Cascade Amplifiers of Intracellular Reactive Oxygen Species Based

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Biological and Medical Applications of Materials and Interfaces

Cascade Amplifiers of Intracellular ROS based on MitochondriaTargeted Core-Shell ZnO-TPP@D/H Nanorods for Breast Cancer Therapy Xiao Liang, Shu-Mao Xu, Jun Zhang, Jing Li, and Qi Shen ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b12590 • Publication Date (Web): 19 Oct 2018 Downloaded from http://pubs.acs.org on October 19, 2018

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ACS Applied Materials & Interfaces

Cascade Amplifiers of Intracellular ROS Based on Mitochondria-Targeted Core-Shell ZnO-TPP@D/H Nanorods for Breast Cancer Therapy Xiao Liang, Shumao Xu, Jun Zhang, Jing Li, and Qi Shen* School of Pharmacy, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, China. E-mail: [email protected] KEYWORDS: mitochondrial targeting, reactive oxygen species, zinc oxide, NRs, breast cancer

ABSTRACT: Tumor cells are vulnerable to reactive oxygen species (ROS). However, it is still a challenge to induce ROS efficiently in tumor cells. In this study, cascade amplifiers of intracellular ROS based on charge-reversible mitochondria-targeted ZnO-TPP@D/H nanorods were firstly developed for breast cancer therapy. The core-shell ZnO-TPP@D/H nanorod with particle size of 179.60 ± 5.67 nm was composed of a core of ZnO nanorod, an inner shell of triphenyl phosphonium (TPP), and an outer shell of heparin. DOX was loaded on ZnO-TPP@D/H nanorods with high drug loading efficiency of 22.00 ± 0.18%. The zeta potential of ZnO-TPP@D/H nanorods varied from 24.00 ± 0.83 mV to -34.06 ± 0.87 mV after heparin coating, protecting ZnOTPP@D/H nanorods from nonspecific adsorption in circulation. Mitochondrial targeting was achieved after the degradation of heparin. Cellular uptake assays showed that ZnO-TPP@D/H nanorods could accumulate in mitochondria. ROS generation assays showed that ZnO-TPP@D/H

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nanorods could triple the intracellular ROS in 4T1 cells (highly metastatic breast cancer cells) than free DOX. Western blot demonstrated that ZnO-TPP@D/H nanorods dramatically induced cell apoptosis in 4T1 cells. In vivo experiments suggested the anti-tumor potential of ZnO-TPP@D/H nanorods.


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1. INTRODUCTION

Tumor cells usually show high vulnerability towards ROS due to the deficiency of antioxidant systems,1,2 Over-production of ROS in tumor cells has been considered as an effective strategy for cancer treatment.3-5 However, inducing ROS efficiently in tumor cells is still a challenge.6 Mitochondria are of great importance in energy production and cell apoptosis.7 The majority of endogenous ROS is stored in mitochondria. Previous studies showed that ROS stimulation around mitochondria could cause the leakage of small solutes including mitochondrial ROS into cytosol, forming an amplified intracellular ROS signal.8 Thus, mitochondria-targeted nano-systems have great potential in cancer therapy. Cationic molecules, such as triphenyl phosphonium (TPP), have good affinity to mitochondria by electrostatic adsorption owing to the negative charge of mitochondrial membrane (-150 ~ -170 mV).9-11 Although cationic mitochondria-targeted nano-systems are proved to accumulate around mitochondria, their application is maximally restricted due to the easy combination with negatively charged plasma proteins.12,13 Thus, it is essential to develop a charge-reversible nano-system that stays negative in circulation but reverses to cationic particles in tumor.14,15 Heparanase, the only endo-β-D-glucuronidase in mammal, is over-expressed in most malignancies including breast cancer, and can cleave the saccharide chains of heparin, making it a valuable tumor microenvironment trigger to activate prodrug or promote drug release.16,17 The negatively charged heparin shell of nanoparticles can be degraded by heparanase in tumor microenvironment, achieving delivery of nanoparticles.18 In clinic, chemotherapy is still one of the most common strategies for breast cancer.19 However, the severe side effects and poor pharmacological effects of chemotherapy agents extremely restrict their application. Investigation of effective tumor-targeting carriers for chemotherapy agents is


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urgently needed to reduce side effects and enhance anti-cancer efficiency.20,21 ZnO has attracted more attention in drug delivery field for its high drug loading potential and biological effects.22,23 Meanwhile, ZnO-based nanomaterials exhibit cytotoxicity towards tumor cells by generating ROS.24,25 Mitochondria-targeted ZnO nanomaterials have not been reported yet. It is worthy investigating whether mitochondria-targeted ZnO nanomaterials could amplify the ROS level in tumor cells for enhanced breast cancer therapy. Herein, charge-reversible mitochondria-targeted ZnO-TPP@D/H nanorods (NRs) were designed and synthesized to improve anti-cancer efficiency. ZnO NRs were synthesized with diameter of 20.20 ± 2.24 nm in width and 129.21 ± 44.77 nm in length. TPP molecules were modified on the surface of the as-obtained ZnO NRs to form the mitochondria-targeted ZnO-TPP NRs. DOX molecules were then loaded on the cationic ZnO-TPP NRs to form ZnO-TPP@D NRs. Heparin was introduced on the surface of ZnO-TPP@D NRs to form ZnO-TPP@D/H NRs. ZnOTPP@D/H NRs could be internalized into the mitochondria of cancer cells resulting from the exposure of TPP molecules after degradation of heparin by heparanase. The aim of this study was to (i) fabricate and characterize ZnO-TPP@D/H NRs for mitochondrial targeting; (ii) evaluate the cytotoxicity, mitochondrial targeting efficiency, apoptosis induction and cell migration inhibition ability of ZnO-TPP@D/H NRs at cellular level; (iii) estimate whether the mitochondria-targeted ZnO-TPP@D/H NRs could act as cascade amplifiers of ROS in tumor cells; (iv) evaluate the antitumor effect of ZnO-TPP@D/H NRs in vivo. The synthesis of ZnO-TPP@D/H NRs was confirmed by transmission electron microscopy (TEM), energy-dispersive X-ray spectroscopy (EDS) and zeta potential analysis. The drug release behaviors of ZnO-TPP@D/H NRs were evaluated using dialysis method. The mitochondrial targeting property of ZnO-TPP@D/H NRs in 4T1 cells was estimated by laser scanning confocal microscope (LSCM). The whole intracellular


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ROS generation and mitochondrial superoxide generation induced by ZnO-TPP@D/H NRs were demonstrated by LSCM and flow cytometry (FCM). The mitochondrial membrane potential (∆ψm) depolarization of 4T1 cells was also investigated by FCM. The induction of apoptosis was confirmed by western blot. In addition, the inhibition of cell migration by ZnO-TPP@D/H was evaluated in 4T1 cells using wound healing, cell migration and invasion experiments. The in vivo anti-tumor effect of ZnO-TPP@D/H NRs was evaluated using 4T1 tumor-bearing Balb/c nude mice models. Results of these experiments highlighted the potential of ZnO-TPP@D/H NRs for efficient breast cancer treatment.

2. EXPERIMENTAL SECTION

2.1. Preparation of ZnO-TPP@D/H NRs. ZnO NRs were synthesized by solvothermal method.26 Zinc acetate (0.01 moL) and octylamine (1 mL) were dissolved in 60 mL triethylene glycol and transferred to a Teflon-lined reaction kettle, heated at 190℃ for 8 h. ZnO NRs were obtained by vacuum drying after washing the precipitates thrice with methanol. 20 mg ZnO NRs were dispersed in 20 mL DMSO and added with 30 μL (3-aminopropyl) triethoxysilane (APTES). The above solution was stirred at 120℃ for 0.5 h and washed with absolute ethyl alcohol thrice. ZnONH2 NRs were obtained by vacuum drying. Carboxyl-activated TPP solution was added dropwise to the DMSO solution of ZnO-NH2 NRs and stirred for 24 h under N2. After washing with methanol thrice, ZnO-TPP NRs were obtained by vacuum drying. 0.8 mL DOX·HCl (1 mg mL-1) was added dropwise to 1 mL ZnO-TPP NRs (1 mg mL-1), and stirred in the dark for 15 h. After centrifuging at 12000 rpm for 15 min, the obtained ZnO-TPP@D NRs were washed with double distilled water until the superabundant was colorless to remove unloaded DOX. The above ZnO-


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TPP@D NRs were re-dispersed and added to heparin sodium solution and stirred at 50 oC for 1 h. Finally, the obtained ZnO-TPP@D/H NRs were centrifuged and dried by vacuum. 2.2. Characterization of ZnO-based NRs. TEM, EDS, Fourier Transform Infrared Spectroscopy (FT-IR), and UV-Vis spectrophotometer were used to demonstrate the synthesis of ZnO, ZnONH2 and ZnO-TPP NRs. The size distribution and zeta potentials of different NRs were characterized using Zetasizer-ZS90. ZnO-TPP@D/H NRs were stored for one week for stability assay. To evaluate the drug loading capacity and encapsulation efficiency, 1 mL ZnO-TPP NRs (1 mg mL-1) was stirred with 0.8 mL DOX·HCl (1 mg mL-1) in the dark for 15 h and washed with double distilled water. All washings were measured by UV-Vis spectrophotometer at 480 nm. The drug loading (DL%) and encapsulation efficiency (EE%) were measured using the following equations: DL% = EE% =

Wtotal ― 𝑊𝑓𝑟𝑒𝑒 𝑊𝑁𝑅𝑠 𝑊𝑡𝑜𝑡𝑎𝑙 ― 𝑊𝑓𝑟𝑒𝑒 𝑊𝑡𝑜𝑡𝑎𝑙

× 100%

(1)

× 100%

(2)

where Wtotal, Wfree and WNRs represent total DOX, unloaded DOX, and ZnO-TPP@D/H NRs, respectively. 2.3. The pH-triggered drug release behaviors of ZnO-TPP@D/H NRs, ZnO-TPP@D/H NRs were investigated by dialysis. ZnO-TPP@D/H NRs were added into membrane bags (800-14000 kD) and set in 20 mL different PBS (pH = 5.5 and 7.4), shaking at 37 oC for 50 h. Dialysis soution was replaced by equal PBS at specified intervals, and determined by multifunctional enzyme marking instrument. In addition, ZnO-TPP@D/H NRs were incubated with PBS at pH 5.5 and 7.4 for 48 h, respectively. 2.4. Cytotoxicity Assays. 4T1 Cells were seeded in 96-well plates for 24 h. Then, cells were exposed to ZnO, ZnO-TPP, DOX, ZnO-TPP@D and ZnO-TPP@D/H NRs from 0.5 to 4 μg mL-1


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for 48 h. Untreated cells were as control. MTT (0.5 mg mL-1) was added to form formazan crystals. The absorbance of formazan crystals at 570 nm was measured by multifunctional enzyme marking instrument. Other cytotoxicity assays of DOX, ZnO-TPP, ZnO-TPP@D and ZnO-TPP@D/H NRs against 4T1 cells and MCF-7 cells were similarly measured. Cell viability was calculated as follows: 𝐴𝑠𝑎𝑚𝑝𝑙𝑒 ― 𝐴𝑃𝐵𝑆

Cell Viablility% = 𝐴𝑐𝑜𝑛𝑡𝑟𝑜𝑙 ― 𝐴𝑃𝐵𝑆 × 100%

(3)

where APBS, Acontrol and Asample refer to the absorbance values of PBS, control cells and treated cells, respectively. 2.4. Cellular Uptake. 4T1 cells were seeded for 24 h, and further incubated with DOX and ZnOTPP@D/H NRs (5 μg mL-1) for 12 h, respectively. Mito-tracker green was used as mitochondria probe. Hoechst 33342 was introduced as nucleic probe. All fluorescence signals were observed by LCSM. The co-localization and Pearson’s correlation coefficient of green fluorescence (generated by Mito-tracker green) / red fluorescence (generated by DOX) were calculated by Image J software. 2.5. ROS Generation. 4T1 cells were seeded as described in cellular uptake assays, and then incubated with DOX, ZnO-TPP NRs, ZnO-TPP@D NRs or ZnO-TPP@D/H NRs (2 μg mL-1) for 24 h. DCFH-DA was added for ROS detection. The green fluorescence was observed by LCSM. For quantification of intracellular ROS, 4T1 cells were seeded and treated as above. The fluorescence of DCF-DA (generated after the oxidation of DCFH-DA by ROS) was measured by flow cytometry. To further investigate whether mitochondrial superoxide was induced by ZnOTPP NRs, 4T1 cells were seeded and incubated with ZnO, ZnO-TPP NRs for 24 h, respectively. Then cells were stained with MitoSOX Red for the detection of mitochondrial superoxide. The red fluorescence was detected by flow cytometry.


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2.6. Mitochondria Membrane Potential Detection. 4T1 cells were seeded and treated as described in ROS generation assays. Rhodamine 123 was employed to indicate mitochondria membrane potential. The green fluorescence was detected by flow cytometry. 2.7. Wound Healing. 4T1 cells were cultured for 24 h and equal wounded gaps was created with 10 μL pipette tip. Cells were treated with free DOX, ZnO-TPP NRs, ZnO-TPP@D NRs and ZnOTPP@D/H NRs (0.5 μg mL-1) for 48 h. Independent fields of the wounded gaps were photographed and analyzed. Untreated cells were noted as control. The wounding ratio was calculated as follows. Wound healing rate% =

𝐴𝑐𝑜𝑛𝑡𝑟𝑜𝑙 0 ℎ ― 𝐴48 ℎ 𝐴𝑐𝑜𝑛𝑡𝑟𝑜𝑙 0 ℎ

× 100%

(4)

where Acontrol 0 h and A48 h refer to the wound area of control group at 0 h and the wound of every group at 48 h, respectively. 2.8. Cell Migration and Invasion Assay. 4T1 cells were seeded in the upper transwell chamber without serum after 24 h incubation with DOX, ZnO, ZnO-TPP, ZnO-TPP@D and ZnOTPP@D/H NRs (2 μg mL-1). The lower chamber well was added with 600 μL medium containing 10% fetal bovine serum (FBS). After 24 h incubation, cells that stayed in the upper chamber were removed. The bottom cells were stained with 0.1% crystal violet and photographed in independent 10× fields of microscope. Cell invasion assays were similarly conducted except that the upper transwell chambers were coated with 10% Matrigel in advance. 2.9. Western Blot. 4T1 cells were seeded and treated respectively with free DOX, ZnO NRs, ZnO-TPP NRs, ZnO-TPP@D NRs and ZnO-TPP@D/H NRs for 24 h. Untreated cells were as control. 4T1 cells were lysed in NP-40 lysis buffer and centrifuged at 12000 rpm for 10 min. The proteins in supernatant were collected and separated by 10% SDS-PAGE gels, and then transferred to Nitrocellulose (0.22 mm pore size). Primary antibodies against β-actin, caspase-9, caspase-3 and cytochrome c were incubated overnight with these Nitrocellulose membranes after the


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membranes were blocked with 5% nonfat milk. Protein bands were analyzed by Odyssey1 infrared imaging system and quantified with Image J software. 2.10. In vivo Anti-tumor Effect. Tumor-bearing Balb/c nude mice models were established to investigate the anti-tumor effect of ZnO-TPP@D/H NRs. 4-week-old Balb/c nude mice were adaptive fed for one week, and then subcutaneously injected with 4T1 cells to establish tumor models. Four days later, tumor-bearing mice were randomly divided into three groups (n=4) and respectively treated every three days for totally 4 times with saline (negative control), DOX (positive control) and ZnO-TPP@D/H NRs at the concentration of 5 mg kg -1 (equivalent to DOX). The tumor volume and body weight of mouse in every group were evaluated every three days after first administration. Tumor-bearing mice were sacrificed at day 18 after first administration.The picture of tumors was photographed. The weight of tumor was measured and the tumor inhibiting rate (TIR%) was calculated as follows: 𝑊test

(5)

TIR% = (1 - 𝑊𝑠𝑎𝑙𝑖𝑛𝑒) × 100%

2.11. Statistical Analysis. All values were presented as mean ± SD. Every experiment was performed thrice. Statistical analysis was conducted by Student’s t-tests.

3. RESULTS AND DISCUSSION

3.1. Synthesis and Characterization of ZnO-TPP NRs. The strategy used for the fabrication of core–shell ZnO-TPP NRs was illustrated in Figure 1a. ZnO NRs were prepared by solvothermal method,26 followed by a surface grafting process using APTES to form ZnO-NH2 NRs.27 The surface of ZnO-NH2 NRs was further modiﬁed with TPP molecules through amidation reaction, forming ZnO-TPP NRs.28 The morphologies of the as-prepared ZnO, ZnO-NH2, and ZnO-TPP NRs were characterized by TEM. As shown in Figure 1b,c, the as-obtained ZnO NRs were club-


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shaped, with diameter of 20.20 ± 2.24 nm in width and 129.21 ± 44.77 nm in length. The lattice fringe spacing of 0.25 nm (Figure 1d) corresponding to the (101) plane of the hexagonal wurtzite structure of ZnO NRs suggested the successful formation of ZnO NRs. After APTES grafting, ZnO-NH2 NRs remained stick-like morphology and the organic shell with weaker contrast was observed (Figure S1a of Supporting Information). To further confirm the structure of ZnO-NH2 NRs, high resolution transmission electron microscopy (HRTEM), EDS mapping and liner scanning were also employed. APTES shell with thickness of ~4 nm was uniformly coated on the surface of ZnO NRs (Figure S1b). The existence of Si and N elements on the outer layer of ZnONH2 NRs was confirmed by EDS mapping (Figure S1c) and EDS liner scanning (Figure S1d), indicating the successful grafting of APTES on the surface of ZnO NRs. The APTES grafting could introduce active amino groups29,30 for further linkage of TPP molecules. As shown in Figure 1e, ZnO-TPP NRs also had core-shell morphology. The thickness of the coatings of ZnO-TPP NRs is ~17 nm (Figure 1f), much thicker than that of ZnO-NH2 NRs, indicating the modification of TPP molecules on ZnO-NH2 NRs. The existence of additional P element in EDS mapping (Figure 1g) also confirmed the successful modification of TPP molecules on the surface of ZnO-NH2 NRs. The characteristic absorption band of TPP appeared at ~260 nm in UV-Vis spectra of ZnO-TPP NRs further demonstrated the existence of TPP molecules on ZnO-NH2 NRs (Figure S2b). As shown in Figure S2c, the intense peaks located between 400 and 500 cm-1 and the other strong peak centered at ~1632 cm-1 in FTIR spectra of ZnO NRs could be ascribed to the vibration of ZnO bonds and hydroxyl groups, respectively. The weak peaks centered at 1647 cm-1 and ~1570 cm-1 in the FTIR spectra of ZnO-TPP NRs attributable to the vibration of amide I band and -NH2 bands, respectively, revealing that the surface of ZnO NRs was grafted with APTES and TPP via chemical conjugations.


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3.2. Synthesis and Characterization of ZnO-TPP@D/H NRs. Dynamic light scattering (DLS) measurements were employed to characterize the size distributions of ZnO, ZnO-TPP@D and ZnO-TPP@D/H NRs in aqueous solution. As shown in Figure 2a, the hydrodynamic diameter was 142.40 ± 0.25 nm (PDI=0.135) for ZnO NRs. After surface modification and DOX loading, the particle size soared to 156.40 ± 1.42 nm (PDI=0.253) for ZnO-TPP@D NRs, and further increased to 179.60 ± 5.67 nm (PDI=0.113) for ZnO-TPP@D/H NRs due to the surface absorption of heparin. The size distribution of ZnO-TPP@D/H NRs in RPMI 1640 medium supplemented with 10% FBS was evaluated (Figure 2b), suggesting the good stability of ZnO-TPP@D/H NRs in bioenvironment. Physicochemical characteristics, such as size, zeta potential and stability are very important for drug delivery systems, which directly affect the bio-behavior of nano-systems in vivo. Thus, the appropriate particle size (< 200 nm), and the stability in cell culture supplemented with FBS indicated the stable transportation of ZnO-TPP@D/H NRs in circulation. The drug loading radio and encapsulation efficiency of ZnO-TPP@D NRs were 21.70 ± 1.80% and 34.70 ± 3.61% (Table S1), respectively, proving that ZnO-TPP NRs could be efficient carriers of DOX. The mechanism of DOX loading by ZnO-TPP NRs was ascribed to be the chelation between Zn2+ and the phenolic oxygen in quinone structure of DOX molecules31 as well as the π-π stacking formed by the aromatic moiety on DOX and TPP molecules. The charge reversal was observed in Figure 2c, indicating the electrostatic adherence of heparin on the surface of ZnO-TPP@D NRs. It was suggested that the negatively charged coating layer made ZnO-TPP@D/H NRs negative in circulation to avoid the nonspecific protein adsorption and the quick elimination.32 After the degradation of heparin by over-expressed heparanase in tumor environment,33 TPP molecules on ZnO-TPP@D/H NRs were exposed, consequently resulting in the mitochondrial targeting. In addition, ZnO-TPP@D/H NRs were stored in room temperature and photographed every day to


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investigate the storage stability. As shown in Figure S3, aqueous solution of ZnO-TPP@D/H NRs was clear and had little precipitate during one week. To evaluate the acid-triggered drug release behavior, TEM images of ZnO-TPP@D/H at different pH (pH= 5.5 and 7.4) were evaluated. As shown in Figure 2d,e, ZnO-TPP@D/H NRs completely degraded at pH 5.5 after 48 h incubation, while the coll-shell rod-like morphology of ZnO-TPP@D/H NRs was still kept. The drug release profiles of ZnO-TPP@D/H NRs in vitro were shown in Figure 2f, demonstrating that ZnOTPP@D/H NRs were degraded and thus more DOX was released at lower pH. The pH value in tumor cells is always lower than that in normal cells, the pH-triggered drug release is appreciated to increase the overall concentration of drugs in tumor cells and decrease the needed dosage.34 3.3. Mitochondrial Targeting Properties of ZnO-TPP@D/H in 4T1 Cells. To confirm whether ZnO-TPP@D/H NRs could indeed accumulate around mitochondria, LSCM was employed to show the intracellular uptake of ZnO-TPP@D/H NRs in 4T1 cells. Free DOX was also studied as a control group. No fluorescence probe was needed in the experiment since the inherent red fluorescence of DOX was strong enough. As shown in Figure 3a-h, the subcellular localization of free ZnO-TPP@D/H or free DOX in 4T1 cells was detectable according to the relative location of nuclei (blue), mitochondria (green), ZnO-TPP@D/H(red), and free DOX (red). After incubation with 4T1 cells for 12 h, the red fluorescence of ZnO-TPP@D/H NRs was luminous, suggesting the successful cellular uptake. Meanwhile, extensive yellow color (the combination of red and green fluorescence) appeared in the overlapping image (Figure 3d), suggesting the majority of ZnO-TPP@D/H NRs accumulated in the mitochondria rather than nucleus. However, the distinct purple regions (the combination of red and blue fluorescence) appeared in the overlapping image (Figure 3h) revealed that free DOX molecules preferred to stay in nucleus. Image J software was introduced to further identify the colocalization of mitochondria with ZnO-TPP@D/H NRs or free


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DOX. The gray values of Mito-tracker and ZnO-TPP@D/H NRs changed synchronously (Figure S4a), which clearly verified the colocalization of mitochondria and ZnO-TPP@D/H NRs. Conversely, the gray values of Mito-tracker green and free DOX changed oppositely, suggesting that free DOX and mitochondria were not in the same relative location (Figure S4b). This phenomenon was also discussed in other work.35 Moreover, the Person’s R value between Mitotracker green and ZnO-TPP@D/H NRs was 0.85, more than two times than that between Mitotracker green and free DOX (0.35) (Figure S4c), further confirming the effective mitochondrial targeting capacity of ZnO-TPP@D/H NRs. 3.4. Cytotoxicity Assays. MTT assays were applied to investigate the cytotoxicity of ZnO NRs and ZnO-TPP NRs. After incubation with 4T1 cells (highly metastatic mouse breast cancer cells) for 48 h, it was apparent that both ZnO NRs and ZnO-TPP NRs showed anti-cancer properties (Figure 4a). Meanwhile, the cytotoxicity of ZnO-TPP NRs was significantly higher than that of ZnO NRs at all concentrations, especially at concentrations larger than 1 μg mL-1, suggesting the enhanced anti-cancer ability was achieved by mitochondrial targeting. ROS producing was considered as the main mechanism of the cytotoxicity caused by ZnO NRs in many previous works.36,37 Mitochondrion is one of the most ROS-sensitive organelles in cells. The mitochondriatargeted ZnO-TPP NRs showed stronger cytotoxicity than ZnO NRs in 4T1 cells. To further investigate the cytotoxicity of ZnO-based NRs, four groups of 4T1 cells were cultured, and individually incubated with free DOX, ZnO-TPP, ZnO-TPP@D and ZnO-TPP@D/H NRs for 24 h (Figure S5a) and 48 h (Figure 4b). ZnO-TPP@D/H NRs exhibited significantly higher cytotoxicity than free DOX and ZnO-TPP NRs, showing that delivering DOX to mitochondria by ZnO-TPP@D/H NRs could achieve a better anti-cancer outcome. Similar results were also found in MCF-7 cells (Figure S5b,c).


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3.5. Intracellular ROS Generating. DCFH-DA, a fluorescence probe for ROS detection, was employed to evaluate the level of intracellular ROS. 4T1 cells were respectively incubated with free DOX, ZnO-TPP NRs, ZnO-TPP@D NRs and ZnO-TPP@D/H NRs for 24 h, and further incubated with DCFH-DA for 30 minutes to mark the induced intracellular ROS. The green fluorescence signal of DCF-DA (generated through the oxidation of DCFH-DA by ROS) was detected by LSCM (Figure 5a-d) and the relative fluorescence intensities of all groups compared with background were calculated using Image J software (Figure 5e). Compared with free DOX, the stronger green fluorescence generated by ZnO-TPP NRs showed significant ROS generation (P < 0.05). Compared with ZnO-TPP NRs, more ROS were detected after 4T1 cells were incubated with ZnO-TPP@D NRs, showing that the further amplification of intracellular ROS was achieved by the delivery of DOX-loaded ZnO-TPP@D NRs to mitochondria. ZnO-TPP@D/H NRs showed the highest ROS level in 4T1 cells, which might attribute to the soft anti-cancer property of heparin.38 Flow cytometry was also introduced to detect intracellular ROS. 4T1 cells were incubated with free DOX, ZnO NRs, ZnO-TPP NRs, ZnO-TPP@D NRs and ZnO-TPP@D/H NRs for 6 h, and further incubated with DCFH-DA. ZnO-TPP NRs showed stronger DCF-DA signal than ZnO NRs (Figure 5f), indicating that more ROS was induced by the delivery of ZnO-TPP to mitochondria. Similar with LSCM images, ZnO-TPP@D/H NRs showed the strongest fluorescence of DCF-DA (Figure 5g), proving that delivery of DOX by ZnO-TPP@D/H NRs to mitochondria of tumor cells would significantly increase intracellular ROS in 4T1 cells. Although more intracellular ROS was generated in 4T1 cells after ZnO-TPP NRs treatment when compared with ZnO NRs, whether ZnO-TPP NRs could induce mitochondria ROS was still unknown. Therefore, 4T1 cells were treated with ZnO or ZnO-TPP NRs for 24 h and then stained with MitoSOX Red, a selective fluorescence probe for the detection of mitochondrial superoxide. The


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fluorescence was detected by flow cytometry. As shown in Figure 5h, the fluorescence of MitoSOX Red was strongest in 4T1 cells after ZnO-TPP NRs treatment, showing that mitochondria-targeted ZnO-TPP NRs could efficiently increase the status of mitochondrial ROS. 3.6. Mitochondrial Membrane Potential (∆ψm) Depolarization. It has been proved that the increasing level of ROS around mitochondria promotes ∆ψm depolarization and thereby small solutes (less than 1.5kD, including mitochondrial ROS) could leak out from the mitochondrial membrane.8,39 To confirm whether ∆ψm depolarization occurred after ZnO-TPP@D/H NRs treatment, 4T1 cells were classified into four groups and treated with free DOX, ZnO-TPP, ZnOTPP@D and ZnO-TPP@D/H NRs, respectively. Then, cells were stained with Rhodamine 123, a cationic dye, which preferentially locates in mitochondria owing to electrical interaction with relatively negative mitochondrial membrane. Once ∆ψm was depolarized, the interaction between Rhodamine 123 and mitochondria was relieved and green fluorescence disappeared. Normal cultured 4T1 cells were used as negative control while 4T1 cells only stained with Rhodamine 123 were used as positive control. Results and the corresponding quantitative analysis were shown in Figure 6a-g. The fluorescence intensity of normal cultured 4T1 cells was lower than 102 at 100%, while the positive control group (only stained by Rhodamine 123) showed a fluorescence intensity larger than 102 at 91.2%. Therefore, 102 was introduced as a threshold to distinguish the negative and positive staining. Compared with positive control groups, the negative staining percentage of 4T1 cells that treated with ZnO-TPP NRs increased to 24.0%, suggesting that ZnO-TPP NRs could notably induce the depolarization of ∆ψm. Compared with free DOX, 4T1 cells treated with ZnOTPP@D/H NRs showed a more serious ∆ψm depolarization, resulting in the increase of negative staining percentage from 20.4% to 64%. This result demonstrated that the delivery of DOX by ZnO-TPP@D/H NRs had a stronger effect on mitochondrial integrity and functions, which might


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be the main reason for enhanced cytotoxicity and ROS generating capacity of ZnO-TPP@D/H NRs observed in MTT assays and ROS generation experiments. ZnO-TPP@D/H NRs could act as cascade amplifiers of intracellular ROS. The mechanism of ROS amplification could be summarized as Figure 6h. Once the ZnO-TPP@D/H NRs accumulated into tumor microenvironment, the heparin shells of NRs would be degraded by heparanase, and then the positively charged TPP molecules were exposed, driving NRs to mitochondria of cancer cells. After that, ZnO-TPP@D/H NRs would slowly dissolve at acidic cell matrix, releasing DOX and generating ROS around mitochondria. The generated ROS would further lead to ∆ψm depolarization, resulting in the leakage of mitochondria ROS that originally stored in mitochondrial matrix. As like dominoes, nearby mitochondria would further be attacked by the already released mitochondrial ROS. Thus, the amplification of intracellular ROS was formed, causing death of cancer cells. 3.7. Influence on Apoptosis Pathway. In intrinsic apoptosis pathway, cytochrome c is released from mitochondria under appropriate stimulations, and then recruits apoptotic protease-activating factor-1 (Apaf-1) and caspase-9 to form apoptosis bodies, which consequently activates caspase3, the direct performer of apoptosis.40 The expression levels of proteins involved in intrinsic apoptosis pathways were evaluated by western blot in this work. Two groups of 4T1 cells were seeded and incubated with ZnO NRs and ZnO-TPP NRs for 24 h, respectively. No treated cells were used as control. The amount of cytochrome c, caspase-9, caspase-3 and β-actin was measured, respectively (Figure 7a-d). The expression level of cytochrome c in the cytoplasm of 4T1 cells was evidently increased after ZnO or ZnO-TPP treatment. ZnO-TPP showed a stronger ability to increase cytochrome c content in 4T1 cells than ZnO. i.e., ZnO-TPP NRs had a stronger ability to promote the release of cytochrome c into cytosol, which was due to the ∆ψm depolarization as


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demonstrated above. Similarly, the expression levels of caspase-9 and caspase-3 were also upregulated and followed the order: ZnO-TPP > ZnO > Control, suggesting stronger activation of cell apoptosis by ZnO-TPP NRs. These phenomena also verified the close relationship between mitochondrial targeting and the stronger induction of cell apoptosis. In order to demonstrate the facilitation of apoptosis caused by ZnO-TPP@D/H NRs, 4T1 cells were incubated and treated with free DOX, ZnO-TPP@D and ZnO-TPP@D/H for 24 h, respectively. Compared with free DOX, the levels of cytochrome c, caspase-9, and caspase-3 proteins were also significantly up-regulated after ZnO-TPP@D/H treatment (Figure 7e-h), suggesting that the delivery of ZnO-TPP@D/H to the mitochondria of 4T1 cells would further induce cell apoptosis. Note that the expression of caspases in 4T1 cells after ZnO-TPP@D/H treatment was higher than ZnO-TPP@D treatment, which was consistent with the results in MTT assays. This phenomenon was also found in our previous study.41 The mechanism of strong induction of apoptosis caused by ZnO-TPP@D/H NRs could be explained as follows. When DOX-loaded ZnO-TPP@D/H NRs arrived at mitochondria of tumor cells and caused ∆ψm depolarization, the leakage of mitochondrial ROS and cytochrome c would induce downstream effector proteins such as caspase-9 and caspase-3, resulting in enhanced apoptosis. 3.8. Influence on Cell Migration. Wound-healing assays were employed to evaluate the migration potential of 4T1 cells. As shown in Figure 8a and 8b, the wound closure followed the order: control > DOX > ZnO-TPP > ZnO-TPP@D ≈ ZnO-TPP@D/H. Compared with the almost total healing of wound in control, the wound was still open with ZnO-TPP@D/H NRs treatment. The wound area in ZnO-TPP@D/H NRs group was approximate triple of that in free DOX group, showing that ZnO-TPP@D/H NRs were potential for inhibition of cell migration. It was worth mentioning that the better anti-migration effect of ZnO-TPP@D and ZnO-TPP@D/H NRs than


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that of ZnO-TPP NRs might result from the cell growth inhibition of DOX. Cell migration and invasion assays were also conducted in transwell. As shown in Figure 8c-f, analogous to the results of wound healing assays, the number of migrated cells and invaded cells followed this order: Control > ZnO > ZnO-TPP > ZnO-TPP@D ≈ ZnO-TPP@D/H, demonstrating ZnO-TPP@D/H NRs could strongly inhibit cell migration and invasion of 4T1 cells. 3.9. In vivo Anti-cancer Effect. 4T1 tumor-bearing Balb/c nude mouse model was established to investigate the anti-tumor effect of ZnO-TPP@D/H NRs in vivo. As shown in Figure 9a, compared with the saline group, the body weight of tumor-bearing mice after DOX administration obviously decreased, indicating the toxicity of DOX in mice. The tumor volume curves after administration with saline, DOX and ZnO-TPP@D/H NRs were shown in Figure 2b. In saline group, the tumor volume increased to 5.9 folds at day 18 after first administration, while that in ZnO-TPP@D/H NRs group was only increased to 3.0 folds. The picture of tumor and the weight of tumor at day 18 after administration were shown in Figure 9c and Figure 9d, respectively. The tumor inhibiting rate of DOX and ZnO-TPP@D/H NRs were 18% and 46%, respectively. These results suggested ZnO-TPP@D/H NRs had significantly (P < 0.05) anti-tumor effect, showing their potential in breast cancer therapy.

4. CONCLUSIONS

In summary, mitochondria-targeted core-shell ZnO-TPP@D/H NRs were synthesized for the first time and evaluated both in vitro and in vivo. Results showed the effective anti-tumor effects by ZnO-TPP@D/H NRs. Laser scanning confocal images identified that ZnO-TPP@D/H NRs could accumulate around mitochondria. MTT assays showed the enhanced cytotoxicity of ZnOTPP@D/H NRs, suggesting its great anti-cancer ability. ROS generation assays and the detection


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of mitochondrial membrane potential depolarization identified that ZnO-TPP@D/H NRs could act as cascade amplifiers of intracellular ROS by releasing mitochondrial ROS into cytosol. Western blot assays showed that ZnO-TPP@D/H NRs had significant advantages in induction of apoptosis. Would healing and cell migration/invasion assays revealed that ZnO-TPP@D/H NRs had great potential to inhibit migration and invasion of 4T1 cells. In vivo studies confirmed that ZnOTPP@D/H NRs could significantly inhibit tumor growth in 4T1 tumor-bearing Balb/c nude mice model. All in all, the utilization of ZnO-TPP@D/H NRs is promising for effective breast cancer therapy.


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Figures

Figure 1. Synthesis and structural identification of ZnO-TPP NRs. (a) Graphical synthetic routes of ZnO-TPP NRs. (b,c) TEM images of ZnO NRs. (d) HRTEM image of ZnO NRs. (e) TEM and (f) HRTEM images of ZnO-TPP NRs. (g) Corresponding EDS element mapping images of ZnOTPP NRs.


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Figure 2. Characterization of ZnO-TPP@D/H. (a) The size distributions of ZnO, ZnO-TPP and ZnO-TPP@D/H NRs in (a) aqueous solution. (b) The size distribution of ZnO-TPP@D/H NRs in serum-containing cell culture. (c) Zeta potentials of ZnO, ZnO-TPP@D and ZnO-TPP@D/H NRs in aqueous solution. TEM images of ZnO-TPP@D/H NRs after incubation in PBS at (d) pH 5.5 and (e) pH 7.4 for 48 h. (f) The pH-sensitive drug release curves of ZnO-TPP@D/H in PBS at pH 5.5 and 7.4.


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Figure 3. Typical laser scanning confocal microscope images of 4T1 cells after incubation with (a-d) ZnO-TPP@D/H NRs and (e-h) free DOX for 12 h, respectively. Concentration: 5 μg mL-1. Scale bars: 15 µm.


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Figure 4. (a) Cell viability of 4T1 cells after incubation with ZnO, ZnO-TPP NRs for 48 h. (b) Cell viability of 4T1 cells after incubation with free DOX, ZnO-TPP, ZnO-TPP@D and ZnOTPP@D/H NRs at different concentrations for 48 h.


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Figure 5. Detection of ROS generation by DCFH-DA in 4T1 cells after incubation with (a) ZnOTPP@D/H, (b) ZnO-TPP@D, (c) ZnO-TPP and (d) DOX for 24 h. Concentration: 2 μg mL-1 Scale bars: 15 μm. (e) Corresponding quantitative analysis. (f) Flow cytometry results of ROS generation by DCFH-DA in 4T1 cells after incubation with ZnO and ZnO-TPP NRs for 6 h. Concentration: 2 μg mL-1. (g) Flow cytometry results of ROS generation by DCFH-DA in 4T1 cells after incubation with ZnO, ZnO-TPP, ZnO-TPP@D, ZnO-TPP@D/H NRs for 6 h. *P < 0.05, **P < 0.01. (h) Detection of mitochondrial superoxide in 4T1 cells by flow cytometry using MitoSOX Red after incubation with ZnO, ZnO-TPP for 24 h. Concentration: 3 μg mL-1.


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Figure 6. Mitochondrial membrane potential (∆ψm) depolarization detected by flow cytometry. Flow cytometry results of (a) untreated cells (negative control), (b) 4T1 cells stained with Rhodamine 123 (positive control), 4T1 cells stained with Rhodamine 123 after incubation with (c) DOX, (d) ZnO-TPP, (e) ZnO-TPP@D and (f) ZnO-TPP@D/H NRs. (g) The corresponding quantitative analysis of ∆ψm. *p < 0.05, **p < 0.01. (h) The mechanism of amplification of intracellular ROS caused by ZnO-TPP@D/H NRs.


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Figure 7. (a) The protein expressions in 4T1 cells after incubation with ZnO and ZnO-TPP NRs, suggesting mitochondria-targeted ZnO-TPP had a stronger ability to induce apoptosis. Concentration: 5 μg mL-1. (b-d) The corresponding quantitative analysis of (a). (e) The protein expressions in 4T1 cells after incubation with ZnO-TPP@D, ZnO-TPP@D/H NRs and free DOX, suggesting the delivery of DOX-loaded ZnO-TPP@D/H NRs to 4T1 cells could induce stronger apoptosis than the delivery of free DOX. (f-h) The corresponding quantitative analysis of (e). Concentration: 1 μg mL-1. *p < 0.05, **p < 0.01.


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Figure 8. (a) Wound healing images (4×) of 4T1 cells after incubation with free DOX, ZnO-TPP NRs, ZnO-TPP@D NRs and ZnO-TPP@D/H NRs for 48 h. Concentration: 0.5 μg mL-1. (b) The corresponding quantitative analysis of the wound healing radio. (c) Transwell migration assay images (10×) of 4T1 cells and (d) the corresponding quantitative analysis of migration rates after


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incubation with free DOX, ZnO, ZnO-TPP, ZnO-TPP@D and ZnO-TPP@D/H NRs. Concentration: 2 μg mL-1. (e) Transwell invasion assay images (10×) of 4T1 cells and (f) the quantitative analysis of invasion rate after incubation with free DOX, ZnO, ZnO-TPP, ZnOTPP@D and ZnO-TPP@D/H NRs. Concentration: 2 μg mL-1. **p < 0.01.


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Figure 9. In vivo anti-tumor effect of ZnO-TPP@D/H NRs in subcutaneous tumor-bearing nude mice established with 4T1 cells. (a) Body weight and (b) tumor volume after intravenous injection of saline, DOX and ZnO-TPP@D/H NRs. Concentration of DOX or ZnO-TPP@D/H NRs was 5 mg kg-1. (c) Picture of tumors and (d) weight of tumors at day 18 after first administration. n=4. *p < 0.05. ASSOCIATED CONTENT Supporting Information. Supporting Information is available free of charge on the ACS Publications website. Details of materials, instruments; TEM images and EDS mapping of ZnONH2 NRs; UV-Vis spectra and FTIR spectra of ZnO and ZnO-TPP NRs; Stability of ZnOTPP@D/H NRs; Colocalization analysis; MTT assays in 4T1 cells and MCF-7 cells; Table of drug


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loading radio and encapsulation efficiency of ZnO-TPP@D/H NRs (Figure S1-5 and Table S1) (PDF) AUTHOR INFORMATION Corresponding Author * E-mail: [email protected] (S.Q.) ORCID Qi Shen: 0000-0003-1509-5391 Notes The authors declare no competing financial interest. ACKNOWLEDGMENT The authors thank Prof. Kai-Xue Wang (School of Chemistry and Chemical Engineering, SJTU) for assistance with the synthesis and characterization of ZnO NRs. Thanks for Dr. Qi. Deng (Bio-X Institutes, Ministry of Education, SJTU) for assistance with the in vivo experiments. ABBREVIATIONS: TPP

triphenyl phosphonium

DOX

docetaxel

NRs

nanorods

LSCM

laser scanning confocal microscope

ROS

reactive oxygen species

EDS

energy-dispersive X-ray spectroscopy

∆ψm

mitochondrial membrane potential

FCM

flow cytometry

TEM

transmission electron microscopy

APTES

(3-aminopropyl) triethoxysilane


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FTIR

fourier transform infrared spectroscopy

DL%

drug loading

EE%

encapsulation efficiency

FBS

fetal bovine serum

TIR%

tumor inhibiting rate

HETEM

high resolution transmission electron microscopy

DLS

dynamic light scattering

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ABSTRACT GRAPHIC

Mitochondrial targeting Apoptosis

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ROS Cascade


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Cascade Amplifiers of Intracellular Reactive Oxygen Species Based

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