Mitochondria-Targeted Hydroxyapatite Nanoparticles for Selective

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Mitochondria-Targeted Hydroxyapatite Nanoparticles for Selective Growth Inhibition of Lung Cancer in Vitro and in Vivo Yi Sun, Yaying Chen, Xiaoyu Ma, Yuan Yuan, Changsheng Liu, Joachim Kohn, and Jiangchao Qian ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b06094 • Publication Date (Web): 07 Sep 2016 Downloaded from http://pubs.acs.org on September 8, 2016

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ACS Applied Materials & Interfaces

Mitochondria-Targeted Hydroxyapatite Nanoparticles for Selective Growth Inhibition of Lung Cancer in Vitro and in Vivo Yi Sun,† Yaying Chen,‡ Xiaoyu Ma,‡ Yuan Yuan,‡ Changsheng Liu,†,‡ Joachim Kohn,§ and Jiangchao Qian*,† †

State Key Laboratory of Bioreactor Engineering, East China University of Science

and Technology, Shanghai 200237, China ‡

Engineering Research Center for Biomedical Materials of Ministry of Education,

East China University of Science and Technology, Shanghai 200237, China §

New Jersey Center for Biomaterials, Department of Chemistry and Chemical

Biology, Rutgers, The State University of New Jersey, NJ 08855, USA

KEYWORDS: Hydroxyapatite nanoparticles, lung cancer, mitochondrion-targeted, cytotoxicity, uptake

ACS Paragon Plus Environment


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ABSTRACT: Lung cancer is the leading cause of cancer-related mortality worldwide. Most patients have metastases at the time of diagnosis, thus demanding development of more effective and specific agents. In this study, the specific anti-cancer effect of hydroxyapatite nanoparticles (HAPNs) to human lung cancer cells (A549) and the underlying mechanisms were investigated, using normal bronchial epithelial cells (16HBE) as the control. Rod-shaped HAPNs (about 10 nm in width and 50 nm in length) were prepared by aqueous precipitation method. Without any further functionalization and drug loading, HAPNs selectively inhibited cancer-cell proliferation. Their efficient mitochondrial targeting correlated strongly with decreased

mitochondrial

membrane

potential

and

induction

of

mitochondria-dependent apoptosis in A549 cells. Caveolae-mediated endocytosis via lysosome trafficking was observed to be a prominent internalization pathway for HAPNs in both A549 and 16HBE cells. However, more nanoparticles were taken up into A549 cells. HAPNs triggered a sustained elevation of intracellular calcium concentration ([Ca2+]i) in cancer cells, but only a transitory increase in normal control cells. In a nude mouse lung cancer model with xenotransplanted A549 cells, HAPN treatment demonstrated nearly 40% tumor growth inhibition without apparent side effect. These results demonstrated that the enhanced cellular uptake and mitochondrial targeting of HAPNs, together with the prolonged elevation of [Ca2+]i in A549 cells, could result in the cancer-specific cytotoxicity of HAPNs. Thus HAPNs might be a promising agent or mitochondria-targeted delivery system for effective lung cancer therapy.


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1. INTRODUCTION

According to the International Agency for Research on Cancer's GLOBOCAN 2012 report, lung cancer is the most common cancer in the world with the highest incidence and mortality rates.1 Non-small cell lung cancer (NSCLC) is the most common type of lung cancer, accounting for >80% of all cases. Since less than 16% of patients are diagnosed at the local stage,2 chemotherapy forms the foundation of treatment for the disease.3 Platinum-based combination chemotherapy is the first-line treatment of NSCLC. The combination regimens have been shown to be superior to single agent treatment in terms of overall survival.4-5 However, combination chemotherapy was associated with increased toxicity, including a 3.6-fold increase in the risk of treatment-related death.6 On the other hand, a significant proportion of patients exhibit and acquire resistance to the currently available agents.3 There thus remains a significant demand for improved effectiveness and specificity of chemotherapy for lung cancer. The development of nanomedicines based on nanoparticles holds the potential to improve the effectiveness and specificity of cancer therapy by enhancing targeted drug delivery.7 Recently, besides being used as carriers for anti-cancer drugs, some nanoparticles have been discovered to selectively kill cancer cells.8-10 Our previous work demonstrated that hydroxyapatite nanoparticles (HAPNs) inhibit cell proliferation of hepatoma cells specifically while exhibiting negligible impact on normal liver cells in vitro.11 However, whether HAPNs have a cancer-specific



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cytotoxic effect on lung cancer cells, and importantly, whether they can inhibit tumor growth in vivo without toxic side effects, have not yet been explored. Hydroxyapatite (HAP, Ca10[PO4]6[OH]2) is the major inorganic constituent of the hard tissue of humans and animals. Due to its excellent biocompatibility and bioactivity, HAP has been widely employed in repairing hard tissue injury.12 It is also used as a vehicle for drug, protein and gene delivery.13-15 Recently, some studies have reported that HAPNs exhibited significant cytotoxicity to some types of cancer cells including breast cancer cells,16 osteosarcoma cells,17 gastric cancer cells18 and glioma cells.19 It has been proposed that the toxic effects of HAPNs on cancer cells may be caused by induction of apoptosis through the mitochondria-dependent pathway,16 resulting from oxidative stress,19 or from inhibition of protein synthesis due to abundant internalized HAPNs in cancer cells around endoplasmic reticulum.20 Our previous studies have demonstrated that differential cell death mechanisms might be involved in the cytotoxicity of HAPNs to various cancer cells,21 and nuclear localization of nanoparticles was related to their selective anti-tumor activity.11 However, most of previous studies did not use matching control cells to investigate the selective effects and mechanisms of HAPNs. Although cytotoxicity of HAPNs has been demonstrated to be particle-property and cell-type dependent,22-24 the underlying mechanisms of selective cytotoxicity to cancer cells are still poorly understood. Here we chose human lung alveolar adenocarcinoma cells (A549) to investigate the anti-cancer effects of HAPNs in vitro and in vivo with the rod-shaped nanoparticles synthesized using the aqueous precipitation method. HAPN-induced cytotoxicity and


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apoptosis were compared between A549 cancer cells and normal bronchial epithelial cells (16HBE). Cellular uptake and trafficking of FITC labeled HAPNs were investigated. We also measured intracellular calcium levels to explore the mechanisms of cancer-cell-specific cytotoxicity of HAPNs. The selective anti-cancer activity of HAPNs observed in vitro was confirmed by a preliminary in vivo study, using the BALB/c nu/nu nude mouse lung cancer model.

2. MATERIALS AND METHODS 2.1.

Reagents

and

3-aminopropyltriethoxysilane

Cell

(AMPTES),

4',6-diamidino-2-phenylindole 5-diphenyltetrazolium

Lines.

bromide

Fluorescein

(DAPI), (MTT),

Dimethysulfoxide isothiocyanate

(DMSO), (FITC),

3-(4,5-dimethylthiazol-2-yl)-2,

methyl-β-cyclodextrin

(MβCD)

and

Chlorpromazine were purchased from Sigma-Aldrich (MO, USA). BCA protein assay kit, Annexin V-FITC/PI assay kit and fluo-3 were purchased from Beyotime Institute of Biotechnology (Haimen, China). Caspase-3 and -9 fluorescence assay kit were purchased from Biovision (CA, USA). MitoTracker Red CMXRos and LysoTracker Red DND-99 were purchased from Invitrogen (CA, USA). Human lung alveolar carcinoma cells (A549) were obtained from the Cell Bank of Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). And normal human bronchial epithelial cell (16HBE) was obtained from Cell Bank in Peking Union Medical College. Cell culture medium and fetal bovine serum (FBS) were from Gibco (CA, USA).



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2.2. Synthesis and Labeling of HAPNs. HAPNs were synthesized using the aqueous precipitation method as follows: 400 mL of the Ca(NO3)2 solution (0.2 M) was added to 240 mL (NH4)2HPO4 (0.2 M) with stirring thoroughly at a constant pH value of 10 at 10°C for 30 h. After that, the resultant precipitate was collected, washed in deionized water and anhydrous ethanol several times before freeze-drying for 24 h. The nanoparticles were calcined in a furnace at 600°C for 2 h. HAPNs were sterilized by autoclaving at 121°C for 30 min and bath-sonicated prior to cell culture experiments. HAPNs were labeled by FITC for cellular uptake and trafficking studies.25 1 mL of 3-aminopropyltriethoxysilane (AMPTE) was allowed to react with 0.05 g of HAPNs in 50 mL anhydrous ethanol under stirring at 74°C for 3 h. Subsequently, 0.025 g of FITC was added and the reaction was kept for 6 h. The synthesized particles were washed several cycles with anhydrous ethanol and deionized water until no free FITC was remained. Finally, the labeled nanoparticles (FITC-HAPNs) were freeze-dried for 24 h.

2.3. Nanoparticle Characterization. The phase composition of prepared HAPNs was confirmed by X-ray diffraction (XRD, Bruker D8 Focus, Karlsruhe, Germany) with Ni-filtered Cu Kα irradiation. The morphology and size of HAPNs were observed under a transmission electron microscope (TEM, JEOL JEM-2100, Tokyo, Japan) operating at an accelerating voltage of 120 kV. HAPNs were dispersed in anhydrous ethanol followed by ultrasonic treatment for 10 min, and droplets of the suspension were applied to carbon-coated copper grids. The Image J software was


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used to analyze the particle size from the TEM images. Nanoparticle composition was analyzed by Fourier transform infrared (FT-IR) spectroscopy (Nicolet 6700, Thermo Fisher Scientific, USA). The surface charge and hydrodynamic size of HAPNs was measured using the Zetasizer Nano ZS (Malvern, Worcestershire, UK).

2.4. Cell Culture. A549 and 16HBE cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) and RPMI-1640 growth medium respectively, with 10% FBS at 37°C under a humidified atmosphere containing 5% carbon dioxide. Cells were passaged upon attaining 80% confluence in cell culture flasks.

2.5. In Vitro Cytotoxicity Assay. Cytotoxicity of HAPNs to A549 and 16HBE cells was measured by MTT assay. The exponentially growing cells (5×103 cells per well) were seeded in 96-well culture plates followed by 24 h incubation. Then, the culture medium was replaced by the fresh complete medium containing specified amounts of HAPNs (62–1000 µg/mL) and the cells were treated for 48 or 72 h. Thereafter, 30 µL of filtered MTT stock solution (5 mg/mL) was added to each well. After 4-h incubation, the supernatant medium was aspirated and 200 µL of DMSO was added to dissolve the dye for 10 min at 37°C. Finally, 150 mL of dye solution was transferred to a fresh plate, and the absorbance at 490 nm was measured by a microplate reader (Molecular Devices SpectraMax M2, Sunnyvale, USA). Cell viability was calculated as percent relative to the control without HAPN treatment.

2.6. Detection of Nuclear Morphology. Cells were treated by HAPNs at the concentration of 500 µg/mL for 48 h, and then stained with DAPI to detect the nuclear



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morphological change as previously described.21 The samples were subjected to fluorescence imaging (excitation wavelength: 330-380 nm; emission wavelength: 430-460 nm).

2.7. Cell Apoptosis Assay. The Annexin V-FITC/PI dual staining assay was performed to identify apoptotic cells as per manufacturer’s instructions. Cells were seeded in a 6-cm culture dish overnight and then incubated with 250 or 500 µg/mL of HAPNs for 48 h. Then, cells were collected for Annexin V and PI staining after rinsing with PBS, and analyzed with the flow cytometer (FACSAriaTM, BD Biosciences, CA, USA).

2.8.

Analysis

of

Mitochondria

Permeability.

5,5,6,6'-tetrachloro

-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine (JC-1) was used to measure the polarization status of mitochondria.26 Cells (1×104 cells/mL) were seeded in 20 mm glass bottom culture dishes and kept for 24 h, then treated by HAPNs at 250 or 500 µg/mL for 48 h, followed by incubation with JC-1 containing medium for 20 min. After washing with PBS for three times, cells were observed using an A1R confocal laser scanning microscope (Nikon, Tokyo, Japan). The emission at 530 nm (green) or 590 nm (red) with the same excitation of 488 nm was used to detect the JC-1 monomer or aggregate, respectively. The green/red ratios were analyzed and quantified by NIS-Element AR Analysis (4.00.06).

2.9. Caspase Activity Assay. The activity of intracellular caspase-3 and caspase-9 was determined as per manufacturer’s instructions. Briefly, A549 and 16HBE cells


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were exposed to 500 µg/mL of HAPNs for 72 h in 6 cm culture dishes. Cells were harvested, washed twice with PBS, resuspended and lysed in chilled cell lysis buffer (200 µl) with gentle shaking for 10 min on ice. Protein concentration was estimated utilizing the BCA protein assay kit. Cell lysates containing 6 µg of protein were added to 50 µl of 2×reaction buffer (containing 10 mM DTT) and incubated with 5 µl of fluorogenic substrates specific for caspase-3 or caspase-9. After incubation at 37°C for 2 h, the relative fluorescence unit was measured with a SpectraMax M2 microplate reader (excitation: 400 nm; emission: 505 nm), and normalized to the value obtained with the same cell line cultured without HAPN treatment.

2.10. Cellular Internalization and Localization of HAPNs. Internalization of HAPNs was quantitatively analyzed via flow cytometry. Cells were exposed to FITC-HAPNs at the concentration of 250 µg/mL for the indicated time in 6 cm culture dishes. After that, cells were washed once with PBS to eliminate floating particles, detached with trypsin, and then resuspended in PBS to a final concentration of 1×106 cells/mL for flow cytometry analysis at the emission wavelength of 488 nm for FITC. To observe the co-localization of FITC-HAPNs within the intracellular compartment, lysosomes or mitochondria were stained with LysoTracker Red DND-99 or MitoTracker Red CMXRos at 37°C for 30 min, respectively. Cells were seeded on 20 mm glass bottom culture dishes. After incubation for 24 h, cells were exposed to 250 µg/mL HAPNs in culture medium for 24 h. The treated cells were subsequently washed several times with PBS, stained with the LysoTracker Red



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DND-99 or MitoTracker Red CMXRos, then fixed, stained with DAPI and observed with a confocal laser scanning microscope as described earlier.

2.11. Detection of Intracellular Calcium Ion Concentration. The fluorescent dye Fluo-3 AM was used to determine intracellular calcium concentration ([Ca2+]i). Cells were seeded at approximately 5×103/well in 96-well black plates overnight, and then incubated with 250 µg/mL of HAPNs. After incubation for pre-determined durations, the medium was replaced with Fluo-3 (dissolved in dimethyl sulphoxide (1:1000)) diluted in Hank's Balanced Salt Solution (HBSS) with bovine serum albumin (1 mg/mL) to reach the final concentration of 4 µM. Cells were incubated with Fluo-3 solution in the dark for 30 min at 37°C, followed by washing twice with HBSS-BSA and a further incubation of 30 min. The fluorescence intensity was measured with a SpectraMax M2 microplate reader (excitation: 488 nm; emission: 525 nm), and normalized to the value obtained from the same cell line cultured without HAPN treatment.

2.12. In Vivo Toxicity and Anti-tumor Activity. Female BALB/c nu/nu nude mice (8–10 week old, weighing about 22–24 g) were purchased from the Shanghai SLAC Laboratory Animal Co., Ltd. All animal experiments were performed in compliance with the Guidelines for the Care and Use of Research Animals established by the East China University of Science and Technology Animal Studies Committee. For in vivo toxicity studies, different concentrations of HAPNs (N=4 per group) in physiological saline were tested. Mice were subcutaneously injected with 0.2 mL/mouse of saline or


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varying doses of HAPNs (10, 20, 30, 40 mg/kg) three times a week. Before each injection, mice were weighed. On day 26, mice were killed by CO2 asphyxiation, major organs were collected and processed routinely into paraffin. Five-micrometer paraffin sections were mounted on a glass slide for hematoxylin and eosin (H&E) staining. The solid A549 tumors established in nude mice were used to generate the human tumor xenograft model. When the tumor volume in donor mice was about 1000 mm3, tumor fragments were harvested, cut into uniform pieces about 30–40 mg, and implanted subcutaneously to the recipient nude mice. When tumor volume reached ~150 mm3 (Day 0), mice with similar mean tumor volume were randomly assigned to 2 groups (N=4). HAPNs were administered as around-tumor injection (0.2 mL per mouse) at a dose of 40 mg/kg three times a week. The mice in the control group were received injections of physiological saline at the same volume. On the day of the treatment, tumor size was measured by a caliper. The formula, width2×length×0.5, was applied to estimate the volume of the tumor as described.27

2.13. Statistical Analysis. Data were presented as means ± standard deviation from three independent experiments. Statistical differences were evaluated using the one-way ANOVA followed by Dunnett’s post-hoc test, and considered significant when p

Mitochondria-Targeted Hydroxyapatite Nanoparticles for Selective

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