Accepted Manuscript Title: Zinc oxide nanoparticles: a promising nanomaterial for biomedical applications Authors: Pawan K. Mishra, Harshita Mishra, Adam Ekielski, Sushama Talegaonkar, Bhuvaneshwar Vaidya PII: DOI: Reference:

S1359-6446(17)30077-6 http://dx.doi.org/10.1016/j.drudis.2017.08.006 DRUDIS 2069

To appear in: Please cite this article as: Mishra, Pawan K., Mishra, Harshita, Ekielski, Adam, Talegaonkar, Sushama, Vaidya, Bhuvaneshwar, Zinc oxide nanoparticles: a promising nanomaterial for biomedical applications.Drug Discovery Today http://dx.doi.org/10.1016/j.drudis.2017.08.006 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Zinc oxide nanoparticles: a promising nanomaterial for biomedical applications Pawan K. Mishra1,5, Harshita Mishra2,5, Adam Ekielski3, Sushama Talegaonkar2, and Bhuvaneshwar Vaidya4 1Department

of Wood Science, Mendel University in Brno, Brno, Czech Republic of Pharmaceutics, Jamia Hamdard, New Delhi, India 3Department of Production Management and Engineering, Warsaw University of Life Sciences, Faculty of Production Engineering, Warsaw, Poland 4School of Pharmacy, Texas Tech University Health Science Center, Amarillo, TX, USA 5These authors contributed equally. Corresponding author: Vaidya, B. ([email protected], [email protected]) 2Department

Highlights: 

ZnO nanoparticles are an emerging novel nanomaterial.



It exhibits effects by inducing ROS generation and causing apoptosis

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It shows synergistic and enhanced therapeutic efficacy when combined with other therapeutic agents.

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ZnO nanoparticles have also been explored as drug carriers for anti-cancer drugs.

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Toxicity concern needs to be explored before clinical applications.

Teaser: Zinc oxide nanoparticles: how useful will they be in the biomedical sciences? Zinc oxide (ZnO) nanoparticles (NPs) are a promising platform for use in biomedical research, especially given their anticancer and antimicrobial activities. These activities are associated with the ability of ZnO NPs to generate reactive oxygen species (ROS) and induce apoptosis. In addition, ZnO NPs have been successfully exploited as drug carriers for loading and transporting drugs to target sites, thereby reducing unwanted toxicity and off-target effects, and resulting in amplified synergistic effects. Here, we discuss the synthesis and biomedical applications of ZnO NPs.

Keywords: zinc oxide nanoparticles; drug delivery; nanocarriers; biomedical applications; anti-cancer; anti-bacterial; wound dressing

Introduction

Over the past few decades, nanotechnology has emerged as a promising technique for various biomedical applications. Among the nanomaterials currently available, metal NPs have been explored as standalone biomedical agents as well as novel carriers for the delivery of therapeutic agents for a variety of disorders. One member of the metal oxide NP family, ZnO NPs, has excellent ultraviolet (UV)-absorbing properties and transparency for visible light, make such NPs excellent sunscreen agents [1,2]. Other properties, such as their antibacterial and anticancer activities, have also been explored, which result from their ability to induce the generation of ROS [3]. In addition to their inherent biomedical properties, ZnO NPs are also excellent drug carrier

systems. Moreover, the US Food and Drug Administration (FDA) has recognized bulk ZnO as a generally recognized as safe (GRAS) substance, and ZnO NPs larger than 100 nm are considered to be relatively biocompatible, which supports their use for drug delivery [4]. Furthermore, the inherent anticancer and antimicrobial activities of ZnO NPs render them superior to other commonly used drug carriers, such as lipid and polymeric NPs. Although other metal NPs, such as iron oxide NPs, also have anticancer activity, they have not been reported to show well-defined antimicrobial or UV-absorbing actions. In addition, ZnO is comparatively inexpensive, biocompatible, and relatively less toxic compared with other metal oxide NPs, which further supports its application potential [5,6]. In addition, Sahdev et al. [7] also reported that zinc does not interact with the majority of pharmaceutically active molecules available. Mechanism of action of ZnO NPs

ZnO is a conventional wide band-gap semiconductor. Its unique properties make it appropriate for various biomedical applications, including anticancer, antibacterial, and antifungal uses [8]. Most of the biomedical applications of ZnO NPs stem from their ability to generate ROS, which results in cell death if the antioxidative capacity of the cell is suppressed [9]. The semiconductor properties of ZnO affect their ability to generate ROS. The electrons (e−) in semiconductors contain energies within certain bands and the band gap, a void region extending from top of the filled valence band to the bottom of vacant conduction band that measures approximately 3.3 eV for crystalline ZnO. UV rays contain sufficient energy and promote e− to the conduction band, leaving behind holes (h+). e− and h+ migrate to the surface of the NPs and react with oxygen and hydroxyl ions, respectively. This leads to the formation of superoxide and hydroxyl radicals [8,10]. In ZnO NPs, a large number of valence-band h+ and/or conduction-band e− are present even in the absence of UV light because of the crystal defects of nanosized materials. The various ROS produced then trigger redox-cycling cascades in the cells, causing irreparable oxidative damage [11]. Necrosis and apoptosis are other mechanisms for the biomedical activity of ZnO NPs [12,13]. DNA damage resulting from ROS induces apoptotic pathways by causing the release of apoptogenic factors from the mitochondrial intermembrane space [14]. This leads to the formation of apoptosomes, which in turn activate executioner enzymes [15]; cleavage of their specific substrates leads to apoptosis and cell death. An alternative mechanism has also been proposed, whereby ZnO induces apoptosis by its rapid dissolution within the acidic lysosomes of the macrophages after uptake in their particulate agglomerated form [12]. Synthesis of ZnO NPs

Several methods have been reported for the preparation of ZnO NPs. The primary focus of each of the methods is the development of stable and uniform nanosized particles. Precipitation method

The precipitation method involves a reaction between a zinc precursor and a precipitating reagent. Typically, a solution of precipitating agent (e.g., sodium hydroxide, ammonium hydroxide, urea, etc.) is added drop wise to the aqueous solution of a zinc precursor (e.g., zinc nitrate, zinc sulfate, etc.). Mixing of these solutions results in the formation of an intermediate product, which ultimately converts to ZnO after calcination at high temperature [16]. Kumar et al. [17] synthesized ZnO NPs of 64 nm using the precipitation method by the reaction of zinc sulfate and sodium hydroxide in a molar ratio of 1:2. The NPs obtained were very pure, as revealed by X-ray diffraction analysis; in addition, the crystallinity of the NPs increased as the calcination temperature increased. Wet chemical synthesis

The wet chemical synthesis of ZnO NPs is a modification of the precipitation method. In this approach, an additive is used to stabilize the NPs formed. The method utilizes the precipitation reaction between zinc nitrate and sodium hydroxide [18]. Briefly, an aqueous solution of starch is mixed with a zinc nitrate solution, whereby sodium hydroxide solution is added drop wise until the solution attains a pH of 12. The precipitate obtained by this reaction (zinc hydroxide) is then converted into ZnO by calcining at 80ºC (Equation 1). Zn2+ + 2OH– → Zn(OH)2 [1]. Addition of excess hydroxyl ions (from sodium hydroxide) should be avoided because these solubilize zinc hydroxide and convert it to zincate ions [Zn(OH)42–], which are soluble in aqueous medium (Equation 2). Zn(OH)2 + 2OH– → Zn(OH)42– [2]. Starch, which is used as a stabilizing agent, is adsorbed at the surface of NPs, providing stability. The mechanism of stabilization involves either increasing the viscosity of the solution or forming a complex with metal ions via the hydroxyl groups [19]. Sharma et al. [11] synthesized ZnO NPs using the wet chemical method and obtained particle sizes in the range of 400 nm. The NPs synthesized were found to have significant anticancer activity and were also demonstrated to be appropriate drug carriers for anticancer agents. Solid-state pyrolytic method

The solid-state pyrolytic method, reported by Wang et al. [20], is a simple, rapid, and cost-effective method. Typical solid-state pyrolytic synthesis involves mixing zinc acetate and sodium bicarbonate. The mixture is then pyrolyzed. Zinc acetate converts to ZnO NPs, while sodium bicarbonate converts to sodium acetate, which is then washed away with deionized water. The particle size of the NPs can be controlled by adjusting the pyrolytic temperature.

The byproducts also have an important role in controlling particle growth and agglomeration. Sodium acetate can become distributed on the surface of the NPs, preventing them from agglomerating. These nanocomposites can then be converted into NPs via the dissolution of sodium acetate. Wang et al. [20] synthesized ZnO NPs of different sizes, ranging from 8 nm to 35 nm, by varying the pyrolysis temperature of the reactant mixture. Synthesized NPs exhibited polycrystalline hexagonal wurtzite structures and showed strong UV emissions, which were a result of the high quality of ZnO produced. Sol-gel method

The sol-gel method of synthesizing ZnO NPs was first developed by Spanhel et al. [21] and later modified by Meulenkamp [22]. Zinc acetate dihydrate is usually used as the precursor because it offers easier control of hydrolysis. Sodium hydroxide is used to adjust the pH of the reaction mixture, given that the pH controls the rate of ZnO formation, affects the size and stable state of the resulting NPs [23]. This method has four stages: solvation, hydrolysis, polymerization, and transformation. Zinc acetate dihydrate is solvated in a solvent such as methanol or ethanol, and is then hydrolyzed, which aids the removal of any intercalated acetate ions. This leads to the formation of a colloidal–gel of zinc hydroxide [24]. Zinc hydroxide then splits into Zn2+ cations and OH– anions, followed by polymerization of the hydroxyl complex to form ‘Zn–O–Zn’ bridges, converting sol into gel. This gel is then transformed into ZnO (Equations 3–5) [24,25]. Zn(CH3COO)2.2H2O + 2NaOH → Zn(OH)2 + 2CH3COONa + 2H2O [3] Zn(OH)2 + 2H2O → Zn(OH)42– + 2H+ [4] Zn(OH)42– ↔ ZnO + H2O + 2OH– [5]. Hayat et al. [26] synthesized 25-nm ZnO NPs using a modified sol-gel method and successfully utilized them for the photocatalytic oxidation of phenol. Biosynthesis

Surface-active biosurfactants produced by culturable microbes offer various advantages over synthetic polymers, such as higher specificity, biodegradability, and biocompatibility. Rhamnolipids (RLs) (cyclic lipopeptide surfactants) are one such biosurfactant that can also be used for capping, stabilizing, and dispersing NPs. Singh et al. reported the synthesis of RL-stabilized ZnO NPs in the range of 35–80 nm by using Pseudomonas aeruginosa. The proposed mechanism of synthesis is as follows: RLs in aqueous solution disperse in the form of typical spherical core-shell micelles. The hydrophobic alkyl chains of RLs attach to the surface of primary ZnO crystallites. The high surface energy of ZnO crystallites begins to form RL-stabilized ZnO (RL@ZnO) NPs. Synthesis of the ZnO NPs proceeds inside the core of the micelles via the nucleation and growth of ZnO NPs. Zn(OH)2 is produced by adding NaOH to the ZnNO3 solution, resulting in the formation of ZnO nucleates by dehydration of Zn(OH)2 [27]. Recently, Surendra et al. [28] synthesized 40–45-nm ZnO NPs from Moringa oleifera (the drumstick tree). Similarly, Gunalan et al. [29] prepared ZnO NPs from aloe leaf extract and showed that the biosynthesized NPs had higher antimicrobial activity compared with chemically synthesized ZnO NPs. Fate of ZnO NPs

When ZnO NPs of two different sizes (20 nm and 70 nm) were administered orally to study their gastro intestinal absorption, absorption parameters, such as AUC, Tmax, and t1/2, were found increase dose dependently. No significant difference was observed for NPs of different particle sizes. However, negatively charged NPs (i.e., coated with negative groups) had higher absorption through the intestinal wall compared with positively charged NPs, implying that negatively charged NPs are more suitable for biological applications, whereas positively charged NPs would pose lower toxicity [30–32]. The distribution of ZnO NPs depends on the route of administration and their physicochemical properties. After intraperitoneal injection, zinc was found to accumulate in heart, liver, spleen, lung, and kidney, with liver being the major site of deposition [33]. Distribution patterns for different administration routes along with effects of other variables on absorption and excretion patterns are summarized in Table 1 [31,33]. Determining the excretion kinetics of ZnO NPs is important to understanding the process and extent of their elimination from the body. Generally, particles with a diameter Aspergillus flavus > Aspergillus nidulans > Trichoderma harzianum [29]. Hence, the authors suggested that ZnO NPs could be used in food safety and the agricultural industries. Lipovsky et al. observed a concentration-dependent effect of ZnO NPs on the viability of Candida albicans. ZnO NPs (o.1 mg/ml) inhibited >95% of the viability of C. albicans. Exciting ZnO NPs by visible light further increased yeast cell death [66]. Anti-Inflammatory activity

Given the known anti-inflammatory activity of ZnO, Ilves et al. compared the efficacy of nano-ZnO and bulk-ZnO and found that only nano-ZnO was able to penetrate the deep layers of allergic skin. Nano-ZnO was also able to better suppress local skin inflammation and induced the systemic production of IgE antibodies. The authors suggested that this effect is the result of nonspecific reactions caused by released Zn2+ affecting the IgE production abilities of B cells [67]. Wound healing

ZnO NPs have also been successfully used in wound dressings owing to their strong antimicrobial properties and the epithelialization-stimulating effect of zinc [68]. Recently, ZnO NP-loaded-sodium alginate-gum acacia hydrogels (SAGA-ZnONPs) were prepared that had a healing effect at low concentrations of ZnONPs in sheep fibroblast cells. High concentrations of ZnONPs were toxic to the cells, whereas SAGA-ZnONPs hydrogels significantly reduced the toxicity and preserved the beneficial antibacterial and healing effects [69]. Karahaliloglu et al. [70] combined chitosan/silk sericin scaffolds with lauric acid and ZnO NPs. The diameter of the inhibition zone increased from 2 ± 0.4 to 7 ± 0.1 mm for E. coli, and from 2.5 ± 0.2 to 6 ± 0.4 mm for S. aureus after addition of ZnO NPs. Kumar et al. [71] prepared chitosan hydrogel/nano ZnO composite bandages that demonstrated an enhanced swelling ratio

and also showed blood-clotting and antibacterial activity. In vivo evaluations in Sprague–Dawley rats revealed enhanced wound healing and faster re-epithelialization and collagen deposition. Concluding remarks

Similar to other metal oxide NPs, ZnO NPs have promising biomedical potential because of their inherent ability to induce ROS generation and cause apoptosis. These attributes of ZnO NPs make them a suitable as anticancer, antibacterial, and antifungal agents. ZnO NPs have also been known to produce synergistic actions when loaded and administered with other therapeutic agents. Although ZnO is considered as a comparatively safe metal oxide and has been approved by the FDA for cosmetic preparations, the toxicity of ZnO NPs remains a concern owing to their large surface area and metallic nature (Box 1). While the biomedical potential of ZnO NPs is currently being explored, detailed studies are warranted to explore their toxicity profile to obtain maximal benefits from this promising metal oxide.

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Box 1. Toxicity concerns Toxicity concerns and the risk:benefit ratio of metal NPs, including ZnO NPs, have remained a topic of debate. Although ZnO is generally considered to be a material with low toxicity, ZnO NPs can be a health hazard because of their minute size. In human bronchial epithelial BEAS-2B cells, nanosized ZnO caused a significant increase in DNA damage after 3 h and 6 h treatments, whereas fine ZnO did not. However, fewer differences were observed between the two ZnO forms after longer treatment times. These findings can be explained by the better uptake or faster intracellular dissolution of nanosized ZnO [89]. Exposure of mice to ZnO NPs resulted in bodyweight loss and a higher level of total cell number, total protein, and hydroxyproline content. Nitric oxide and malondialdehyde levels in lung homogenates also increased. In addition, inflammatory and hyperplastic changes in the lungs were observed [90]. High doses (500 mg/kg) of positive and negative ZnO NPs (100 nm) were found to produce significant changes in hematological and blood biochemical analyses, which could correlate with anemia-related parameters. Significant adverse effects were observed in the stomach, pancreas, eye, and prostate gland, and the particle charge did not affect the tendency or the degree of the lesions [91]. The harmful effects of ZnO NPs to normal body cells can be decreased by the surface functionalization of the NPs with targeting proteins or chemical groups. This improves their selectivity, rendering them benign to normal cells without any loss of activity against the target tissue [92].

Figure 1. Biomedical applications of zinc oxide (ZnO) nanoparticles (NPs). For further details of these applications, please see the main text.

Figure 2. Mechanism of action of zinc oxide (ZnO) nanoparticles (NPs). (a) ZnO NPs induce the generation of reactive oxygen species (ROS). ROS damage DNA, which in turn causes the release of apoptogenic factors from the mitochondrial membrane. Apoptogenic factors result in the formation of apoptosomes, which ultimately leads to apoptosis. (b) ZnO NPs bear negative charge on their surface because of partially bonded oxygen atoms. At lower pH, protons from the environment are transferred to the particle surface, resulting in a positively charged surface (ZnOH2+). These positive particles interact with negative phospholipids on the outer membrane of the cell, which results in their uptake by the cell. ZnO NPs then dissolve in the acidic lysosomes and release Zn+, which inhibits the action of respiratory enzymes, causing cell death.

Table 1. Pharmacokinetic parameters of ZnO NPs Parameter

Variable

Effect

Conclusion

Absorption (oral administration)

Dose Particle size Surface charge

Surface charge rather than particle size has a major role in absorption

Tissue distribution

Route of administration

Directly proportional No significant effect Higher absorption of negatively charged particles compared with positively charged particles Oral More accumulation in kidneys, liver, and lungs Intraperitoneal More accumulation in liver, spleen, kidneys, lungs, and heart Inhalation Severe toxicological effects in liver and lung Independent Very small particles eliminated through urine, while larger particles eliminated through feces Most orally administered particles excreted via feces Independent

Fecal excretion has major role in elimination

Particle size Excretion

Particle size Route of administration Surface charge

Liver and kidneys are common sites of biodistribution

Table 2. Summary of studies investigating ZnO NPs as standalone anticancer agents and as carriers for chemotherapeutic agents Type of particle

Type of cancer/cell line

Important findings

ZnO NPs as standalone anticancer agents or in combination with chemotherapeutic agents ZnO NPs Hepatocellular carcinoma Significantly reduced elevated serum levels of tumor markers alpha-fetoprotein and alpha-l-fucosidase; HepG2, human prostate also decreased elevated levels of hepatocyte integrity and oxidative stress markers PC3, non-small cell lung cancer A549 Melanoma B16F10, A375 Maximum efficacy among other metal oxide NPs. Inhibited ERK enzyme and several other cancerassociated kinases (AKT, CREB, p70S6K) Human colon carcinoma Entered cells by passive diffusion, endocytosis or both, depending on agglomeration state of LoVo nanomaterial Contact with acid pH of lysosomes altered organelle structure, resulting in release of Zn2+ Generate ROS at mitochondrial and nuclear levels, inducing severe DNA damage HeLa mRNA expression of apoptotic gene p53 and level of ROS increased in dose-dependent manner Hep-G2 Dose-dependent cytopathic effects Caspase-3 activation and DNA fragmentation assays confirmed apoptosis Caco-2 ZnO and TiO2 NPs both produced ROS and increased 8-oxodG levels in Caco-2 cells; but only ZnO NPs induced micronuclei and DNA damage Caco-2 cells exposed to ZnO NPs unable to repair oxidative DNA damage PC3 ZnO NPs showed more effective anticancer activity than Ag NPs, (70% versus 62% apoptosis, respectively) Human skin melanoma Significant decrease in cell viability A375 Cells treated with NPs had lower density and rounded morphology, revealed by phase-contrast imaging Generation of ROS and depletion of the antioxidant, glutathione Apoptosis confirmed by chromosomal condensation assay and caspase-3 activation Dose-dependent DNA damage Caco-2 Higher toxicity of ZnO NPs than Ag NPs in same concentration range Dose-depended toxicity Significant depletion of SOD level, variation in GSH level, and release of ROS HepG2, MCF-7 Concentration-dependent cytotoxic action Quantitative RT-PCR results demonstrated significant upregulation of mRNA expression level of Bax, p53, and caspase-3 and downregulation of antiapoptotic gene Bcl2 Caco-2 Significant reduction in glutathione Increase in ROS and lactate dehydrogenase Deletion of cells in G1 phase, accumulation of cells in S and G2/M phases Size-dependent cytotoxic effects Malignant human T98G Highly toxic to T98G cancer cells, moderately effective against KB cells, and least toxic against normal gliomas, KB, HEK normal human HEK cells non-malignant kidney cells Sensitized T98G cells by increasing mitotic (linked to cytogenetic damage) and interphase (apoptosis) death ZnO:Ag Human malignant Incorporation of Ag significantly improved photo-oxidation capabilities of ZnO NPs nanocomposites melanoma HT144, HCEC ZnO/SiO2 core- Human prostate Under X-ray irradiation (200kVp), NPs scintillate emitting luminescence in 350–700 nm range shell NPs adenocarcinoma LNCaP, Enhanced radiation-induced reduction in cell survival Du145 Radiosensitizing effect attributed to X-ray radiocatalysis induced by NPs Pure and AlMCF-7 Al-doping increased band gap energy of ZnO NPs, and also enhanced cytotoxicity and oxidative stress doped ZnO NPs response of ZnO NPs Upregulation of apoptotic genes (e.g., p53, bax/bcl2 ratio, CASP3 and CASP9) and loss of mitochondrial membrane potential Al-doping did not change benign nature of ZnO NPs towards normal cells ZnO NPs as carriers for drug delivery DaunorubicinA549 ZnO NPs incorporated in liposomes demonstrated pH-responsive release of anticancer drug ZnO NPs No premature drug leakage while maintaining relevant therapeutic concentrations Sensitive leukemia: K562; Drug-resistant cell line more sensitive to ZnO NPs than K562 cell line resistant leukemia: Presence of ZnO NPs efficiently enhanced accumulation of daunorubicin, improved permeation of cell K562/A02 membrane, and enhanced uptake of daunorubicin into both sets of cells DOX-ZnO NPs MCF-7 Combination of ZnO NPs and Dox resulted in higher cytotoxicity than either alone Drug-loaded NPs produced even higher cytotoxicity Isoorientin-DOX HepG2 Dose- and time-dependent cytotoxicity NPs Combined treatment demonstrated greater cytotoxicity than single treatment NPs synergistically potentiated isoorientin to induce apoptosis through mitochondrial dysfunction, inhibiting phosphorylation of Akt and ERK1/2, and enhancing phosphorylation of JNK and P38 Cell uptake via endocytic pathway, enhanced uptake of isoorientin; no significant injury to normal liver cells

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Photofrin-ZnO NPs FAfunctionalizedPTX-ZnO NPs

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NPs excited intracellularly with 240 nm UV light; resultant 625 nm red light emitted in presence of Photofrin activated chemical reaction that produced ROS and led to cell death MCF-7 and MDA-MB-231 Simultaneous passive and active targeting of PTX Released ~75% drug within 6 h in acidic pH Switching of fluorescence from blue to green and tenfold increase in fluorescence intensity Combined pH and folate-receptor targeting reduced accumulation at nontarget sites Improved efficacy of PTX against subcutaneous tumors in vivo DOX-ZnO/PEG HeLa Remarkable improvement in antitumor activity nanocomposites Photodynamic activity considerably increased cancer cell injury mediated by ROS under UV irradiation Increased intracellular concentration of DOX and enhanced its potential antitumor efficiency 91% drug released within 26 h of incubation of conjugates in vitro in an acidic environment DOX-loadedHuman breast cancer Stimulative effect of heat, pH-responsive, and sustained drug release properties FAMDA-MB-231, Human Maximum rate of death in breast cancer cells compared with single chemotherapy or photothermal functionalized breast cell line HBL-100 therapy PEG-coated Minimum systemic toxicity in mouse model system ZnO nanosheet Advanced chemophotothermal synergistic targeted therapy and good drug release properties effectively avoid frequent and invasive dosing

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