Exploration of Zinc Oxide Nanoparticles as a Multitarget and

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Exploration of Zinc Oxide Nanoparticles as a Multitarget and Multi-functional Anticancer Nanomedicine Jiao Wang, Jung Seok Lee, Dongin Kim, and Lin Zhu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b11219 • Publication Date (Web): 27 Oct 2017 Downloaded from http://pubs.acs.org on October 27, 2017

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ACS Applied Materials & Interfaces

Exploration of Zinc Oxide Nanoparticles as a Multi-target and Multi-functional Anticancer Nanomedicine Jiao Wang,1 Jung Seok Lee,2 Dongin Kim,1 and Lin Zhu*,1 1

Department of Pharmaceutical Sciences, Irma Lerma Rangel College of Pharmacy, Texas A&M University Health Science Center, Kingsville, Texas 78363, United States 2

Department of Biomedical Engineering, School of Engineering & Applied Science, Yale University, New Haven, CT 06511, United States

* Corresponding Author: Lin Zhu Department of Pharmaceutical Sciences, Irma Lerma Rangel College of Pharmacy, Texas A&M University Health Science Center, Kingsville, Texas 78363, United States Tel.: 3612210740 E-mail address: [email protected]

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Abstract Due to the complexity of cancer, an ideal anticancer strategy is better to target both cancer cells and the tumor microenvironment (TME). In this study, for the first time, we demonstrated that zinc oxide nanoparticles (ZnO NPs) were able to target multiple cell types of cancer, including cancer cells, cancer stem cells (CSCs) and macrophages, and simultaneously perform several key functions, including inhibition of cancer proliferation, sensitization of drug-resistant cancer, prevention of cancer recurrence and metastasis, and resuscitation of cancer immunosurveillance. As a nanocarrier, the chemotherapy drug, doxorubicin (Dox), could be loaded to ZnO NPs and the Dox-loaded ZnO NPs (ZnO/Dox) possessed excellent physicochemical and pH-responsive drug release properties. ZnO/Dox could be effectively internalized by both drug-sensitive and multi-drug resistant (MDR) cancer cells and penetrate more efficiently through 3D cancer cell spheroids compared to free Dox. As a cytotoxic agent, ZnO NPs were more efficient to kill MDR cancer cells. Interestingly, neither ZnO nor Dox showed high cytotoxicity in the 3D cancer cell spheroids, while ZnO/Dox showed the remarkable synergistic anticancer effects. More important, we demonstrated that ZnO NPs could effectively downregulate CD44, a key CSC surface marker, and decrease the stemness of CSCs, leading to the sensitization of the Dox treatment, inhibition of the cancer cell adhesion and migration, and prevention of the tumor (3D cancer cell spheroids) formation. As an immunomodulator, ZnO NPs could protect macrophages from the Dox-induced toxicity and boost the Dox-induced macrophage polarization towards an M1-like phenotype. The macrophage-conditioned medium could promote the cancer cell apoptosis in both cancer cell monolayers and 3D spheroids. The findings in this study indicated that ZnO NPs were a multi-functional and multi-target nanocarrier and nanomedicine that would have more profound effects on cancer treatment.

Keywords: Zinc oxide nanoparticles; Cancer stem cells; CD44; Immunotherapy; Drug resistance.


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1. Introduction Rather than a mass of cancer cells, cancer is regarded as a complex organ containing cancer cells and the associated tumor microenvironment (TME). The TME of solid tumor is characterized with a heterogeneous cell-protein matrix, surrounding blood vessels, and a dense hypoxic core. Cancer cells, fibroblasts, immune cells, adipocytes, etc., reside in the extracellular matrix and communicate via various cell surface markers, proteins, cytokines and chemokines. The TME plays a crucial role in cancer initiation and development.1 It is known that cancer tissues are subject to chronic inflammation and various immune cells, including tumor-associated macrophages (TAMs), are recruited by cancer cells and infiltrate into tumorigenic tissues. After tumor formation, however, the immune response in the tumor is impaired. For example, TAMs fail to clear apoptotic cancer cells via the classical activation to an M1 phenotype. Instead, the abundant apoptotic signals, like phosphatidylserine,2 on cancer cells induce the polarization of TAMs toward an M2 phenotype. The M2-like TAMs suppress the host adaptive immunosurveillance. In most solid tumors, predominant infiltration of neoplastic tissue by M2 TAMs, has been correlated with poor cancer prognosis, indicating the importance of TAMs in tumor angiogenesis, proliferation, invasion, and metastasis.3 Cancer stem cells (CSCs) or cancer initiating cells (CICs) are a small group of cancer cells, which share some common features with normal stem cells, such as drug resistance and self-renewal. Current radio and pharmacological interventions kill the bulk of cancer cells, but fail to eradicate the CSCs which are protected by specific resistance/repair mechanisms, resulting in drug resistance, metastasis and relapse.4 Although various anticancer therapeutics have been developed, they are commonly cancer cell-oriented and can’t effectively influence other important cells/aspects of the TME, resulting in unsatisfactory outcomes. Chemotherapy drugs are the most widely used cytotoxic agents against cancer cells, while their off-target toxicity and acquired multidrug resistance (MDR) compromise the therapeutic outcome.5 Molecular targeted therapeutics which are designed to target cancer cell-specific intracellular pathways are believed to have higher drug response and tolerance and lower toxicity compared to chemotherapeutics. Unfortunately, their anticancer spectra are relatively narrow and also show severe side effects in clinical applications.6 The emerged immunotherapy using host’s immune system



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to fight cancer is considered a potent TME-targeted approach and gains a lot of attentions. However, recent clinical trials indicated that immunotherapy drugs are not perfect and face certain issues as other types of drugs.7-8 All these facts suggest that an ideal anticancer strategy is better to target both cancer cells and cancer microenvironment, especially the critical cancer-supporting cells, such as TAMs and CSCs, and simultaneously perform multiple functions, such as inhibiting cancer growth, overcoming drug resistance, preventing cancer recurrence and metastasis, and resuscitating cancer immunosurveillance. In fact, some clinical drug combination approaches have used the multi-target strategy,9-10 whereas, they are usually the simple drug combination, without many considerations of pharmacokinetics (PK), tumor target ability, and formulation factors, which may sometimes lead to the compromised therapeutic outcomes and undesired side effects. An elegant and sophisticated multi-target/multi-functional medicine will be a critical need for dealing with the complex malignant disease in the future. Nanomedicine that formulates traditional drugs by nanotechnology, has proved its advantages over naked drugs and conventional formulations in many aspects. Most nanomaterials are inert and work only as drug carriers, while others may induce certain biological responses and contribute to overall therapeutic effects. Zinc oxide nanoparticles (ZnO NPs), an FDA-approved pharmaceutical excipient, are widely used in drug formulations and cosmetics, due to their stability, biocompatibility and safety.11 ZnO NPs have been investigated as the nanocarriers for delivery of a variety of cargoes, including drugs, genes, proteins, and imaging agents.11-12 Because ZnO NPs are readily dissolved at low pH, they have been used as an excellent pH-sensitive nanocarrier for tumor-targeted drug delivery and intracellular drug release.13-14 In fact, ZnO NPs were not inert and showed photocatalytic and photo-oxidizing abilities against chemical and biological species.15 The growing evidence indicated that ZnO NPs were able to kill many types of cancer cells via the generation of hydroxyl radicals (OH·), superoxide anion (O2-), and perhydroxyl radicals (HO2·) from the surface of ZnO, suggesting their potential as an anticancer agent.16-17 The pro-inflammatory properties of ZnO NPs have been well known for decades and considered as the undesired side effects.18-23 However, recently, coadministration of ZnO NPs and vaccines were found to boost the specific immune response against the vaccines, suggesting ZnO NPs might be an efficient immunologic adjuvant.24-26


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These evidences suggest that ZnO NPs can be not only a nanocarrier but also a multitarget therapeutic agent. Unfortunately, so far, these many properties of ZnO NPs have not been fully explored especially for cancer treatment. In the current study, for the first time, we demonstrated that ZnO NPs were able to simultaneously inhibit cancer cell proliferation, overcome drug resistance, sensitize cancer stem cells, prevent cancer cell migration and tumorigenesis, and stimulate macrophages to exert anticancer immune response. We hypothesized that all these properties contributed to the anticancer activity of ZnO NPs as well as their loaded drugs. Here, the chemotherapy drug, doxorubicin (Dox), was used as an example to test our hypothesis. 2. Results and Discussion 2.1. ZnO NPs as a Drug Nanocarrier 2.1.1. Morphology, Particle Size, Zeta Potential, and Drug Release In this study, the well-characterized commercially available ZnO NPs were used although ZnO NPs could be prepared by various methods and showed different particle size and morphology. These ZnO NPs were >99% pure and nearly spherical with a claimed aerodynamic particle size (APS) of 10-30 nm (US Research Nanomaterials). Here, the appearance and morphology of ZnO NPs and ZnO/Dox were examined and confirmed. As shown in Figure S1A, the ZnO NPs dry powder was white and the Dox powder was red. However, after loading of Dox, the ZnO/Dox NPs turned to dark purple. Both ZnO NPs and ZnO/Dox could be well dispersed in water and aqueous buffers. In the TEM micrographs (Figure S1B), both ZnO NPs and ZnO/Dox were tiny and near-spherical, which was consistent with the data provided by the vendor. The TEM results also indicated that the loading of Dox didn’t significantly change the size and morphology of ZnO NPs. Furthermore, the hydrodynamic particle sizes of ZnO NPs and ZnO/Dox were measured by dynamic light scattering (DLS) (Table 1). In water, the ZnO NPs had a hydrodynamic particle size of around 90 nm. It has been reported that ZnO was an amphoteric oxide and underwent hydrolysis in water resulting in formation of a hydroxide coating on its surface (Zn-OH). This hydroxide surface of the hydrolyzed ZnO NPs led to the chemical and physical adsorption of water molecules.27 We also could not rule out the possibility of the agglomeration in the presence of water molecule.28 While, in the DMEM complete medium containing 10% FBS, the hydrodynamic size of ZnO NPs was a little bit smaller (around 60



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nm). This was mostly due to the serum protein adsorption onto the nanoparticles, so as to stabilize the ZnO colloid and decrease the agglomeration.29 The zeta potential of ZnO NPs in pure water (neutral pH) was around +35 mV because of their isoelectric point (8.8–9.8).29 In the DMEM complete medium, it changed to around -18 mV because of the adsorption of negatively charged serum proteins.28-29 Compared with ZnO NPs, the hydrodynamic size of ZnO/Dox in water didn’t significantly change (Figure 1A), while their zeta potential was decreased to around +20 mV (Figure 1B), suggesting the successful Dox loading on the surface of ZnO NP. When dispersed in the DMEM complete medium, the size and zeta potential of ZnO/Dox were similar to those of ZnO NPs, indicating that the protein corona significantly influenced the surface properties of nanoparticles. Our data also indicated that bare ZnO NPs might not be a good in vivo drug delivery carrier since they would be readily opsonized and caught by the mononuclear phagocytic system.30-31 The surface modification of ZnO NPs might be needed for tumor targeting. Here, the ZnO/Dox weight ratio was ~5/1. The drug loading efficiency (DL) was ~16.67% and the encapsulation efficiency (EE) was ~30%. Although the exact drug loading mechanisms were not very clear, it has been proposed that the materials including Dox,32 proteins,33 and polymers,34 were able to attach to ZnO NPs’ surface through various strong interactions, such as covalent bonds,27 hydrogen bonds,34 electrostatic interactions,33 and chelation,35 rather than weak adsorption. As shown in Figure 1C, the loaded Dox could be rapidly released from the Dox-loaded ZnO NPs (ZnO/Dox) at pH 5.0 due to the acidmediated decomposition, while the ZnO/Dox showed slow drug release at pH 7.4, in agreement with the pH-dependent drug release property of ZnO NPs.36 Table 1. Hydrodynamic size and zeta potential of ZnO and ZnO/Dox in different media. Sample

Medium

Size (nm)

PDI

ZnO NPs

Water

95.43 ± 1.73

0.19 ± 0.01

35.24 ± 2.83

ZnO NPs

DMEM/10% FBS

56.74 ± 1.11

0.37 ± 0.02

-18.64 ± 3.50

ZnO/Dox

Water

92.41 ± 5.83

0.19 ± 0.01

20.98 ± 2.35

ZnO/Dox

DMEM/10% FBS

62.02 ± 0.98

0.31 ± 0.03

-16.06 ± 0.09


Zeta potential (mV)

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Data are presented as the mean ± SD. PDI, polydispersity index.

2.1.2. Cellular Uptake The cellular uptake of Dox-loaded ZnO NPs (ZnO/Dox) was investigated in various cancer cells including the Dox-sensitive (MDA-MB-231 and HeLa) and Dox-resistant (NCI/ADR-RES and MES-SA/Dx5) MDR cells. As shown in Figure 1D and S2, on the sensitive cells, both free Dox and ZnO/Dox showed similar cellular uptake, as evidenced by the similar mean fluorescence intensity, indicating that the ZnO NP-mediated endocytosis was comparable to the passive diffusion-mediated uptake of free drug. However, on the MDR cells, ZnO/Dox had a remarkably higher cellular uptake (>4 folds) than free Dox. We would like to point out that in this study, the control (free drug) was the water-soluble form of doxorubicin (hydrochloride salt) and should have higher cellular uptake compared to the insoluble Dox base. For ZnO/Dox, their nanoscale size and positive charge (Figure 1A-B) might account for the efficient endocytosis. The results were confirmed by the intracellular drug localization determined by confocal microscopy. As shown in Figure 1E, ZnO/Dox showed stronger red fluorescence than Dox in NCI/ADR-RES cells, while in MDA-MB-231 cells, ZnO/Dox and Dox had the similar fluorescence intensity. It is well known that both NCI/ADR-RES and MES-SA/Dx5 cells overexpress drug efflux transporters, such as P-glycoprotein (Pgp).37-38 The data indicated that ZnO/Dox might be able to overcome the Pgp-mediated drug efflux via the enhanced cellular internalization, drug-holding capability, and delayed intracellular drug release, leading to the drug accumulation in the cells. Besides, the microscopy data also showed that the red fluorescence was co-localized with the blue fluorescence, suggesting that, after endocytosis, Dox was released from ZnO NPs (in response to acidic endosomal pH) and mainly accumulated in the cell nuclei. 2.1.3. Penetration through 3D Tumor Cell Spheroids In addition to epigenetic and genetic alterations in cancer cells that influence the uptake, metabolism, or efflux of drugs, the TME mediates responses of solid tumors to drug treatments.39 The cells distant from blood vessels are likely to be resistant to systemic therapy because of poor tissue penetration.40 Furthermore, the dense extracellular matrix and stromal components of solid tumors increase interstitial fluid pressure, which limits the



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ability of large molecules, e.g. antibodies or nanomedicine, to penetrate through the tumor tissue by convection. These heterogenic features may serve as a “wall-like” barrier against drug delivery and promote aggressive behavior of cancer.41 In this regard, to better mimic the tumor tissue, we developed tumor cell spheroids to further challenge ZnO NPs. Two types of cancer cell spheroids were generated from NCI/ADR-RES and MDA-MB231 cells, respectively. The NCI/ADR-RES spheroids had a heterogeneous structure, as evidenced by a dense core and smooth surface, while the MDA-MB-231 spheroids seemed to have a homogenous structure and the “core-surface” structure was not observed (Figure 2). We found that both Dox and ZnO/Dox could penetrate through the established 3D cell spheroids. In the NCI/ADR-RES (Dox-resistant) spheroids, after 1 h incubation, ZnO/Dox had a shorter penetration depth but higher cellular uptake, as evidenced by strong fluorescence surrounding the dark spheroid core, indicating that the 3D cell spheroids increased drug resistance.42 In contrast, free Dox had a deeper penetration but lower cellular uptake, as evidenced by the evenly distributed weak fluorescence (Figure S3B). The data suggested that the Dox salt might be able to freely diffuse/penetrate through the spheroids due to its small size and good solubility. However, the up-regulated Pgp on the NCI/ADRRES cells decreased the intracellular drug concentration. The data were consistent with the previous reports that the nanoparticles around or less than 100 nm were able to accumulate in the tumor, while smaller nanoparticles or molecules were more efficient to penetrate in the tumor tissue.43-44 The increase in the incubation time from 1 h to 4 h (Figure 1F and S3B), both penetration depth and cellular uptake of ZnO/Dox were significantly increased, while the uptake of free Dox in spheroids was still weak, suggesting that the ZnO NPs could overcome Dox efflux in both cell monolayers and 3D structures. As shown in Figures 1F and S3A, in the MDA-MB-231 (Dox-sensitive) spheroids, both Dox and ZnO/Dox could penetrate into the spheroid core, as evidenced by the evenly distributed fluorescence, indicating that the homogenous, loose structure of the MDA-MB231 spheroids (compared to the NCI/ADR-RES spheroids) might not be a hindrance to the penetration of ZnO NPs. The increase in the incubation time increased the cellular uptake of ZnO/Dox, but didn’t significantly increase the uptake of Dox in the spheroids (4 h vs. 1 h), probably because the sustained drug release of ZnO/Dox (Figure 1C) increased the drug residence time in the spheroids.


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The results indicated that the 3D cell structures were more challenging to the drug treatment compared to the cell monolayers. The 3D cell-cell and cell-matrix communication influenced the properties of the spheroids, including their shape, size, morphology, cell proliferation, and distribution, etc., so as to influence the drug penetration and uptake.42 Based on the above results, we might conclude that as a nanocarrier, ZnO NPs could load drugs on their surface. The drug loaded ZnO NPs were stable in normal physiological pH, but could rapidly and completely release the loaded drug in the acidic environment. The drug-loaded ZnO NPs were able to penetrate the cell monolayers as well as the 3D multicellular spheroids, which effectively overcame the efflux and 3D cell structure induced drug resistance.

Figure 1. (A) Particle size determined by dynamic light scattering in water. (B) Zeta potential determined in water. (C) Dox release from ZnO/Dox at pH 7.4 and pH 5.0. (D) Cellular uptake of Dox and ZnO/Dox after 1 h incubation, determined by flow cytometry.



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(E) Intracellular localization after 1h drug incubation, determined by confocal microscopy. The scale bar represents 25 µm. (F) Drug penetration through cell spheroids after 4 h incubation. The scale bar represents 100 µm. Data are presented as the mean ± SD. ***P< 0.001. 2.2. ZnO NPs as a Cytotoxic Agent 2.2.1. Cytotoxicity on Cancer Cell Monolayers The cytotoxicity of the free Dox, ZnO NPs, and ZnO/Dox were compared in various types of cancer cells, including both Dox -sensitive and -resistant cells. After 24 h incubation, in the sensitive cells (MDA-MB-231 and HeLa), free Dox showed the highest cytotoxicity, while ZnO NPs showed the lowest cytotoxicity among the treatments. Loading of Dox to the ZnO NPs decreased the efficacy of Dox (Figure 2A and S4). Since both Dox and ZnO NPs showed the comparable cellular uptake efficiency in the monolayers of sensitive cells (Figure 1D and S2), the decreased efficacy of ZnO/Dox might be mainly attributed to the delayed drug release (Figure 1C). In contrast, in the Dox-resistant cells (NCI/ADR-RES and MES-SA/Dx5), free Dox showed much lower cytotoxicity due to the elevated drug efflux, while ZnO NPs showed higher cytotoxicity. Compared with ZnO NPs, loading of Dox to ZnO NPs slightly increased the cytotoxicity (Figure 2B and S4), indicating that these cells might lack the resistant mechanisms against ZnO NPs-induced cytotoxicity. But we could not rule out the contribution of the increased drug uptake (Figure 1D-E and S2) to the cytotoxicity. After analysis of their IC50 (Table S1), we might conclude that Dox was more efficient in killing the Dox-sensitive cells, while ZnO NPs were more efficient in killing the MDR cells. 2.2.2. Intracellular Reactive Oxygen Species (ROS) To understand the cytotoxicity of ZnO NPs, we measured the intracellular ROS levels of the treated NCI/ADR-RES cells by 2, 7-dichlorofluorescein diacetate (H2DCFDA).16 After 24 h incubation, ZnO NPs significantly induced cells’ ROS production, as evidenced by strong intracellular green fluorescence (Figure S5). The ROS induction by ZnO NPs was dose-dependent, in agreement with previous reports.16, 45 In contrast, free Dox didn’t induce ROS but the Dox-loaded ZnO NPs still induced strong ROS signal, indicating that the drug attachment on the ZnO surface didn’t significantly change their ROS induction (i.e. toxic


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nature) capability. The results were consistent with the previous reports on the Dox-loaded ZnO NPs.13, 32 2.2.3. Caspase 3/7 Levels The caspase activity of the treated NCI/ADR-RES cells was evaluated. The ZnO NPsinduced ROS production significantly increased the caspase 3/7 level (Figure S6), in agreement with a previous report,45 while free Dox didn’t increase the caspase activity due to its insufficient intracellular concentration in the MDR cells (Figure 1D-E). ZnO/Dox slightly decreased the caspase level compared to ZnO NPs (250% vs. 200%), however ZnO/Dox showed a slightly higher cytotoxicity than ZnO NPs (Figure 2B), indicating that the surface properties influenced the cytotoxicity of ZnO NPs, in agreement with previous reports.46-47 Here, ZnO NPs might impact multiple apoptosis signaling pathways other than just caspase cascade.48 2.2.4. Cytotoxicity on Cancer Cell Spheroids To further challenge ZnO NPs, the tumor spheroids were used as model. The cell viability and morphology of the spheroids were determined after treatments (Figure 2C-F). In the NCI/ADR-RES spheroids, free Dox didn’t significantly influence the cell viability and spheroid morphology (Figure 2D-F), because of the cells’ Dox resistance. However, ZnO NPs and ZnO/Dox increased the “apparent” size of spheroids and induced a significant shedding of cells/debris (Figure 2D and S7). Interestingly, ZnO NPs alone couldn’t cause cell death, whereas ZnO/Dox efficiently killed the cancer cells in the spheroids (Figure 2F). In the MDA-MB-231 spheroids, the similar changes in the spheroid size and morphology were observed after ZnO or ZnO/Dox treatments (Figure 2C and S7). Free Dox showed some cytotoxicity but its level was much lower than that of ZnO/Dox (Figure 2E), suggesting that the 3D cell structure increased drug resistance although the MDA-MB-231 cells in the monolayer were sensitive to free Dox (Figure 2A). In contrast, ZnO/Dox showed the highest cytotoxicity among the treatments, indicating the synergistic effects between ZnO NPs and Dox. Our results confirmed that the spheroids were more challenging to drug treatments and suggested that the combined use of ZnO NPs and Dox might be an effective and simple way to overcome drug resistance.



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Figure 2. Cytotoxicity of Dox, ZnO NPs, and ZnO/Dox on (A) MDA-MB-231 and (B) NCI/ADR-RES monolayer cells. Morphology and cytotoxicity of Dox, ZnO NPs, and ZnO/Dox on MDA-MB-231 (C and E) and NCI/ADR-RES (D and F) cell spheroids. C and D were micrographs at 40×. E and F were the cell viability of the spheroids measured by the CellTiter-Blue® Cell Viability Assay. The scale bar represents 100 µm. Data are presented as the mean ± SD. *P< 0.05, **P< 0.01. All P-values shown are vs the untreated group.

2.3. ZnO NPs as a Drug Sensitizer against Stem Cell-like Cancer Cells The ZnO-induced ROS and Dox-induced DNA intercalation might work “cooperatively” to kill cancer cells in cell monolayers.32 Obviously, this “cooperation” might not be adequate to explain the high cytotoxicity of ZnO/Dox because neither Dox nor ZnO NPs could kill cancer cells particularly in drug-resistant spheroids. The ZnO NP-induced cell shedding from tumor cell spheroids at high doses (1000 µM) has been reported and


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attributed to the ROS-induced toxicity/inflammatory response.49 However, at our tested low doses, ZnO NPs didn’t kill cancer cells of the spheroids (Figure 2D-F). Our results suggested that some unknown mechanisms might exist. To have a better understanding, we studied the impact of ZnO NPs on cancer stem cells/stem cell-like cancer cells that can remain as viable and self-renewable “seeds” during treatments for cancer initiation, recurrence and metastasis.50 2.3.1. Downregulation of CD44 CD44 is a transmembrane glycoprotein and works as a receptor for hyaluronic acid (HA), a major component of the extracellular matrix, and a co-receptor for many growth factors and cytokines. Recent evidence showed that CD44 is one of the most common CSC surface markers and plays a key role in the cell-cell and cell-matrix interaction, particularly in cancer dissemination, and regulating CSC stemness.51 Here, we measured the CD44 expression of MDA-MB-231 and NCI/ADR-RES and found that both cell lines were CD44+ (Figure 3A), in agreement with a previous report.52 The NCI/ADR-RES cells expressed much higher CD44 than the MDA-MB-231 cells (>1000 folds vs. 500 folds compared to untreated cells), indicating their higher stemness and drug resistance, in consistent with the cellular uptake and cytotoxicity data (Figure 1D-F and 2A-B). After incubating with 20 µg/mL of ZnO NPs for 24 h, the cell surface CD44 was remarkably downregulated in both cell lines (~50% left in NCI/ADR-RES and ~60% left in MDA-MB-231) (Figure 3B). The ZnO NPs-induced CD44 downregulation was dosedependent and 30 µg/mL ZnO NPs could decrease the CD44 expression to as low as ~35% in the NCI/ADR-RES cells (Figure 3C). On the contrary, Dox (4 µg/mL) treatment increased the CD44 level for about 10-15% (Figure 3B), in agreement with.53-54 It was reported that downregulation of CD44 by anti-CD44 siRNA, shRNA or miRNA could decrease the expression of anti-apoptotic proteins (e.g. Bcl-xL, Bcl2, etc.) but increase the expression of pro-apoptotic proteins (e.g. Bax, caspases, etc.), so as to sensitize resistant cancers to chemotherapy.32,

55-56

Here, ZnO NPs could inhibit the Dox-induced CD44

upregulation (Figure 3B) and the effect was comparable to that of the reported anti-CD44 siRNAs,54 indicating their potential as a CD44 inhibitor. ZnO NPs seemed to be more efficient in downregulation of CD44 in the MDR cancer cells than in the drug-sensitive cells (Figure 3C), which might account for their higher efficacy in the MDR cancer cells



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(Figure 2B vs. 2A). In the cell spheroids, due to the increased resistance, the ZnO NPs at 20 µg/mL couldn’t significantly kill cancer cells (Figure 2E-F). However, they effectively downregulated the CD44 level (stemness) of cancer cells/CSCs, leading to remarkable spheroid dissociation (expansion and cell shedding) (Figure 2C-D and S7). The reduction of CD44 expression sensitized the CD44+ cancer cells to Dox, resulting in the substantial cell death in the ZnO/Dox- treated spheroids (Figure 2E-F).

Figure 3. CD44 downregulation determined by flow cytometry. (A) Cell surface levels of CD44 in MDA-MB-231 and NCI/ADR-RES cells. (B) Comparison of CD44 levels after 24 h cell incubation with Dox, ZnO NPs, or ZnO/Dox. (C) Dose-dependent CD44 downregulation by ZnO NPs after 24 h cell incubation.


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2.3.2. Inhibition of Cancer Cell Adhesion and Migration It was known that the adhesion and migration of the CD44+ cancer cells were highly dependent on the interaction between CD44 and hyaluronic acid, a major component of the extracellular matrix.57 Blocking CD44 by anti-CD44 antibody or downregulation of CD44 by siRNA resulted in the inhibition of cell adhesion and migration. Here, we performed the cell adhesion assay57 and scratch wound healing assay58 to evaluate the influence of ZnO NPs on cell adhesion and migration. As demonstrated in Figure 4A and S8, the untreated CD44+ cells (NCI/ADR-RES) bound with the HA coating very well, while the ZnO NPs pretreatment downregulated the CD44 on the cell surface (Figure 3), leading to the substantial reduction of the number of the bound cells. The ZnO-induced inhibition of cell adhesion was dose-dependent. For cell migration study, the scratch was made in the NCI/ADR-RES cell monolayer in the presence of Dox, ZnO, or ZnO/Dox at subtoxic doses (Figure 4B and S9). The results indicated that Dox failed to stop the cell migration towards the scratch, as evidenced by a significantly narrowed gap (~53%) similar to that of the untreated wound (~49%). In contrast, ZnO NPs and ZnO/Dox effectively hindered the cell migration and almost completely inhibited the wound healing (~90%), confirming that CD44 played a key role in the process and its downregulation slowed down cancer cell migration.57 Therefore, ZnO NPs might be able to interfere with the CD44-mediated tumor metastasis via inhibition of cancer cell adhesion and migration. 2.3.3. Inhibition of Tumor Cell Spheroid Formation CD44 is also highly associated with tumorigenesis. CD44+ cancer cells showed high spheroid colony formation capability compared to CD44- cells.51 Blocking CD44 by antiCD44 antibody or Rho-associated kinase (ROCK) inhibitor prevented the multicellular spheroid formation and decreased the stemness gene expression.59 To study the impact of the treatments on the tumor spheroid formation, the NCI/ADRRES cells were seeded in the presence of Dox, ZnO NPs, or ZnO/Dox and incubated for 48 h. We found that Dox at all concentrations didn’t influence the spheroid formation, however, the treated spheroids shrank and the cell density was increased at high Dox concentration, as evidenced by a smaller and darker spheroid. In contrast, ZnO NPs and



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ZnO/Dox significantly inhibited the formation of spheroids. At low ZnO concentration, the spheroids were formed with several separate small cell aggregates, while at high ZnO concentration, the compact spheroid could not be formed, as evidenced by a big loose cell aggregate with several scattered small cell aggregates (Figure 4C and S10). The data indicated that substantial CD44 downregulation by ZnO NPs significantly interfered the cell-cell interaction/adhesion and hindered the formation of a tight and compact spheroid. The data also suggested that the remaining CD44 or other adhesion molecules might be still functional but they could only help cancer cells to form loose aggregates rather than regular spheroids.60 In the presence of Dox particularly at ZnO/Dox (20/4 µg/mL), the effect of ZnO NPs was dramatically augmented. The loose cell aggregates seemed to undergo further disintegration and the 3D structure was gone. Under this condition, the synergistic anticancer activity of Dox and ZnO NPs could be enhanced (Figure 2) due to the increased drug uptake in the loose 2D-like structure (Figure 1D-E). All results suggested that ZnO NPs could effectively downregulate CD44, so as to inhibit cancer cell adhesion, migration and tumorigenesis, facilitate its ROS-induced cytotoxicity, and sensitize the Dox treatment. Delivery of CD44-stimulating drugs/agents by ZnO NPs might be a promising combination strategy to decrease drug resistance and improve drug efficacy.


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Figure 4. (A) Cancer cell adhesion on the hyaluronic acid (HA) coating. The NCI/ADRRES cells were incubated with ZnO NPs for 24 h, followed by incubating with the HA coating for 30 min. (B) Wound healing assay. The NCI/ADR-RES cells were incubated with Dox, ZnO NPs, or ZnO/Dox for 24 h. After generation of the scratch, the cells were washed and incubated in the serum-free medium for 24 h. (C) Cancer cell spheroid formation. The NCI/ADR-RES were seeded in the 1.5% agarose-coated plate and incubated with Dox, ZnO NPs, or ZnO/Dox for 48 h.

2.4. ZnO NPs as a Drug Immunological Adjuvant Although ZnO NPs have been known to stimulate immune response for decades,18-26 their immunological properties against cancer have not been fully explored. Here, we studied if ZnO NPs could polarize macrophages, the front line of host immune defense, to exert anticancer activity.



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2.4.1. Phagocytosis and Cytotoxicity of ZnO NPs We have demonstrated that ZnO NPs could be endocytosed by various cancer cells (Figure 1D-E and S1). Here, the phagocytosis of ZnO NPs was investigated using Dox as a fluorescence indicator. Like the results in cancer cells, ZnO/Dox could be internalized by the murine macrophages, RAW264.7 cells, and showed the similar uptake efficiency as free Dox after 1 h incubation (Figure S11). After 24 h cell incubation, Dox showed much higher cytotoxicity compared to ZnO/Dox in RAW264.7 cells, while ZnO/Dox showed the similarly low cytotoxicity as ZnO NPs (Figure S12). It seemed like that loading of Dox to ZnO NPs decreased the Dox’s toxicity against the RAW264.7 cells. Because macrophages are a crucial player against pathogens, this “protective” effect of ZnO NPs might be beneficial to the macrophage-oriented anticancer immunotherapy. 2.4.2. Polarization of Macrophages towards M1 Phenotype It is known that macrophages undergoing M1 (classically activated) polarization exhibit anticancer activity, via the phagocytic cell clearance, production of pro-inflammatory cytokines/chemokines (IL-12, IL-6, TNF-α, etc.), and expression of costimulatory markers (CD80, CD86, MHCII, etc.) that can activate T cells. In contrast, the M2 (alternatively activated)-like macrophages as characterized by the upregulation of IL-10, CD206, CD163, etc. fail to clear cancer cells and inhibit T cell immune responses.61 In the solid tumor, the increased infiltration and recruitment of macrophages are usually observed, as an indicator of poor prognosis. Unfortunately, the majority of these tumor-associated macrophages (TAMs) are M2 phenotype in malignant tumors, which promotes tumor angiogenesis, proliferation, invasion and metastasis, and protects the tumor from the damage induced by adaptive immunosurveillance.3 Here, the pro-inflammatory cytokines, TNF-α and IL-6, were measured by ELISA after incubation of RAW264.7 cells with the formulations for 24 h (Figure 5A). We found that ZnO NPs alone just slightly increased the TNF-α level, while ZnO/Dox significantly increased TNF-α level, particularly at 10 µg/mL ZnO and 2 µg/mL Dox. It has been reported that certain chemotherapeutics, such as vinblastine, paclitaxel, mitomycin C, and Dox, could induce the anticancer immune response at non-cytotoxic concentrations via the increase of antigen production.62 To decrease the influence of the Dox’s toxicity on the macrophages (Figure S12), in this experiment, we seeded a higher number of RAW264.7


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cells (2×105 cells/well in a 12-well plate). However, at high Dox concentrations, free Dox still induced modest cell apoptosis, resulting in the reduction of the TNF-α production. For IL-6, all treatments could increase the IL-6 production. Among them, ZnO/Dox had the highest effect at 10 µg/mL ZnO and 2 µg/mL Dox. Similarly, the Dox’s toxicity decreased the IL-6 production at high Dox concentration. The cytokine production induced by ZnO or ZnO/Dox was dose-dependent. The data indicated that the combined use of Dox and ZnO NPs could synergistically boost the pro-inflammatory cytokine production. Furthermore, the costimulatory markers on macrophages were analyzed by flow cytometry (Figure 5B). ZnO or Dox alone only slightly increased the expression of CD80 and CD86, while ZnO/Dox increased the levels of CD80 for 2.5 folds and CD86 for 2 folds, respectively. Both ZnO and ZnO/Dox could also increase the MHCII level dramatically rather than Dox. The data suggested that ZnO NPs might be an immunomodulator or adjuvant which would be able to activate T cell-mediated immune responses. All these data implied that ZnO or ZnO/Dox-incubated RAW264.7 cells most likely underwent a M1-like polarization.



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Figure 5. Polarization of the macrophages. (A) Production of the pro-inflammatory cytokines, TNF-α and IL-6, was determined by ELISA after 24 h incubation with Dox, ZnO NPs, or ZnO/Dox at various concentrations. (B) Expression of the costimulatory factors, CD80, CD86, and MHCII, was determined by flow cytometry after 24 h incubation with Dox (2 µg/mL), ZnO NPs (10 µg/mL), or ZnO/Dox (10/2 µg/mL). The RAW264.7 cells were seeded at 2×105 cells/well in 12-well plates. Data are presented as the mean ± SD. *P< 0.05, **P< 0.01, and ***P< 0.001. All P-values shown are vs the untreated group.

2.4.3. Killing Monolayer Cancer Cells by Macrophage-conditioned Media Current studies on ZnO NPs’ anticancer activity mainly focused on their direct cytotoxicity against cancer cells.16-17 The immunological properties of ZnO NPs suggested that ZnO or ZnO/Dox-activated macrophages might be able to exert immunogenic modulation on cancer cells. Here, to explore the combinatory effects between ZnO and Dox regarding their immunological properties, we measured cancer cell viability in the presence of the macrophage-conditioned medium (CM). The CM was prepared according to Figure 6B. The medium from the untreated (“immature”) macrophages [CM (untreated)] could slightly inhibit the cancer cell proliferation (82% viability) compared to the cancer cells in the normal cell culture medium (NM), while the medium from the drug-activated (“mature”) macrophages could induce significant cell apoptosis/death and the effects were dose-dependent, especially for ZnO and ZnO/Dox (Figure 6B). The medium from the Doxtreated macrophages showed mild cytotoxicity against cancer cells and the highest effect was observed at 0.5 µg/mL (58% viability). Low concentration of Dox (0.1 µg/mL) might not be sufficient to polarize the macrophages, whereas high Dox concentrations (1 and 2 µg/mL) substantially killed the macrophages (Figure S12), leading to the reduction of cytokine production (Figure 5A). In contrast, ZnO/Dox had a broader safe activation dose range on the macrophages, which might be benefited from the “protective” effect of ZnO NPs (Figure S12). The medium from the ZnO/Dox-treated macrophages could effectively kill cancer cells, as evidenced by 42% viability at 10 µg/mL ZnO and 2 µg/mL Dox. The data suggested that the ZnO/Dox-activated macrophages were able to produce sufficient pro-inflammatory cytokines to inhibit cancer proliferation. The results also confirmed that


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Dox-induced immune response was mild and it might be improved if combined with an immunomodulatory,63 such as ZnO NPs. 2.4.4. Enhancement of Cytotoxicity in Cancer Spheroids by Macrophage-conditioned Media The effects of the macrophage CM were further studied on the NCI/ADR-RES cell spheroids. First, we compared the anticancer effect of the drug treatments in the absence and presence of the CM (Figure 6C). In line with the results obtained in cancer cell monolayers, the medium of untreated macrophage [CM (untreated)] just showed a slight cytotoxicity (90% viability compared to the NM). The medium from the Dox-treated macrophages failed to help Dox in killing cancer cells in the spheroids, as evidenced by the similar cytotoxicity between Dox + NM and Dox + CM (Dox), indicating that the tumor inhibition capability of Dox was mainly due to its cytotoxicity rather than immunogenicity.62 Interestingly, unlike in the cell monolayers, ZnO NPs didn’t cause significant cell death in the spheroids even in the presence of the conditioned medium from the ZnO-treated macrophages [~96% in ZnO + CM (ZnO) vs. ~100% in ZnO +NM]. In contrast, ZnO/Dox alone substantially killed cancer cells (~60% in ZnO/Dox + NM) and the addition of the conditioned medium obtained from the ZnO/Dox-treated macrophages could further bring down the cell viability to ~42% [ZnO/Dox + CM (ZnO/Dox)]. Then, we analyzed the influence of the CM obtained from different concentrations of ZnO/Dox-treated macrophages on the cell spheroids since ZnO/Dox showed the highest cytotoxicity in cancer cells (Figure 2) as well as induced the highest pro-inflammatory cytokine production in macrophages (Figure 5A). Although the CM from the ZnO/Doxtreated macrophages alone could substantially kill cancer cells in the cell monolayers (Figure 6B), it hardly exerted cytotoxicity on the spheroids (Figure 6D), most likely due to the increased drug resistance in the 3D cell structures. In contrast, the cytotoxicity directly induced by ZnO/Dox was more significant in the cell spheroids (Figure 6C). However, the CM could further promote the ZnO/Dox’s anticancer activity and its effect on the ZnO/Dox efficacy was dose-dependent (Figure 6D). All these data suggested that the combined use of ZnO NPs and Dox could induce both direct cytotoxicity and anticancer immune response via activation of macrophages. These two aspects could work cooperatively to enhance the anticancer activity of ZnO NPs and



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Dox. We might also speculate that, in the in vivo condition, in addition to the macrophageexerted anticancer immune response, the enhanced expression of costimulatory factors would activate T cells to exert more profound anticancer effects.

Figure 6. (A) The diagram of the preparation of various conditioned media (CM). (B) Killing monolayer NCI/ADR-RES cells by macrophage-conditioned media induced by Dox, ZnO NPs, and ZnO/Dox. NM, normal cell culture medium. (C) Effect of NM and CM on the NCI/ADR-RES cell spheroids in the presence of Dox, ZnO NPs, or ZnO/Dox. The medium of the RAW264.7 cells was collected after incubation with Dox (2 µg/mL), ZnO (10 µg/mL), or ZnO/Dox (10/2 µg/mL) for 24 h. Then, the medium was added to the spheroids and incubated in the presence of Dox (4 µg/mL), ZnO (20 µg/mL), and ZnO/Dox (20 /4 µg/mL), respectively for 48 h. (D) Dose-dependent effect of CM (ZnO/Dox, 5/1 µg/mL or 10/2 µg/mL) on the NCI/ADR-RES cell spheroids in the presence of ZnO/Dox (20/4 µg/mL). Data are presented as the mean ± SD. *P< 0.05, **P< 0.01, and ***P< 0.001.


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Figure 7. The diagram showing the proposed mechanism of ZnO NPs that can be served as a potential multi-target and multi-functional anticancer nanomedicine.

3. Conclusions In this study, for the first time, we demonstrated that (i) ZnO NPs could work as a pHdependent drug nanocarrier and showed enhanced cellular uptake and tumor spheroid penetration; (ii) ZnO NPs could kill cancer cells and showed the synergistic anticancer activity with the loaded Dox; (iii) ZnO NPs could effectively downregulate CD44 expression of cancer (stem-like) cells to inhibit cell adhesion, migration, and tumorigenesis, as well as sensitize drug treatment; and (iv) ZnO NPs could polarize macrophages towards the M1-like phenotype and boost Dox’s immunogenicity and anticancer activity. Based on current results, ZnO NPs might have great potential to be a multi-target and multi-functional nanocarrier and nanomedicine for anticancer research (Figure 7). 4. Experimental Section 4.1 Materials Zinc oxide nanoparticles (ZnO NPs, 99+%, 10-30 nm) were purchased from US Research Nanomaterials, Inc (Houston, TX, USA). Doxorubicin hydrochloride salt (Dox, >99%) was purchased from LC Laboratories (Woburn, MA, USA). 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium

bromide

(MTT),

2´,7´-dichlorodihydrofluorescein


diacetate


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(H2DCFDA), and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (St Louis, MO, USA). Dulbecco’s modified Eagle’s medium (DMEM), penicillin streptomycin solution 100× (PS), Hoechst 33258, and trypsin-EDTA were from Invitrogen Corp. (Carlsbad, CA, USA). Fetal bovine serum (FBS) and Hank’s balanced salt solution (HBSS) were from Mediatech (Manassas, VA, USA). The human breast cancer (MDA-MB-231), human cervical cancer (HeLa), MDR human ovarian cancer (NCI/ADR-RES), MDR human uterine sarcoma (MES-SA/Dx5), and murine macrophage (RAW264.7) cells were grown in DMEM supplemented with 100 U/mL penicillin, 100 µg/mL streptomycin, and 10% FBS at 37 °C in a 5% CO2. To minimize the risk of contamination during the experiments, ZnO NPs were stored in anhydrous ethanol as the stock suspension and Dox was dissolved in water (sterilized by autoclave) as the stock solution. 4.2. Preparation of Dox-loaded ZnO NPs (ZnO/Dox) After removal of ethanol, ZnO NPs (22.5 mg) were dispersed in the Dox aqueous solution (5 mg/mL, 3 mL). After stirring for 24 h in the dark, the mixture was centrifuged at 10,000 rpm and washed with water (5 mL) for three times. The product (ZnO/Dox) was freeze-dried in a tabletop lyophilizer overnight. The unentrapped Dox in the supernatant was determined by a UV-vis spectrophotometer at the wavelength of 480 nm. The Dox concentration (C) was calculated according to an obtained standard curve of Dox (Abs = 0.01823C - 0.01116, r = 0.99987). The drug loading efficiency (DL) and drug encapsulation efficiency (EE) were calculated as follows:

DL =

weight of Dox in ZnO/Dox × 100% weight of ZnO/Dox

EE =

weight of Dox in ZnO/Dox × 100% weight of Dox fed initially

4.3. Morphology, Particle Size, and Zeta Potential Measurement The samples (0.5 mg) were dispersed in 1 mL of water or DMEM complete media (DMEM containing 100 U/mL penicillin, 100 µg/mL streptomycin, and 10% FBS). After sonication for 10 min, the particle size of ZnO NPs and ZnO/Dox was measured by dynamic light scattering (DLS) on a NanoBrook 90Plus PALS (Brookhaven Instruments) at


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25 ºC. The zeta potential of ZnO NPs and ZnO/Dox was determined in water by the same instrument. For morphology examination, 5 µl of the nanoparticle suspension was placed on a 400-mesh copper-carbon grid and dried at room temperature. Then, samples were elucidated by the transmission electron microscopy (TEM, FEI Tecnai Osiris 200kV, Hillsboro, USA). 4.4. Drug Release Two buffer solutions were prepared for the experiment, PBS (pH 7.4) and acetic acid/sodium acetate buffer (pH 5.0). Briefly, ZnO/Dox (2 mg) was dispersed in 0.6 mL of pH 7.4 or pH 5.0 buffer solutions and immediately dialyzed (MWCO: 12,000-14,000 Da) in 80 mL of pH 7.4 and 5.0 buffer solutions, respectively, at 37 °C for 24 h. The Dox in the outside medium was determined by microplate reader at λ ex = 480 nm and λ em = 590 nm at the indicated time intervals. 4.5. Cellular Uptake The cellular uptake of Dox or ZnO/Dox was investigated by fluorescence-activated cell sorting (FACS). In brief, Dox-sensitive cancer cells (MDA-MB-231 and HeLa), MDR cancer cells (NCI/ADR-RES and MES/SA-Dx5), or murine macrophages (RAW264.7 cells) were seeded into 12-well plates at 2×105 cells/well and incubated overnight before experiments. Then, the cells were washed and incubated with Dox or ZnO/Dox at 8 µg/mL Dox for 1 h in the cell culture medium. For FACS analysis, the cells were trypsinized and collected by centrifugation at 2,000 rpm for 2 min. After washing with ice-cold PBS, the cells were re-suspended in 500 µL of PBS and immediately applied on a BD Accuri™ C6 flow cytometer. Totally, a number of 2.0×104 cells were recorded. The cells were gated using the forward vs. side scatter to exclude the dead cells and cell debris. The untreated cells were served as the negative control. The mean fluorescence intensity (MFI) was analyzed by the CFlowPlus Software. 4.6. Intracellular Drug Distribution MDA-MB-231 or NCI/ADR-RES cells were seeded on glass coverslips in a 35×10 mm dish at 2.5×105 cells/dish and incubated overnight before experiments. The cells were treated with Dox or ZnO/Dox at an equal Dox concentration (8 µg/mL) for 1 h. The coverslips were washed 3 times with HBSS, and then the cells were fixed with 4% paraformaldehyde for 15 min at room temperature. After washing with HBSS, the cells



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were stained with Hoechst 33258 (4 µg/mL) for 15 min in the dark. The coverslips were mounted onto microscope slides and analyzed by a Nikon Eclipse Ti confocal microscope system at 200×. 4.7. Establishment of Cancer Cell Spheroids The cancer cell spheroids were established by a widely used liquid overlay method.42 Briefly, MDA-MB-231 or NCI/ADR-RES cells were seeded in the agarose (dissolved in DMEM, 1.5%, v/v)-coated 96-well plates at 1×104 cells/well and incubated at 37 °C for spheroid formation. The formed spheroids were identified by their size and shape. The 2-5 day spheroids were used in the penetration and cytotoxicity study. 4.8. Drug Penetration through Cancer Cell Spheroids Dox or ZnO/Dox was incubated with the spheroids at an equal Dox concentration (8 µg/mL) for 1 h and 4 h and analyzed by confocal microscopy at 100×. Z-stack images were obtained at a fixed interval as shown in the results. Fluorescence intensity of the selected images were analyzed using NIS-Elements AR software (version 4.20.01). 4.9. Cytotoxicity on Cancer Cell Monolayers Cancer cells or RAW264.7 cells were seeded into 96-well plates at 6×103 cells/well and incubated at 37 °C overnight before experiments. Then, Dox, ZnO NPs, or ZnO/Dox was incubated with cells at various Dox concentrations (0.1-100 µg/mL) for 24 h, followed by the MTT assay. In brief, the wells were added into MTT (20 µL, 5 mg/mL, dissolved in PBS). After incubating for 4 h, the medium was removed and DMSO (150 µL) was added. The absorbance was determined at 570 nm with a reference wavelength of 630 nm by a microplate reader. 4.10. Cytotoxicity on Cancer Cell Spheroids Briefly, Dox, ZnO NPs, or ZnO/Dox was incubated with the cell spheroids at an equal Dox concentration (8 µg/mL) for 48 h. The images (morphology) of the spheroids were recorded at 0, 18, and 48 h by microscopy at 40×. The cell viability in the 5-day spheroids after 48 h incubation was determined by the CellTiter-Blue® Cell Viability Assay (Promega, USA). In brief, the regent (40 µL) was added to each well and incubated for 24 h. The fluorescence intensity was recorded at λ

ex

= 560 nm and λ

em

= 590 nm on a microplate

reader. 4.11. Intracellular Reactive Oxygen Species (ROS) Measurement


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The ROS production was determined using 2´,7´-dichlorodihydrofluorescein diacetate (H2DCFDA) according to the previous report.16 In brief, NCI/ADR-RES cells were seeded in 12-well plates at 1.5×105 cells/well and incubated at 37 °C overnight before experiments. Then, ZnO NPs (10, 20, or 30 µg/mL), Dox (4 µg/mL), or ZnO/Dox (20/4 µg/mL) were incubated with the cells for 24 h. To detect the ROS, the cells were washed with HBSS and incubated with H2DCFDA (10 µM) for 30 min at 37 °C, followed by analysis by a Nikon Eclipse Ti confocal microscope system at 100×. 4.12. Caspase 3/7 Measurement NCI/ADR-RES cells were seeded into 96-well plates at 6×103 cells/well and incubated at 37 °C overnight before experiments. Then, the cells were incubated with ZnO NPs (10, 20, or 30 µg/mL), Dox (4 µg/mL), and ZnO/Dox (20/4 µg/mL) for 24 h. The caspase 3/7 activity was measured by the Apo-ONE® Homogeneous Caspase-3/7 Assay. Briefly, the reagent (100 µL) was added to the cells and incubated at room temperature for 40 min. The fluorescence intensity was measured on a microplate reader at λ ex = 485 nm and λ em = 530 nm. 4.13. CD44 Expression MDA-MB-231 or NCI/ADR-RES cells were seeded into 12-well plates at 2×105 cells/well and incubated at 37 °C overnight before experiments. The cells were incubated with ZnO NPs (10, 20, or 30 µg/mL), Dox (4 µg/mL), or ZnO/Dox (20/4 µg/mL) for 24 h. The cells were washed twice by HBSS and stained with PE-labeled anti-human CD44 antibody (eBioscience, CA) at 4 °C in the dark for 1 h. Then, the cells were washed twice with PBS and analyzed by the FACS on a BD Accuri™ C6 flow cytometer. For the FACS, the same conditions as those in “4.5. Cellular Uptake” were used. 4.14. Cell Adhesion Assay The binding of cancer cells with hyaluronic acid (HA) was evaluated by cell adhesion assay.57 In brief, the 96-well plates were pre-coated with HA (16 mg/mL) at 37 °C for 24 h. The coating was air-dried overnight, and then gently washed by HBSS. The NCI/ADR-RES cells were treated with ZnO NPs (10, 20, or 30 µg/mL) for 24 h and then harvested by trypsinization and centrifugation. After washing with DMEM twice, the cells were resuspended in DMEM at 1×105 cells/mL and incubated in the HA-coated plates at 37 °C for



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30 min. The non-adherent cells were removed by washing with HBSS for three times. The adherent cells were counted under a microscope. 4.15. Wound Healing Assay NCI/ADR-RES cells were seeded into 12-well plates at 2×105 cells/well and incubated at 37 °C overnight before experiments. The cells were incubated with ZnO NPs (10 µg/mL), Dox (2 µg/mL), and ZnO/Dox (10/2 µg/mL) respectively for 24 h. Then, the cells were washed twice with HBSS and cultured in serum-free medium. The scratch on the cell monolayer was generated using a 200 µL pipette tip, followed by washing with HBSS to remove the cell debris. The cells were incubated in the serum free medium for 24 h. The pictures were taken at 0 h and 24 h by a Nikon Eclipse Ti fluorescence microscope at 40×. The scratch width at 24 h was measured and the cell migration distance was estimated by comparing with the initial scratch width at 0 h. 4.16. Inhibition of Cancer Cell Spheroid Formation The NCI/ADR-RES cells (1×104 cells/well) were seeded to the agarose-coated 96-well plates in the presence of ZnO NPs (5, 10, or 20 µg/mL), Dox (1, 2, or 4 µg/mL), and ZnO/Dox (5/1, 10/2, or 20/4 µg/mL), respectively and incubated for up to 48 h. The formation of spheroids was observed by the Nikon Eclipse Ti fluorescence microscope system at 40×. 4.17. Determination of Pro-inflammatory Cytokines The pro-inflammatory cytokines from the RAW264.7 cells were determined by ELISA. In brief, the cells were seeded into 12-well plates at 2×105 cells/well and incubated at 37 °C overnight before experiments. The cells were incubated with ZnO NPs (2.5, 5, or 10 µg/mL), Dox (0.5, 1, or 2 µg/mL), and ZnO/Dox (2.5/0.5, 5/1, or 10/2 µg/mL) for 24 h, respectively. Then, the cytokines (TNF-α and IL-6) in the cell medium were analyzed by the ELISA kits (eBioscience, CA). 4.18. Determination of Costimulatory Markers The macrophage surface markers, CD80, CD86, and MHC-II, were analyzed by flow cytometry. Briefly, the RAW264.7 cells were seeded into 12-well plates at 2×105 cells/well and incubated at 37 °C overnight before experiments. The cells were incubated with ZnO NPs (10 µg/mL), Dox (2 µg/mL), and ZnO/Dox (10/2 µg/mL) for 24 h, respectively. Then, the cells were collected and stained with the FITC-labeled (anti-CD80, anti-CD86 or anti-


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MHC-II) antibodies (eBioscience, CA) according to the manufacturers’ protocols, followed by FACS analysis using the same conditions as those in “4.5. Cellular Uptake”. 4.19. Preparation of the Macrophage-conditioned Media (CM) The RAW264.7 cells were seeded into 12-well plates at 2×105 cells/well and incubated at 37 °C overnight before experiments. The cells were incubated with ZnO NPs (0.5, 2.5, 5, or 10 µg/mL), Dox (0.1, 0.5, 1, or 2 µg/mL), and ZnO/Dox (0.5/0.1, 2.5/0.5, 5/1, or 10/2 µg/mL) for 24 h, respectively. Then, the cell medium was centrifuged at 7000 rpm for 6 min to remove the cell debris and other particles. The supernatant, i.e. CM, was collected and kept at -80°C. 4.20. Impact of CM on Cancer Cell Monolayers The NCI/ADR-RES cells were seeded into 96-well plates at 6×103 cells/well and incubated at 37 °C overnight before experiments. Then, the medium was replaced by CM and the cells were incubated for 24 h, followed by the MTT assay. 4.21. Impact of CM on Cancer Cell Spheroids ZnO NPs (20 µg/mL) were suspended in the CM obtained from the ZnO (10 µg/mL)treated macrophages. Dox (4 µg/mL) was dissolved in the CM obtained from the Dox (2 µg/mL)-treated macrophages. ZnO/Dox (20/4 µg/mL) was suspended in the CM obtained from the ZnO/Dox (10/2 µg/mL)-treated macrophages. After 48 h incubation with the 2-day NCI/ADR-RES spheroids, the cell viability in the spheroids was determined by the CellTiter-Blue® Cell Viability Assay. 4.21. Statistical Analysis Data were presented as mean ± standard deviation (SD). Statistical analysis was performed by a one-way ANOVA analysis. P < 0.05 was considered to be statistically significant.

Associated Content Supporting Information Photographic images and TEM micrographs, Cellular uptake in HeLa and MES-SA/Dx5 cells, Drug penetration in cancer cell spheroids, Cytotoxicity in HeLa and MES-SA/Dx5 cells, Intracellular levels of ROS, Intracellular levels of caspase 3/7, Morphology of cancer cell spheroids, Inhibition of cancer cell adhesion, Inhibition of cancer cell migration, Inhibition of



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cancer cell spheroid formation, Cellular uptake in RAW264.7 cells, Cytotoxicity in RAW264.7 cells, IC50 in various cancer cells.

Author Contribution Lin Zhu conceived the idea and supervised the project. Jiao Wang performed most experiments and analyzed data. Jung Seok Lee and Dongin Kim performed the TEM. Lin Zhu and Jiao Wang wrote the paper.

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Graphical Abstract


Exploration of Zinc Oxide Nanoparticles as a Multitarget and

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