomaterials as drug delivery systems that are capable of dissociating in weakly acidic lysosomal environment (pH 4−5) and consequently releasing the payloads into the cytoplasm have been paid extensive attention, but their autophagy-modulating effects are less reported so far. In this study, we report pH-sensitive micelle-like nanoparticles (NPs) that self-assembled from poly(β-amino ester)s to induce cell autophagy. By encapsulation of gold(I) compounds (Au(I)) into hydrophobic domains of NPs, the resultant Au(I)-loaded NPs (Au(I)⊂NPs) shows synergistic cancer cell killing performance. The Au(I)⊂NPs enter cells through endocytosis pathway and accumulate into acidic lysosomes. Subsequently, the protonation of tertiary amines of poly(β-amino ester)s triggers the dissociation of micelles, damages the lysosomes, and blocks formation of autolysosomes from fusion of lysosomes with autophagosomes. In addition, Au(I) preferentially inhibits thioredoxin reductase (TrxR) in MCF-7 human breast cancer cells that directly links to up-regulate reactive oxygen species (ROS) and consequently induce autophagy and apoptosis. The blockade of autophagy leads to excessive depletion of cellular organelles and essential proteins and ultimately results in cell death. Therefore, pH-sensitive polymeric nanoparticles with gold(I) compound payloads can synergistically induce cancer cell death through regulation of autophagy. Identification of the pH-sensitive nanomaterials for synergistically inducing cell death through regulation autophagy may open a new avenue for cancer therapy. KEYWORDS: autophagy, ROS, pH-sensitive, polymer, nanoparticles, cancer terials.18 The pH-sensitive polymeric nanoparticles with tertiary amine groups,19−21 acetals/ketals,22−25 ortho esters,26−32 hydrazine/imide bonds,33−38 etc. have been widely used as drug delivery systems (DDS) because they could dissociate in weakly acidic lysosomal environment (pH 4−5) and eventually release the payloads into the cytoplasm.39−43 We have prepared a series of pH-sensitive polymeric nanoparticles to enhance killing efficacy on cells.44,45 The thioredoxin (Trx) system, composed of thioredoxin reductase (TrxR), Trx, and NADPH, plays critical roles in maintaining cellular redox balance and regulating cell death.46 The inhibition of TrxR can destruct the redox homeostasis of cells47,48 through the elevation of cellular reactive oxygen

1. INTRODUCTION Autophagy is an important and highly conserved metabolic process through the lysosome-based degradation pathway, which plays a critical role in maintaining cellular homeostasis and enhancing cellular defense.1 Basal autophagy as a protective process usually promotes cell survival. Paradoxically, excessive or sustained cell autophagy, particularly in apoptosis-defective cells, promotes cell death.2,3 Therefore, autophagy has been recognized as a type-II programmed cell death (PCD) or autophagic cell death.4 Regarding the regulation of autophagy, variable strategies have been developed and shown potentials in cancer prevention and therapy.5−8 Recently, a variety of nanoparticles, such as quantum dots,9,10 gold nanoparticles,11,12 lanthanide oxide nanocrystals,13,14 fullerene and its derivatives,15,16 dendrimers,17 etc., have been demonstrated as autophagy inducers. Therefore, it would be of great interest to develop a simple yet effective approach to induce/enhance cancer cell death through regulation autophagy by nanoma© XXXX American Chemical Society

Received: January 19, 2015 Revised: June 4, 2015 Accepted: June 23, 2015

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DOI: 10.1021/acs.molpharmaceut.5b00060 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Molecular Pharmaceutics

Scheme 1. Schematic Diagram of Au(I)-Loaded Poly(β-amino ester)s Micelle-like Nanoparticles (Au(I)⊂NPs) and Their Mechanisms on Synergistic Induction of Cell Death

2. MATERIALS AND METHODS 2.1. Materials. Au(I) was prepared according to the previous method,56 and pH-sensitive polymers were prepared as described previously.44,45 Cy 5 was obtained from SigmaAldrich (St Louis, MO, USA). LC3II, cathepsin B, p-p70S6K, p70S6K, p62, and β-actin antibody were purchased from Cell Signal Technology, Inc. LysoTracker Green DND-26 and LysoTracker Green DND-189 were purchased from Invitrogen Corporation. Cell counting kit-8 (CCK-8) and DCFH-DA were purchased from Beyotime Institute of Biothechnology (China). Acid phosphatase assay kit and Earle’s balanced salt solution were purchased from Sigma-Aldrich. MCF-7 cells were obtained from the cell culture center of Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences (Beijing, China). 2.2. Synthesis of pH-Sensitive Copolymers. The pHsensitive mPEG-poly(amino ester)s graft copolymers were prepared by Michael addition as we previous reported.44 mPEG-NH2 2K (0.200g, 0.1 mmol), HDDA (0.226g, 1 mmol), and DBPA (0.117g, 0.9 mmol) were first dissolved into 2 mL of DMSO and then bubbled with N2 for 15 min under stirring. After they incubated for 5 days at 50 °C, polymers were dialyzed against deionized water (MWCO: 3500 Da) to obtain the final solution of polymers. Other copolymers were prepared employing the same method. 2.3. Loading of Au(I) into Au(I)⊂NPs. The Au(I)-loaded nanoparticles were prepared using the dialysis method.44 Au(I) and the copolymers were first dissolved in 1 mL of DMSO under stirring. PBS (2 mL) was added to the mixture solution under stirring. To determine the molecule loading efficiency (MLE) and molecule loading content (MLC) of Au(I), the Au(I)⊂NPs were evaluated through the concentrations of gold

species (ROS) levels and further leading to necrosis or apoptosis of cells.49,50 Growing evidence from the past few years supports the idea that ROS and the occurrence of autophagy are closely linked.51−55 Excessive ROS can induce cell death through autophagy.46 Our group has developed a series of gold(I) phosphine compounds,56 which are capable of up-regulating ROS by inhibiting TrxR.57 By leveraging the advantages of pH-sensitive polymers and gold(I) compounds, we attempt to prepare a novel nanoparticle to achieve the efficient regulation of autophagy that directly leads to the death of cancer cells. Herein, we report pH-sensitive polymeric nanoparticles with gold(I) compound (Au(I)) payloads that synergistically induce cancer cell death through regulation of autophagy. The pHsensitive poly(β-amino ester)s self-assemble into micelle-like nanoparticles with Au(I) payload (named as Au(I)⊂NPs). The Au(I)⊂NPs enter cells through endocytosis pathway and accumulate into acidic lysosomes (pH 4−5). On one hand, under acidic condition, the protonation of tertiary amines of poly(β-amino ester)s triggers the dissociation of micelles, damages the lysosomes, and releases the Au(I) molecule. The poly(β-amino ester)s significantly block the autophagic flux owing to irreversible lysosome impairment. On the other hand, Au(I) molecule inhibits TrxR activity that indirectly increases intracellular ROS. ROS are not only enhancing oxidative stress, but also inducing autophagy. However, the poly(β-amino ester)s damage the lysosomes and block autophagic flux, and subsequently enhance the killing efficacy of Au(I) molecular (Scheme 1). Therefore, synergistically inducing cell death through regulation of autophagy by pH-sensitive polymeric nanoparticles can be a novel strategy in cancer therapy. B


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incubation for 30 min, the loading solution was replaced with PBS, and the cells were observed using flow cytometry analysis and Olympus IX71 fluorescence microscope. 2.10. Acid Phosphatase Assay. MCF-7 cells were incubated with or without Au(I)⊂NPs for 24 h and then were collected by centrifugation, washed, and the cells were ruptured by sonication on ice. Then the supernatant was collected, which contains lysosomal enzymes. The supernatant was used to measure the acid phosphatase activity with an acid phosphatase assay kit. 2.11. Confocal Laser Scanning Microscopy (CLSM) Observation. CLSM was employed to observe endocytosis of Au(I)⊂NPs. MCF-7 cells were plated in 15φ culture dishes at density of 5 × 105 cells/mL and cultured in RPMI1640 medium (10% FBS and 1% penicillin−streptomycin) with 5% CO2 for 15 h. These cells were cultured with Cy5-loaded nanoparticles for 2 h and then incubated with LysoTracker Green DND-26 (10 μM) for 20 min. The cells were imaged by Zeiss LSM710 confocal laser scanning microscope. 2.12. Statistical Analysis. Statistically significant, one-way ANOVA with Bonferroni’s post-test analysis was used. P < 0.05 (∗), P < 0.01 (∗∗), or P < 0.001 (∗∗∗) is marked in the figures.

detected by ICP-AES analysis (ELAN 6100, PerkinElmer SCIEX). At the same measurement conditions, the MLC and MLE were estimated. 2.4. Characterization of Polymeric NPs and Au(I)⊂NPs. The pH-sensitive polymeric NPs and Au(I)⊂NPs (1 mg/mL, pH 7.4 PBS) were first passed through syringe filters (0.45 μm, Millipore) and then performed in a 1.0 mL quartz curet at 25 °C. The hydrodynamic diameter was detected by Zetasizer Nano instrument (Malvern Instruments, Malvern, UK). Morphology and sizes of polymeric NPs and Au(I)⊂NPs were measured by transmission electron microscopy (TEM). 2.5. Cell Viability. The cytotoxicities of Au(I), pH-sensitive polymeric NPs, and Au(I)⊂NPs were analyzed by the CCK-8 assay. Ninety-six-well plates were used to culture MCF-7 cells at 6 × 103 cells/well in RPMI1640 containing 10% FBS and 1% penicillin−streptomycin with 5% CO2. After 15 h, the sample solutions were added in each well. Upon incubation for 4, 12, or 24 h, the RPMI1640 medium was removed, and 100 μL of medium containing CCK-8 (10%, v/v) was added. The absorbance at 450 nm of each well was measured after an incubation period of 2 h at 37 °C using the microtiter plate reader (TECAN Infinite M200, Austria). Cell viability (%) was equal to (Asample − Ablank)/(Acontrol − Ablank) × 100. The experiments were performed in triplicate. 2.6. Isobologram. The isobologram analysis was used to evaluate the interaction of two drugs: drug 1 and drug 2. First, the effect of drug 1 or drug 2 alone was assayed (e.g., IC50). Second, the dose-dependent resultant effect of drugs 1 and 2 (C1, 0) and (0, C2), respectively, was analyzed in coordinate plot. A straight line connecting these two points gives the additive. Finally, the concentrations of the two drugs were denoted as C1, C2, which may appeared below, on, or above the line to denote that the interaction of two drugs is synergic, additive, or antagonistic, respectively. 2.7. Western Blotting. MCF-7 cells were first resuspended in lysis buffer (Tris-HCl (50 mM, pH 8.0), 150 mM NaCl, 1% (v/v) Triton-X 100 and protease inhibitor). Protein content was measured using BCA kit (Applygen) measured protein content, and then the samples (60 μg protein) were subjected to SDS-PAGE. Upon transferring to nitrocellulose membrane, blots were incubated in blocking buffer (5% (wt/v) nonfat milk, 0.1% (v/v) Tween 20 in 0.01 M TBS) for 1 h. Finally, the blots were incubated with primary antibodies overnight at 4 °C and then cultured with secondary antibody (ZSGB-BIO) for 1 h at room temperature. Final blot was scanned on a Typhoon Trio Variable Mode Imager. NIH ImageJ software was used to calculate band density. 2.8. Biological Transmission Electron Microscopy. MCF-7 cells at a density of 1 × 106 cells/mL were seeded in 100 mm plates for 15 h. Then, the cells were coincubated with Au(I), polymeric NPs, and Au(I)⊂NPs for 4 and 24 h. Subsequently, the cells were washed and isolated by centrifugation, and then immediately fixed overnight at 4 °C in 2.5% glutaraldehyde. Samples were prepared for TEM according to standard procedures and then viewed using a JEOL JEM-1400 electron microscope (JEOL, Tokyo, Japan). 2.9. Lysosomal Stability Assay. To measure lysosomal pH, the acidification of lysosomes was assessed with LysoTracker Green DND-189 (Invitrogen). Cells were seeded in six-well plates at a density of 1 × 105 cells/mL and treated with polymeric NPs and Au(I)⊂NPs for 4, 12, and 24 h. The medium was removed, and prewarmed (37 °C) probecontaining medium (10 μM) was added to the cells. After

3. RESULTS AND DISCUSSION 3.1. Synthesis and Characterization of Au(I)⊂NPs. We copolymerized HDDA (1.0 equiv), DBPA (0.9 equiv), and

Figure 1. Characterizations of Au(I)⊂NPs. (a) Number size distribution of Au(I)⊂NPs, as measured by DLS. (b) TEM images of Au(I)⊂NPs. Error bar: 50 nm. (c) Comparison analysis of MCF-7 cell viabilities of Au(I) (1.8 μg/mL), pH-sensitive polymeric NPs (64.7 μg/mL), and Au(I)⊂NPs (66.5 μg/mL). Statistical significance: ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001, one-way ANOVA for indicated comparison. (d) Isobologram analysis of antiproliferactive effects of Au(I) and pH-sensitive polymeric NPs on MCF-7 cells.

mPEG-NH2 (2000) (0.1 equiv) to prepare the copolymers via Michael addition according to our previous report.44,45 GPC and 1H NMR were used to determine the structures and molecule weight of the poly(β-amino ester). The results revealed that calculated repeat units, and the average molecule weights of poly(β-amino ester) were around 30 and 20 500, respectively. The micelle-like nanoparticles were prepared by C


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Molecular Pharmaceutics Table 1. Loading Content and Efficiency of Au(I)-Loaded Nanoparticlesa run

polymer

HDDA:DBPA:DEPA:mPEG-NH2 (2K)

wd/wp (wt %)b

MLC (wt %)c

MLE (wt %)d

1

P1

1.0:0:0.9:0.1

6

2.8

47.4

a

Molecule loading content experiment was detected in PBS (pH 7.4). bwd/wp means the percent molecule/polymer ratio in feed. cMLC, defined as the percentage ratio of molecule in polymer micelles/polymer micelles. dMLE, defined as the percentage ratio of molecule in polymer micelles/ molecule in feed.

Figure 2. Au(I)⊂NPs induce cancer cell death partially through modulation of oxidative stress. (a) Apoptosis analysis. The MCF-7 cells were determined by FACS using Annexin V/PI double staining after 24 h treatments. (b) The corresponding results of quantitative analysis from panel a. Statistical significance: ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001, one-way ANOVA for indicated comparison. (c) Flow cytometry analysis of ROS production. MCF-7 cells were incubated with Au(I) (0.9 μg/mL), NPs (32.1 μg/mL), and Au(I)⊂NPs (33 μg/mL) for 0, 4, 12, and 24 h, respectively.

using the dialysis method.45 Au(I) composed of 4-methylphenyl alkynyl gold and thienyl-diphenylphosphine ligand was synthesized according to the method developed by our group previously.56 The product was obtained as a white solid with a yield of 63%. The structure of the molecule was characterized by 1H, 13C, and 31P NMR, and matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry (MALDI-TOF MS) (Supporting Information, Figure S1). Subsequently, the Au(I) was loaded into the hydrophobic domains of micelles according to previous reported protocol.44 Dynamic light scattering (DLS) measurement exhibited that the hydrodynamic sizes of formed micelle-like nanoparticles (named as polymeric NPs) and Au(I)-loaded nanoparticles (named as Au(I)⊂NPs) were 43 ± 15 and 50 ± 18 nm, respectively (Figures S2a and 1a). The hydrodynamic size of Au(I)⊂NPs in cell culture medium was little changed (Figure S3). TEM images provided the information about morphological details (Figures S2b and 1b). The gold concentration in Au(I)⊂NPs was measured by inductively coupled plasma-atomic emission spectroscopy (ICP-AES). Thus, the MLE and MLC values of Au(I) into the micelles were estimated. The results revealed that the MLE and MLC were 2.8 and 47.4%, respectively (Table 1).

3.2. Au(I)⊂NPs Synergistically Induced Cell Death. To investigate the synergistically induced cell death of the pHsensitive poly(β-amino ester)s, we evaluated the cytotoxicity of Au(I), NPs, and Au(I)⊂NPs in MCF-7 cells by the CCK-8 assay. As can be seen from Figures 1, panel c and S4, the cytotoxicities of Au(I), NPs, and Au(I)⊂NPs exhibited a dosedependent manner. The IC50 values of NPs, Au(I), and Au(I)⊂NPs treated MCF-7 cells for 24 h were ∼160, ∼1.8, and ∼40.0 μg/mL (that composed of 1.1 μg/mL Au(I)), respectively. To eliminate the cytotoxicity of the NPs themsleves, we chose a safe concentration of NPs (64.7 μg/ mL) for further experiments. Under this condition, more than 80% of cells remain alive (Figure 1c). Significantly, upon incubation of Au(I)⊂NPs composed of NPs (64.7 μg/mL) and Au(I) (1.8 μg/mL) with MCF-7 cells for 24 h, about 80% of cells were killed, which was remarkably higher than that of free Au(I) compound (∼50%) at the same condition (Figure 1c). However, when cocultured with Au(I) and unloaded NPs, the cytotoxic effects were lower than that of Au(I)⊂NPs (Figure S5). All these results implied that besides the intrinsic toxicity of Au(I) compound, low-dose NPs in Au(I)⊂NPs contributed to the resultant cell death beyond the apoptosis mechanism. To D


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untreated cells (>90%) but much higher than that of Au(I)⊂NPs (69.0%) (Figure 2a). All the treatments significantly increased the population of early apoptotic cells, for example, 3.44% for Au(I), 3.62% for NPs, and 2.52% for Au(I)⊂NPs compared with 0.994% for untreated cells. However, the percentages of late apoptotic cells were 8.52% and 4.56% induced by Au(I) and polymeric NPs and increased to 18.5% if incubation with Au(I)⊂NPs (Figure 2a). The similar percentage of necrotic/dead cells was detected with Au(I) (4.08%), NPs (2.06%), and Au(I)⊂NPs (10.0%). In comparison with Au(I), the Au(I)⊂NPs promote a much higher cell dead rate of MCF-7 cells with the same dose (Figure 2b). These results suggested that Au(I)⊂NPs can effectively kill the cancer cells compared with Au(I) and NPs alone. To validate that cell death in Au(I) ⊂NPs treated cells resulted from oxidative stress, we measured the intracellular ROS production through flow cytometry analysis. The cells were incubated with Au(I) (1.8 μg/mL), NPs (64.7 μg/mL), and Au(I)⊂NPs (66.5 μg/mL) for 0, 4, 12, and 24 h, respectively, and then labeled with a commercial available nonfluorescent probe DCFH-DA. DCFH-DA is hydrolyzed by intracellular esterases to form dichlorofluorescin (DCFH). DCFH can be oxidized by intracellular ROS producing a fluorescent product DCF. As shown in Figure 2, panel c, the intracellular ROS production was increased after Au(I) and Au(I)⊂NPs treatment for 24 h. However, there is little change in NPs group. In addition, we found that DCF fluorescence of cells treated by Au(I) and Au(I)⊂NPs in 2 h were slightly decreased, which was reasonable due to the cell self-repairing mechanism. To further explore the biochemical effects of ROS, we detected the cytotoxicity of Au(I) and Au(I)⊂NPs when cotreated with antioxidants such as vitamin E by the CCK-8 assay. The results demonstrated that vitamin E restored Au(I) and Au(I)⊂NPs killing effects (Figure S6). These results suggested that the cell death induced by Au(I)⊂NPs partially resulted from up-regulating ROS; pH-sensitive polymer enhances the killing effect though other pathway. 3.4. NPs Induced Autophagosome Accumulation. To further explore the biochemical effects of NPs, we then examined their effect on induction of autophagy. Bio-TEM is one of the most convincing and standard methods to detect autophagy.59 Therefore, we applied this method to observe the ultrastructure and morphology of breast cancer cells (MCF-7). The cells were treated with Au(I) (1.8 μg/mL), NPs (64.7 μg/ mL), or Au(I)⊂NPs (66.5 μg/mL for 24 h. In comparison with untreated controls, a large number of small double/multimembrane vesicles (arrowheads) and huge vacuoles (arrow) were observed in Au(I), NPs, and Au(I)⊂NPs treated cells (Figure 3a). These small double/multimembrane vesicles were autophagosomes, and large vacuoles were the autophagic vacuoles. These morphological features we observed clearly reflect the autophagic characteristics.59 To further confirm autophagy-inducing effect of Au(I), NPs, and Au(I)⊂NPs, we examined the expressions of LC3II, which was regarded as an autophagic marker, and the levels of its expression were correlated to the extent of autophagosome formation59 by using Western blot method. Starvation is widely used to induce autophagy and acted as positive control, which causes LC3II accumulation. As shown in Figure 3, panel b, upon treatment of MCF-7 cells with Au(I) (1.8 μg/mL), NPs (64.7 μg/mL), and Au(I)⊂NPs (66.5 μg/mL) for 24 h, there were much more LC3II accumulation. The ratio of LC3II/βactin that measured by integrated optical densitometry

Figure 3. pH-sensitive polymeric NPs and Au(I) synergistically induced autophagy in MCF-7 cells. (a) TEM images of MCF-7 cells were untreated (control), treated with Au(I) (1.8 μg/mL), polymeric NPs (64.7 μg/mL), and Au(I)⊂NPs (66.5 μg/mL) for 24 h. Nuclei were labeled as N. Autophagic structures, autophagosome (arrowheads), and autophagic vacuoles (arrows) are shown independently. (b) Western blot of the LC3II expressions in MCF-7 cells that were untreated (control), treated with Earle’s balanced salt solution (starve), and treated with Au(I) (1.8 μg/mL), polymeric NPs (64.7 μg/mL), and Au(I)⊂NPs (66.5 μg/mL) for 24 h, separately. The corresponding results of semiquantitative analysis are shown in the lower panel. Statistical significance: ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001, one-way ANOVA for indicated comparison. (c) Western blot of the LC3II expressions in MCF-7 cells that were untreated (control), treated with Earle’s balanced salt solution (starve), and treated with Au(I)⊂NPs (10, 25, 50, and 66.5 μg/mL, respectively) for 24 h. The corresponding results of semiquantitative analysis are shown in the lower panel.

determine whether the anticancer behaviors of NPs and Au(I) in MCF-7 cells were synergistic, the growth inhibitory effects of them were analyzed by the isobologram method.58 For Au(I)⊂NPs, the ratio of Au(I) and NPs was 1:36. They were investigated by comparing with free Au(I) and NPs. The results of the isobologram analysis revealed that the growth inhibitory effect between Au(I) and NPs was strongly synergistic (Figure 1d). This conclusion matched our finding previously that Au(I) and NPs synergistically induced autophagy on MCF-7 cells. Therefore, we predicated that the combination of Au(I) and NPs synergistically induced cancer cell death. 3.3. Au(I)⊂NPs Induce Cancer Cell Death Partially through Modulation of Oxidative Stress. Generally, small molecules kill tumor cells though inducing apoptosis. We used the FITC-Annexin V/propidium iodide (PI) method to determine whether the cancer cells could be killed by Au(I)⊂NPs. MCF-7 cells were first incubated with Au(I), polymeric NPs, and Au(I)⊂NPs for 24 h and then subjected to FITC-Annexin V/PI staining. The flow cytometry analysis shows that the ratio of viable cells of Au(I) (84.0%) and polymeric NPs (89.8%) treated groups was lower than that of E


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Figure 4. High dose of Au(I)⊂NPs treatment induced autophagosome accumulation and then led to cell death. (a) TEM images of MCF-7 cells that were untreated (control) and treated with Au(I)⊂NPs (66.5 μg/mL) for 4, 12, and 24 h. (b) Western blot of the p62 expressions in MCF-7 cells that were untreated (control), treated with Earle’s balanced salt solution (starve), and treated with Au(I) (1.8 μg/mL), polymeric NPs (64.7 μg/mL), and Au(I)⊂NPs (66.5 μg/mL) for 4 and 24 h, separately. The corresponding results of semiquantitative analysis are shown in the lower panel. Statistical significance: ∗p < 0.05; ∗∗p < 0.01, and ∗∗∗p < 0.001.

Figure 5. Impairment of lysosomes by Au(I)⊂NPs. (a) Confocal microscopy of MCF-7 cells. (I) Blank shows cell morphology. (II) Green fluorescence shows lysosomes staining with LysoTracker Green DND-26. (II) Red fluorescence shows the location of Cy5-NPs. (IV) Merged image of I, II, and III. The cells were incubated with Cy5-NPs (5 μg/mL) for 2 h. (b) Acid phosphatase enzyme activity measurement. The MCF-7 cells were incubated with NPs (64.7 μg/mL) and Au(I)⊂NPs (66.5 μg/mL) for 24 h. Statistical significance: ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001. (c) Western blot of the Cathepsin B expression in MCF-7 cells. The cells were incubated with NPs (64.7 μg/mL) and Au(I)⊂NPs (66.5 μg/mL) for 24 h.

illustrated that LC3II/β-actin ratios were increased (Figure 3b). Quantitatively, the LC3II/β-actin ratios of Au(I) and NPs were only 3.35 and 2.73, respectively. However, the LC3II/β-actin ratios of cells treated by Au(I)⊂NPs were increased to 5.25 (Figure 3b). These results implied that autophagosome accumulation was remarkably increased by Au(I)⊂NPs. To

study the concentration-dependent autophagy-inducing effect, MCF-7 cells treated by different concentrations of Au(I)⊂NPs were measured. As shown in Figure 3, panel c, autophagy activation by Au(I)⊂NPs was observed under lower-concentration treatment (10 μg/mL). We also examined the induction of autophagy in 4 h. As shown in Figure S8a, Au(I), polymeric F


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Figure 6. Effects of Au(I)⊂NPs on lysosome alkalinization. (a) Flow cytometry analysis of MCF-7 cells. The cells were treated with Au(I)⊂NPs (66.5 μg/mL) for 24 h and then stained with LysoTracker Green DND-189 for 20 min. (b) The fluorescence microscopy images of MCF-7 cells.

Microscopic examination also revealed the phenotype of cells that were treated with Au(I)⊂NPs for 24 h, and we found that the cells with Au(I)⊂NPs treatment for 24 h became contracted and round (Figure S8). Taken together, we confirmed that Au(I)⊂NPs induced autophagosome accumulation and then led to cell death. 3.6. Au(I)⊂NPs Treatment Caused Impairment of Lysosome. The nanoparticles exhibit remarkable influences on lysosomes owing to their endocytosis cell uptake pathway and high accumulation in lysosome compartment.60−62 To study the subcellular localization of Au(I)⊂NPs, we first prepared the Cy5-loaded micelles. Cy5 as a fluorescence signal molecule was encapsulated into the hydrophobic domains of NPs self-assembled from poly(β-amino ester)s (named as Cy5polymeric NPs). CLSM images exhibited that the most of Cy5 signal colocalized with endosomes/lysosome after incubation for 2 h (Figure 5a), which indicated that NPs were internalized by cellular endocytosis. The lysosome is very closely related to the autophagic process.63,64 Under physiological conditions, lysosomal hydrolases participate in organelles and damaged long-lived proteins turnover through autophagy.65 To test whether lysosome was impaired after Au(I)⊂NPs (66.5 μg/mL) treatment, we measured the activity of lysosomal marker acid phosphatase. Here, NPs (66.5 μg/mL) treatment acted as positive control, and we found that enzyme activity treated by Au(I)⊂NPs was significantly lower than the negative control (Figure 5b). Cathepsins are lysosomal proteolytic enzymes, and release of these enzymes into the cytosol can be defined as a consequence of lysosomal rupture. Western blot analysis also showed decrease of Cathepsin B after Au(I)⊂NPs (66.5 μg/mL) treatment for 24 h (Figure 5c). These results demonstrated that the lysosome was damaged after Au(I)⊂NPs treatment. 3.7. Au(I)⊂NPs Increased Lysosome Alkalinization. The optimal pH of most lysosomal enzymes is ∼4.5. To test whether Au(I)⊂NPs can affect lysosome pH, the cells were labeled with LysoTracker Green DND-189 dye. LysoTracker Green DND-189 is an acid tropic dye that can be accumulated in intracellular acidic organelles and has a well-defined pHdependent increase in fluorescence intensity.12,66 As shown in Figure 6, panel a, flow cytometry analysis showed that fluorescence intensity became weakened after Au(I)⊂NPs

NPs, and Au(I)⊂NPs all can induce autophagy in 4 h. However, Vitamin E restored Au(I) and Au(I)⊂NPs killing effects and deceased autophagy activation in 24 h (Figure S6). These results indicated that autophagy played a critical role in responding to treatment by Au(I)⊂NPs. 3.5. Au(I)⊂NPs Treatment Induce Autophagic Cell Death. To explore whether the high dose of Au(I)⊂NPs treatment can induce autophagic cell death, we examined the autophagic ultrastructure of cells by Bio-TEM. Bio-TEM revealed the ultrastructure and morphology of MCF-7 that were treated by Au(I)⊂NPs (66.5 μg/mL) for 4, 12, and 24 h. In comparison with untreated controls, a large number of small double/multimembrane vesicles (arrowheads) and huge vacuoles (arrow) were observed (Figure 4a), and small double/multimembrane vesicles (arrowheads) and huge vacuoles (arrow) were increased with the time going on. We also found small double/multimembrane vesicles (arrowheads) and large autophagic vacuoles occupied the major cellular space in 12 h and 24 h (Figure 4a). Such massive cellular autophagic structures are probably to induce autophagic cell death. P62 is an autophagy maker that preferentially is degraded by autophagy. Starvation as an inducer of autophagy can cause LC3II accumulation and degradation of P62. The cells were treated with Au(I) (1.8 μg/mL), NPs (64.7 μg/mL), and Au(I)⊂NPs (66.5 μg/mL) for 4 and 24 h, and we found that expression of P62 in 24 h was higher than that in 4 h (Figure 4b). In addition, we found starvation caused rapid degradation of P62; however, Au(I), NPs, and Au(I)⊂NPs treatment did not cause P62 degradation but instead caused accumulation of P62 remarkably (Figure 4b). Semiquantitative analysis results also showed the P62/β-actin ratios of Au(I), NPs, and Au(I)⊂NPs increased from 2.3, 6.9, and 11.3 in 4 h to 15.2, 13.9, and 18.3 in 24 h, respectively (Figure 4b). These results indicated that Au(I) and NPs synergistically induced autophagosome accumulation. For Au(I)⊂NPs, the P62/βactin ratio was raised with Au(I)⊂NPs increasing (Figure S4b), which reflected that Au(I)⊂NPs could effectively regulate autophagy. In addition, when the cells were first cultured with 3-MA (autophagy inhibitor) for 1 h and then treated with Au(I)⊂NPs for 24 h, we found that 3-MA promoted Au(I)⊂NPs killing effects (Figure S7), which means that autophagy played an important role in cell death processes. G



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(66.5 μg/mL) treatment. Flow cytometry analysis also showed that fluorescence intensity became weakened after NPs treatment (Figure S10). All these results indicated that there was alkalinization of lysosomes in Au(I)⊂NPs. In conclusion, our data demonstrated that Au(I)⊂NPs treatment caused impairment of lysosomes through lysosome alkalinization.

ASSOCIATED CONTENT

S Supporting Information *

Preparation and characterization of Au(I). DLS and TEM images of NPs. Cell viability assay. Western blot analysis of the LC3II and P62 expression in MCF-7 cells in 4 h. Effects of polymeric NPs on lysosome alkalinization. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.molpharmaceut.5b00060.

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4. CONCLUSION Regulation of autophagy by nanotechnology might be a powerful tool in cancer therapy. Although nanomaterials with diversified physiochemical parameters have demonstrated the possibility of autophagy regulation, this remains a challenge for cancer therapy owing to the limited affection on autophagic networks. We have prepared Au(I) encapsulated pH-sensitive polymeric micelles for effectively killing cells through regulation of cellular autophagy. The Au(I) as a TrxR inhibitor can upregulate the ROS, which positively contributes to autophagy and apoptosis. Moreover, the protonation of tertiary amines of poly(β-amino ester)s damages the lysosomes and blocks formation of autolysosomes from fusion of lysosomes with autophagosomes, which means pH-sensitive polymers stop autuphagic flux. The blockade of autophagy leads to excessive depletion of cellular organelles and essential proteins, and ultimately results in cell death. By encapsulation of gold(I) compounds into hydrophobic domains of NPs, the resultant Au(I)-loaded NPs show synergistic cancer cell killing performance. Identification of synergistically induced cell death through regulation of autophagy may open a new avenue for cancer therapy in the nanomedicine field.

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Article

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +86-10-82545759. Fax: (+86) 10-82545759. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Basic Research Program of China (973Program, 2013CB932701), the 100Talent Program of the Chinese Academy of Sciences (Y2462911ZX), National Natural Science Foundation (Nos. 21374026, 21304023 and 51303036) and Beijing Natural Science Foundation (2132053).

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ABBREVIATIONS NPs, pH-sensitive micelle-like nanoparticles; Au(I), gold(I) compounds; Au(I)⊂NPs, pH-sensitive polymeric nanoparticles with gold(I) compounds; ROS, reactive oxygen species; TrxR, thioredoxin reductase; LC3, microtubule-associated protein 1A/1B-light chain 3; P62, sequestosome 1 (SQSTM1); TEM, transmission electron microscope; DLS, dynamic light scattering H


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