Targeted delivery of quercetin via pH-responsive zinc oxide nanoparticles for breast cancer therapy

Materials Science & Engineering C 100 (2019) 129–140

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Targeted delivery of quercetin via pH-responsive zinc oxide nanoparticles for breast cancer therapy

T

Pritam Sadhukhana, Mousumi Kundua, Sharmistha Chatterjeea, Noyel Ghosha, Prasenjit Mannab, Joydeep Dasc,⁎,1, Parames C. Sila,⁎⁎,1 a

Division of Molecular Medicine, Bose Institute, P-1/12, CIT Scheme VII M, Kolkata 700054, India Biological Science and Technology Division, CSIR-North East Institute of Science and Technology, Jorhat, Assam 785006, India c School of Chemistry, Shoolini University of Biotechnology and Management Sciences, Bajhol, PO Sultanpur, Distt., Solan 173229, HP, India b

ARTICLE INFO

ABSTRACT

Keywords: Anticancer Quercetin Reactive oxygen species ZnO nanoparticles PBA conjugated nanoparticles

Naturally occurring bioactive compounds are gaining much importance as anti-tumor agents in recent times due to their high therapeutic potential and less systemic toxicity. However, different preclinical and clinical studies have noted significant shortcomings, such as nonspecific tumor targeting and low bioavailability which limit their usage in therapeutics. Therefore, a safe and compatible nanoparticle mediated controlled drug delivery system is in high demand to enable effective transport of the drug candidates in the tumor tissue. Herein, we have synthesized phenylboronic acid (PBA) conjugated Zinc oxide nanoparticles (PBA-ZnO), loaded with quercetin (a bioflavonoid widely found in plants), with zeta potential around −10.2 mV and diameter below 40 nm. Presence of PBA moieties over the nanoparticle surface facilitates targeted delivery of quercetin to the sialic acid over-expressed cancer cells. Moreover, Quercetin loaded PBA-ZnO nanoparticles (denoted as PBAZnO-Q) showed pH responsive drug release behavior. Results suggested that PBA-ZnO-Q induced apoptotic cell death in human breast cancer cells (MCF-7) via enhanced oxidative stress and mitochondrial damage. In line with the in vitro results, PBA-ZnO-Q was found to be effective in reducing tumor growth in EAC tumor bearing mice. Most interestingly, PBA-ZnO-Q is found to reduce tumor associated toxicity in liver, kidney and spleen. The cytotoxic potential of the nanohybrid is attributed to the combinatorial cytotoxic effects of quercetin and ZnO in the cancer cells. Overall, the presented data highlighted the chemotherapeutic potential of the novel nanohybrid, PBA-ZnO-Q which can be considered for clinical cancer treatment.

1. Introduction Breast cancer is a problem of global importance; since its incidence, it is increasing worldwide, and therapeutic options are quite limited. According to the World Health Organization, nearly 1.3 million women develop breast cancer every year [1,2]. There are several lines of conventional therapies including chemotherapy, hormone replacement therapy, radiotherapy and surgical removal. Among these, chemotherapy is considered as the most promising modality for treating breast cancer till date. Ideally, a chemotherapeutic agent should eradicate cancer cells by targeting a particular receptor, protein or DNA specific to those neoplastic cells by its cytotoxic and/or cytostatic effects with minimal collateral damage to adjacent normal cells [3]. Pharmaceutical agents which are currently available in the market for

chemotherapy are non-targeted and cause severe cytotoxicity in other vital organs like kidney, liver and heart [4–6]. Moreover, the use of high drug concentration at the tumor site is limited due to the occurrence of drug resistance and high drug efflux capacity of the cancer cells [7,8]. As a result, side effects attributed to the nonspecific accumulation of these drugs and their overdose are inevitable. Thus, due to low response to conventional therapies, it is of utmost importance to find new therapeutic targets and new molecules with therapeutic potentials for selectively killing cancer cells without being toxic to the normal tissues. In this respect, naturally occurring compounds are considered to be the promising therapeutic agents against cancer due to their anticipated multimodal actions and minimal side effects. Flavonoids are such ingredients found in nature that have shown potent anticancer activities in several in vitro and in vivo studies [9,10]. Within a long list of

Corresponding author. Correspondence to: P.C. Sil, Division of Molecular Medicine, Bose Institute, P-1/12, CIT Scheme VII M, Kolkata 700054, West Bengal, India. E-mail addresses: [email protected] (J. Das), [email protected] (P.C. Sil). 1 P.S. and J.D contributed equally to this work. ⁎

⁎⁎

https://doi.org/10.1016/j.msec.2019.02.096 Received 9 October 2018; Received in revised form 25 February 2019; Accepted 25 February 2019 Available online 28 February 2019 0928-4931/ © 2019 Elsevier B.V. All rights reserved.

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Scheme 1. A schematic diagram representing the synthesis of the nanohybrid and overview of the most probable intracellular mechanism of action of the nanohybrid.

flavonoids, quercetin is one of the most abundant dietary polyphenolic compounds, commonly found in different fruits and vegetables such as capers, lovage, cilantro, onions, apples and berries [11]. Several in vitro and in vivo studies have revealed that quercetin shows antitumor activity due to its anti/pro-oxidant and anti-inflammatory properties, and also to other ways of action less explored [11,12]. Among least explored antitumor actions of quercetin one can highlight its role as GLUT-1 inhibitor [13], an inhibitor of tumor neovascularisation [14], a competitive inhibitor of drug efflux pumps [15] and their ability to arrest cell cycle at multiple phases [16–18]. Despite the antitumor properties, the major limitation has been its poor bioavailability [19]. Quercetin obtained from plant sources as hydrophilic glycosides are not absorbed directly with ease. Studies also suggested that quercetin is metabolised rapidly and excreted through urine with very little accumulation in tissue and biological fluids [20]. This poor bioavailability and its high metabolic concentration indicate an extensive first pass metabolism in the gut and/or liver. Sidewise insolubility in aqueous medium and

lower solubility in alcohol is another concern associated with its intravenous administration [21]. However, the use of nanoparticle mediated drug delivery systems can overcome these challenges by improving their bioavailability and rendering therapeutics completely dispersible in aqueous medium and thus make them intravenously injectable. Quercetin has also been reported as a renewable source of nanocarriers for the delivery of the chemotherapeutic drug, doxorubicin [22]. Other naturally occurring polyphenols, such as green tea catechins are also capable of forming nanoparticles via molecular assembly with keratin proteins and considered as a potential candidate for drug delivery applications [23]. Metal oxides are fantastic choices among the huge pool of nanoparticles due to their tunable size and shape, surface chemistry and ability to express wide range of oxidation states. Recently, one such metal oxides, zinc oxide nanoparticles (ZnO NPs) have been reported to be effective against the tumor cells owing to their attractive chemical and physical properties. Studies have found ZnO NPs exhibiting high 130

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cancer cell selectivity, retention, and controlled release of ligated as well as loaded drugs [24,25]. Zinc being a trace element in the body, ZnO NPs show reduced toxicity and well biocompatibility. ZnO NPs dissolute rapidly to Zn2+ ions at pH lower than 5.5 similar to the acidic tumor microenvironment. It enables ZnO NPs to show cytotoxic effect to tumor cells through mitochondrial dysfunction, ROS outburst, lipid peroxidation, DNA damage and finally apoptosis [26]. Besides, easy detection due to intrinsic fluorescence, simple synthesis techniques and low cost are some of the other reasons behind its suitability as a nanocarrier for cancer treatment. Such nanocarriers can be easily extravasated and accumulated in tumor site due to the leaky vasculatures and immature lymphatics of tumor tissue [27]. This phenomenon, known to be enhanced permeability and retention (EPR) effect, enables nanomedicines to be distributed in tumor region by passive tumor targeting [28]. However, passive tumor targeting based on the pathophysiological characteristics (such as pH, temperature) of the tumor site possesses some pitfalls too. The lack of tumor specificity can hamper the precise delivery of antitumor drugs to the tumor site. Moreover, due to the high density of tumor cells and high interstitial fluid pressure (IFP), nanomedicines can cross only a few layers which cannot ensure their homogenous distribution and proper penetration [29]. Hopefully, this dilemma can be solved through active tumor targeting where the nanocarriers can be modified by linking some ligands specific to the receptors overexpressed specifically on tumor cells. Among several such agents, the use of 3-carboxybenzeneboronic acid (PBA) as a tumor targeting ligand and penetration moiety is highly reasonable [30]. Tumorigenicity and metastasis can alter glycosylation of proteins and lipids on the surface of the neoplastic cells. Notably, increase of sialyltransferases upregulates the expression of terminal sialic acid (SA) residues of glycans on the tumor cells [31]. The formation of reversible cyclic boronate esters between the exocyclic polyol group of SA (expressed in tumor cells) and the boronic acid group of PBA is known as one of main binding mechanisms, facilitating active PBA mediated tumor targeting [32]. Even though PBA can form complexes with other common sugars, they are unstable unless and until formed at higher pH compared to the pKa value of PBA, whereas the complex formed between PBA and SA is stable even at pHs lower than its pKa [33]. This characteristic of controlled pKa value of PBA provides the molecular basis for the specific SA recognition at physiological pH. In addition to selectivity and high binding affinity for SA, PBA is also beneficial for being nontoxic, nonimmunogenic and inexpensive. Herein, for the first time, we have developed quercetin loaded PBA functionalized ZnO NPs for specific targeting of SA epitopes overexpressed on breast cancer cells and monitored their anti-tumor efficacy using in vitro as well as in vivo tumor models. For this purpose, human breast cancer MCF-7 cells overexpressing SA on their surface and Ehrlich's ascites carcinoma (EAC) solid tumor bearing Swiss albino mice were used. Targeted delivery of quercetin in association with ZnO nanocarrier is found to enhance its cellular uptake of this flavonoid in vitro and improved their tumor accumulation and retention in vivo, leading to a superior antitumor effect and suppression of the tumor growth (Scheme 1). Moreover, use of ZnONPs did not show any sign of toxicity, rather found to reduce tumor associated toxicity in liver, kidney and spleen.

(APTES) with purity 99%, quercetin dihydrate were purchased from Sigma-Aldrich. Ethyl alcohol (ETOH), dimethyl sulphoxide (DMSO) was purchased from Merck. RPMI-1640, MEM and other chemicals like trypsin, antibiotics etc. were purchased from HIMEDIA (Mumbai, India). Fetal bovine serum (FBS) was bought from HyClone (United States). H2-DCFDA and Rhodamine 123 was purchased from SigmaAldrich Chemical Company (United States). 3-(4,5-Dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) and other necessary chemicals were purchased from the SISCO Research Laboratory, India.

2. Experimental sections

2.2.4. Characterization of the synthesized nanoparticles The crystal structure of the synthesized nanoparticles was characterized by an X-ray diffractometer (XRD; Siemens D-500). The analysis was performed in the 2θ range from 20 to 90° using copper Kα1 (λ = 1.54056 Å) radiation. UV–visible spectra of the synthesized nanoparticles were obtained using a Shimadzu Spectrophotometer instrument. The zeta potential and hydrodynamic diameter were measured by dynamic light scattering (DLS) (Delsa™ Nano C particle size analyzer Beckman Coulter, Brea, CA, USA). The morphology and the primary size of the nanoparticles were observed by transmission electron microscopy (TEM) (JEM-2100HR-TEM, JEOL, Japan) and field

2.2. Methods 2.2.1. Synthesis of ZnO and NH2-ZnO NPs The preparation of ZnO and NH2-ZnO NPs were carried out following the methods as reported elsewhere [34–36]. Briefly, 2.0 mmol zinc acetate and 44 mg of magnesium acetate were added to 50 mL anhydrous ethanol, and the solution was refluxed for about 5 h at 60 °C. After that, 2.5 mmol KOH was dissolved in 5 mL anhydrous ethanol and was added drop by drop to the previously refluxed solution. Then the final solution was stirred for 2 h at 60 °C to obtain ZnO nanoparticles (NPs). After that, 1 mL of 3-Aminopropyltriethoxysilane (APTES) was added, and the solution was stirred continuously at room temperature for 1 h to achieve NH2-ZnO NPs. Then the solution was washed several times with anhydrous ethanol and was centrifuged at 6000 rpm for 20 min to get the solid product. 2.2.2. Conjugation of 3-carboxybenzeneboronic acid to NH2-ZnO NPs The preparation of PBA conjugated NH2-ZnO NPs was carried out following the methods as reported elsewhere. Briefly, 100 mg PBA was dissolved in 10 mL DMSO. To the above PBA solution, 40 mg of EDC and equivalent NHS were added, and the activation of PBA was carried out for 3 h with continuous stirring. After that, a dispersed solution of NH2-ZnO NPs (in DMSO) was added to the activated PBA solution under a continuous stirring condition for 24 h to achieve PBA-NH2-ZnO NPs (PBA-ZnO). Finally, the product was centrifuged and washed. 2.2.3. Loading of quercetin to PBA-ZnO NPs The loading of quercetin to the PBA-ZnO NPs was carried out following the methods as reported elsewhere. Briefly, PBA-ZnO NPs was dispersed in DMSO. To this dispersed solution, quercetin was added and the reaction was continued for 24 h in stirring condition. Quercetin loaded nanoparticles are denoted as PBA-ZnO-Q. The loading amount of quercetin was calculated with UV–vis absorbance of quercetin at 378 nm and was estimated with the calibration curve produced by a series of quercetin solutions under the same conditions. The calculations of drug loading content and entrapment efficiency were as follows:

Drug loading content (%) = [(weight of drug in nanoparticles) /(weight of nanoparticles taken)] × 100. Drug entrapment efficiency (%) = [(weight of drug in nanoparticles) /(weight of drug injected)] × 100.

2.1. Chemicals Zinc(II) acetate dihydrate with purity 99.5%, magnesium(II) acetatetetrahydrate with purity 99%, 3-Carboxybenzeneboronic acid with purity 97%, N-hydroxyl succinimide (97%), 1-(3Dimethylaminopropyl)-3-Ethyl Carbodiimide Hydrochloride (EDC.HCl) extrapure, 99%, potassium hydroxide pellets, sodium chloride, disodiumdihydrogen phosphate dehydrate, and potassium dihydrogen phosphate, were purchased from SRL. (3-Aminopropyl) triethoxysilane 131

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emission scanning electron microscopy (FESEM) (JSM-7600F, JEOL, Japan). The surface functionalization of the nanoparticles was investigated by Fourier transform infrared spectroscopy (FTIR) using a Fourier transform infrared instrument (NEXUS-470, Nicolet, USA).

70%, the cells were dose dependent exposed to PBA-ZnO (7 and 21 μg/ mL), Free Q (3 and 9 μg/mL) and PBA-ZnO-Q (10 and 30 μg/mL). After 48 h of exposure, the cells were washed with PBS and scraped out into an Eppendorf tube. For the determination of ROS and MMP, the cells were stained with DCFDA and Rhodamine 123 respectively, for 30 min in the dark. Finally, the cells were analysed in flow cytometry System (BD FACS Calibur) using the FITC filter.

2.2.5. Fluorescent micrographs of the nanoparticles The newly synthesized ZnO nanoparticles were characterized with fluorescent microscopy (Fluorescent microscope, Nikon). The nanoparticles were suspended in a 1× PBS solution at a concentration of 2 mg/mL. On a clean glass slide, a drop of the solution was placed with a glass cover slip. The micrographs were captured in a fluorescent microscope using the bright field, DAPI, FITC and RITC filter. The micrographs were captured using a 40× objective lens [37].

2.2.11. Detection of tissue accumulation of quercetin To determine the amount of quercetin accumulated in the tumor tissue per treatment dose, HPLC techniques were employed. A reversephase HPLC assay was carried out using a C-18 column; injection volume of 20 μL, flow rate of 1.7 mL/min, a mobile phase of 2% acetic acid and acetonitrile (60:40, v/v), and a detection wavelength of 375 nm. Ehrlich's ascites carcinoma (EAC) solid tumor bearing animals were developed in male Swiss albino mice. The tumor model was developed according to the protocol described elsewhere [39]. 106 EAC cells were injected in the left flank of the animals. 24 (EAC) solid tumor bearing mice were divided into 2 sets containing 4 groups of animals; group 1: Tumor control (animals received 0.9% NaCl solution); group 2: ZnO-Q; group 3: PBA-ZnO-Q and group 4: Free Q. The equivalent dose of quercetin injected was10 mg/kg. The accumulation of quercetin in the tumor tissue was determined time dependently at 6 and 24 h of intravenous administration. After the respective time intervals, the animals were sacrificed, and the tumors were dissected out. From each tissue, 2 g of tissue was taken and subjected to homogenization in 0.05% DMSO and 75% EtOH. The homogenized mixture was then centrifuged at 10,000 rpm for 15 min. The supernatant was dried in nitrogen evaporator, the dry product was solubilized in MeOH, and the amount of quercetin present in the respective tissues was checked by HPLC instrument with a UV detector set at 375 nm. The quercetin content was calculated using a standard curve.

2.2.6. Determination of pH dependent release behavior of the nanoparticle The release of the loaded quercetin from the nanohybrid was determined using the dialysis diffusion technique (MWCO 3500 Da bags were used). The release behavior of the quercetin loaded nanoparticles was investigated in three different pH conditions (pH-5, 6 and 7.4). Briefly, nanohybrids (with 1 mg/mL of quercetin) were evenly suspended in a PBS solution of the desired pH and incubated at 37 °C with gentle shaking. The absorbance of the released quercetin was measured at 375 nm after a specific time interval between 0 and 48 h. 2.2.7. Determination of intracellular uptake of the nanoparticles MCF-7 cells were plated in a 35 mm cell culture dish at a density of 0.3 × 106 with a sterile glass cover slip. At a confluency of 70% the cells were treated with ZnO nanoparticles and PBA-ZnO nanoparticles for 6 h. After 6 h, the cells were thoroughly washed with 1× PBS to washout the free nanoparticles. Finally, the coverslips were mounted on a glass slide using a mounting media with DAPI. The images were captured in a fluorescent microscope using the FITC filter at a magnification of 40× [38]. 2.2.8. Determination of intracellular release of quercetin MCF-7 and MCF-10a cells were exposed to 30 μg/mL of PBA-ZnO-Q for 48 h. After the incubation period, the cells were then washed using PBS for 3 times and then incubated in a lysis buffer for 10 min and centrifuged at 6000 rpm. The supernatants were collected, and the amount of quercetin was determined by measuring the absorbance at 375 nm.

2.2.12. Investigation of the in vivo antitumor efficacy To investigate the in vivo effect of the newly synthesized nanohybrids, EAC (a spontaneous murine mammary adenocarcinoma) solid tumor bearing animals were developed in male Swiss albino mice according to the protocol described elsewhere [39]. EAC cells induced solid tumor bearing mice is an established model to study breast cancer pathogenesis. After 10 days of the inoculation, the animals were divided into 4 groups (n = 6) as described below:

2.2.9. Determination of cytotoxic potential of the nanoparticles To determine the cytotoxic efficiency of the synthesized PBA tagged ZnO nanoparticle attached to quercetin (PBA-ZnO-Q) on MCF-7 cells and compared its effect with its component like PBA tagged ZnO nanoparticle (PBA-ZnO) and quercetin (Free Q), MTT cell viability assay was performed. The assay was performed according to the protocol described elsewhere [39]. Briefly, the cells were plated in a 24 well culture dish at a density of 0.05 × 106 cells per well. At 70% confluency, the cells were exposed to different concentration of PBA tagged ZnO nanoparticles (3.5–35 μg/mL), free quercetin (1.5–15 μg/mL) and PBA-ZnO-Q (5–50 μg/mL) for 48 h. The media was then replaced with 1× PBS solution containing MTT at a concentration of 0.5 mg/mL. The cells were incubated for 4 h, and the formed formazan crystals were dissolved by the adding DMSO. Finally, the absorbance was read at 570 nm, and cell viability was calculated as percentage to the control value. Cytotoxic potential of the nanoparticle was also determined on MCF-10a cells.

Group 1: Tumor control The animals received 0.9%NaCl solution. Group 2: PBA ZnO

The animals were treated with 23 mg/kg PBA ZnO.

Group 3: Free Q

The animals were treated with 10 mg/kg quercetin.

Group 4: PBA ZnO Q

The animalswere treatedwith 33 mg/kg PBA ZnO Q.

The animals were then intravenously treated with the nanoparticles (PBA-ZnO and PBA-ZnO-Q) and quercetin (Free Q) on alternative days for 14 days. After 14 days the animals were sacrificed, and the tumors were dissected out. The tumor volume was determined according to the ellipsoid volume equation and the mass was determined in an electronic weighing balance. The animal experiments were carried out following to the guidelines of the Institutional Animal Ethical Committee (IAEC), Bose Institute, Kolkata [IAEC/BI/3(I) cert./2010] and the work plan was approved by CPCSEA (Committee for the Purpose of Control and Supervision on Experiments on Animals), Ministry of Environment and Forests, New Delhi, India (1796/PO/Ere/S/14/CPCSEA).

2.2.10. Detection of intracellular reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) To determine effect of nanoparticles on oxidative status of the cell, intracellular ROS and MMP were determined using FACS analysis. FACS analyses were performed according to the protocol described elsewhere [40]. MCF-7 cells were plated in 35 mm culture dish. At confluency of 132

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2.2.13. Detection of systemic toxicity To detect the occurrence of systemic toxicity in the nanoparticles treated animals as described in Section 2.2.10, the serum biomarkers for hepatic and renal toxicity was measured. Blood was collected from each sacrificed animal, and subsequently, serum was isolated using centrifuge as described elsewhere. In the serum, the level of alanine aminotransferase (ALT), alkaline phosphatase (ALP) was measured as hepatic toxicity marker and for nephrotoxicity blood urea nitrogen (BUN) and creatinine was measured. The experiments were done according to the protocol of the kit (Span diagnostics Ltd., India) [41]. Moreover, the tumor associated splenomegaly was observed in all the experimental animals by comparing the morphology of spleen as described elsewhere [42].

the UV-VIS spectrum of pure quercetin and quercetin loaded nanoparticles. Pure quercetin showed a characteristic peak at around 378 nm, but that peak was shifted to lower wavelength region, and a new peak was obtained at around 360 nm for quercetin loaded ZnO NPs, most probably because of the overlap of the two absorption peaks for pure quercetin and ZnO NPs. It may also happen due to the existence of an interaction between quercetin and ZnO [47,48]. The surface functionalization of ZnO NPs was further confirmed by FT-IR analyses (Fig. 2C). As shown in Fig. 2C, the transmission at around 479 cm−1 was assigned for ZneO bond stretching vibration of ZnO NPs. In addition, the spectra of NH2-ZnO exhibited absorption at 1585 cm−1 and 1022 cm−1, which were assigned for NeH bending vibration and SieOeSi stretching vibration respectively, indicating APTES conjugation to ZnO nanoparticles. The characteristic peak of PBA appeared at 1689 cm−1 which is for eC]O bond vibration of the carboxylic acid group. After reaction of PBA with eNH2 group of ZnO, this peak was omitted, and a new peak appeared at 1630 cm−1, conforming the amide bond (eNHeCOe) vibration, which directed that PBA was conjugated to the ZnO nanoclusters by covalent amide interactions. The transmission peaks of pure quercetin appeared at 1670 cm−1, 1201 cm−1, 812 cm−1 which were attributed to the stretching vibration of C]O aryl ketonic, the CeO stretching vibration in phenol and out-of-plane bending vibration of CeH in an aromatic hydrocarbon [49]. All these absorption peaks were obtained in quercetin loaded ZnO nanoparticles with little changes, which indicated the loading of quercetin in the nanohybrid. By using the Dynamic Light Scattering (DLS) experiments, the average hydrodynamic size of ZnO, PBA-ZnO and PBA-ZnO-Q were determined. The hydrodynamic sizes of ZnO, PBA-ZnO and PBA-ZnO-Q, were 140.9 ± 10.1 nm, 278 ± 12.3 nm and 400.3 ± 11.1 nm respectively (Fig. 3A). The size of nanoparticles appeared larger under DLS as compared to TEM analysis because of the extensive solvation/hydration of nanoparticles in water [50]. Successive incorporation of PBA and quercetin onto ZnO surface was further indicated by the successive increase in hydrodynamic diameters. The size of nanoparticles appeared larger under DLS as compared to TEM analysis because of the solvation/hydration of nanoparticles in water. Zeta potential was shown in Fig. 3B. The zeta potential value of ZnO NPs, PBA-ZnO, PBAZnO-Q were 17.8 ± 0.3 mV, −1.8 ± 0.12 mV, −10.2 ± 0.36 mV respectively. Step by step successful tagging of PBA and quercetin were also confirmed by the change in surface potential values. We have further evaluated the stability of the nanohybrid in a solution which mimics in vivo environment. We suspended the nanohybrid (ZnO-PBA-Q) in water containing 10% FBS and checked the timedependent change in the hydrodynamic size and PDI. The hydrodynamic size of PBA-ZnO-Q was found to be 252.65 ± 66.11 nm, 264.43 ± 64.5 nm and 273.97 ± 48.98 nm at day 0, day 7 and day 14 respectively (Table 2). The hydrodynamic size increased by only 4.7% and 8.4% after 7 and 14 days, thereby indicating good stability of the nanohybrid in solution. However, the hydrodynamic size in water containing FBS appeared smaller due to formation of a protective layer (protein corona) over the nanohybrid which prevents interaction with water molecules. Besides, there was no apparent change in the PDI values (0.55–0.60) of the nanosuspension after 7 and 14 days, indicating further stability of the nanohybrid.

2.2.14. Statistical analysis The results were expressed as their mean data ± SD, after carrying out three independent experiments. The statistical analyses were done by one-way analysis of variance (ANOVA), and Tukey test was also performed to compare the mean values between the groups. A p-value of less than 0.05 was considered to be statistically significant. 3. Results and discussion 3.1. Synthesis and characterization of ZnO, PBA-ZnO and ZnO-PBA-Q NPs First, we have prepared water dispersible amino functionalized (NH2-ZnO) nanoparticles following a previously reported method [34]. In order to prepare PBA-ZnO, PBA was conjugated to NH2-ZnO (via amide bond formation) and finally, quercetin was loaded (via metal ion-ligand coordination) to achieve targeted drug delivery to cancer cells. The DLC and DEE were found to be 29.83% and 46.69% respectively (Table 1). TEM and SEM have been used to determine the size and shape of PBA-ZnO-Q (Fig. 1A and B). The morphology of the particles was found to be tetragonal. The average particle size of the nanohybrid was at about 30–40 nm according to the TEM image (Fig. 1B). Magnesium acetate was used in the synthesis of ZnO nanoparticles to distort the host ZnO lattice by the incorporation of foreign Mg2+ impurities, thereby reducing the size of the nanoparticles [43]. EDX spectrum of the nanohybrid has been shown in Fig. 1C. The result showed that the nanoparticles contained about 53.44% Zn, 22.97% O and 15.11% C by weight. It also contained silicon, boron and nitrogen which indicated the successful surface modification of the ZnO NPs. XRD pattern of the synthesized ZnO, PBA-ZnO and PBA-ZnO-Q NPs was shown in Fig. 1D. All obtained peaks were indicative of the hexagonal crystallite structure of ZnO NPs, devoid of any impurities [25,44]. To examine the optical properties of the synthesized nanoparticles, we performed both fluorescence and UV-VIS spectroscopic analyses. The fluorescence spectrum of PBA, ZnO, and PBA-ZnO NPs was given in Fig. 2A. ZnO-PBA exhibited two strong emission peaks at 340 nm and 527 nm when excited at 310 nm. On the other hand, PBA exhibited one strong peak at 340 nm under the same excitation [45]. In case of only ZnO, the intensity of the first peak (i.e. at 340 nm) was lower than that of the second peak (i.e., at 527 nm). The first peak intensity of the ZnOPBA was increased due to the presence of PBA. These results indicated the presence of PBA in the nanohybrid. The UV-VIS absorption spectrum of the nanoparticles was shown in (Fig. 2B). ZnO NPs showed a strong absorption band at about 342 nm, which is consistent with the earlier report [46]. The loading of quercetin was checked by comparing

3.2. Time based pH-responsive quercetin release study Quercetin was loaded onto the PBA-ZnO NPs by the formation of a chelate or metal ion (Zn2+) – ligand (carbonyl and hydroxyl groups of quercetin) coordination bond [51,52]. The Zn2+-Quercetin complex is stable at slightly alkaline condition as the hydroxyl groups (eOH) of quercetin remain in their ionised form (O−), therefore acts as effective ligands towards chelate formation. Quercetin was released from the nanohybrid when the breakdown of the chelate occurred. At low pH, the stability of the Zn2+-Quercetin complex is less as quercetin is

Table 1 Drug loading content (DLC) and drug entrapment efficiency (DEE) of ZnO/ quercetin nanoparticles. Feed ratio, ZnO: quercetin

DLC%

DEE%

1:1

31.83

46.69

133

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Fig. 1. Images obtained from (A) TEM and (B) SEM of PBA-ZnO-Q NPs. (C) EDX analysis of PBA-ZnO-Q NPs. Figure (D) shows the XRD pattern of PBA-ZnO-Q NPs.

predominantly present in its unionised form (poor ligand) as well as the partial dissolution of ZnO NPs. Based on the UV-VIS spectrophotometric analyses, the released percentage of quercetin was measured in different pH values at several time intervals (Fig. 3C). Release percentage of quercetin was almost 35% at pH 5.0 after 6 h, while negligible released occurred at pH 7.4. Three different pH values (endosome pH 5.0, physiological pH 7.4 and intermediate pH 6.0) were taken to experiment. At pH 5.0, the release amount of quercetin was almost 47% after 48 h, while it was almost 28% and 13% at pH 6.0 and pH 7.4 respectively. Therefore, PBA-ZnO NPs could be effectively used as a pH-responsive drug delivery system for quercetin.

3.3. Determination of intracellular uptake efficiency of the PBA conjugated nanoparticles The fluorescent nature of the synthesized nanohybrids was observed under a fluorescent microscope (at a magnification of 40×), which exhibit strong green (FITC) and blue (DAPI) fluorescence (Fig. 4A). Therefore, our synthesized nanohybrids can also be used as a potential bioimaging agent. The intracellular uptake of the ZnO and PBA-ZnO NPs was determined in the MCF-7 cells by performing both fluorescent microscopy and FACS analysis. The fluorescent intensity is supposed to be proportional to the number of nanoparticles internalize in the cells. It was observed that after 6 h of incubation, the PBA conjugated ZnO nanoparticles were internalized by the cells with higher efficiency. An 134

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Fig. 2. (A) Fluorescence spectra of ZnO (blue), PBA (green) and PBA-ZnO (red). (B) UV-VIS spectra of Quercetin (blue), ZnO (red) and ZnO-Q NPs (green). (C) FTIR spectra of Quercetin (Q), PBA-ZnO-Q, ZnO, PBA-ZnO and PBA. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

increase in the green fluorescence intensity in both microscopic, as well as FACS analysis, also confirms the higher uptake efficiency of the PBA conjugated nanoparticles in the cancer cells (Fig. 4B & C). Cancer cells are reported to express sialic acid in its membrane higher than the normal cells [53,54]. The expression of sialic acid is a characteristic feature of cancer cells, and it facilitates cellular proliferation and metastasis. Presence of PBA on the surface of nanoparticle allows a specific interaction of the nanoparticle with the sialic acid present in the cell surface. This property of PBA can be attributed to the higher intracellular uptake efficiency of the PBA tagged nanoparticles compared to the untagged nanoparticles [55].

cells. PBA-ZnO-Q showed dose dependent cytotoxicity from a dose ranging from 3.5 to 35 μg/mL. The LC50 concentration for PBA-ZnO nanoparticles was found to be 33.06 μg/mL, whereas for the free Q treatment group the LC50 concentration was 11.12 μg/mL ZnO. However, exposure of PBA-ZnO-Q (LC50 at 26.97 μg/mL, equivalent to 19 μg/mL PBA-ZnO and 8 μg/mL free Q) showed a combinatorial effect of ZnO-Q and free Q at all indicated concentrations (Fig. 5A). The cytotoxic potential was further confirmed by analysing the bright field micrographs of the nanohybrids and quercetin exposed cells. The occurrence of cell shrinkage, membrane blebbing and the presence of apoptotic bodies was a clear indication of cytotoxicity of the nanohybrids [56]. The cytotoxic lesions in the PBA-ZnO-Q exposed cells were found to be higher compared to PBA-ZnO and free Q exposed cells (Fig. 5B). Exhibition of cytotoxicity is the primary criteria of an anticancer drug candidate. The nanohybrid considered in the study was found to induce significant cytotoxicity in breast cancer cells. Thus, considering the anticancer efficacy of quercetin and ZnO nanoparticles, as shown as shown in our present study, it is worth to carry out further investigation to understand the cytotoxic efficacy of this novel PBA conjugated nanohybrid. The cytotoxicity of the nanoparticles was also investigated in a nontumorigenic human epithelial cell line (MCF-10a). The experimental results suggest no significant toxicity for PBA-ZnO and PBA-ZnO-Q up to a concentration of 35 μg/mL and 50 μg/mL respectively (Fig. 5C), indicating higher biocompatibility of the nanocarriers.

3.4. Intracellular pH responsivity of the nanocarriers The pH responsivity of the nanocarriers was also investigated by checking the intracellular release of quercetin after treatment of the cells with non-targeted ZnO-bound quercetin (ZnO-Q) to ensure similar uptake of ZnO-Q nanoparticles. We treated both MCF-7 (human breast adenocarcinoma cell line) and MCF-10a (a non-tumorigenic human epithelial cell line) cells with equal concentration of ZnO-Q for 48 h and then determined the intracellular level of free quercetin. We observed a higher level of intracellular free quercetin in MCF-7 cells compared to MCF-10a cells probably due to lower intracellular pH (Fig. 4D). This result suggests the pH responsivity of the nanocarriers. 3.5. Determination of the dose dependent cytotoxicity of PBA-ZnO-Q nanohybrids

3.6. Determination of ZnO-quercetin nanohybrid induced oxidative stress

To determine the cytotoxic efficiency of the newly synthesized ZnO nanoparticles MTT cell viability assay was carried out on the MCF-7

To investigate the underlying rationale behind the cytotoxicity of 135

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Fig. 3. (A) Dynamic Light Scattering and (B) Zeta Potential of ZnO (red), PBA-ZnO (green) and PBA-ZnO-Q (orange). (C) Time-dependent release of Quercetin from the nanoconjugate at various pH: 5 (blue), 6 (red) and 7.4 (green). All values are expressed as mean ± SD. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

PBA-ZnO-Q in the MCF 7 cells, intracellular ROS level and MMP was determined by performing FACS analysis with DCFDA and Rhodamine 123 dye respectively. In line with the previous MTT cell viability results, it was observed that PBA-ZnO-Q could significantly enhance the level of intracellular ROS dose dependently (increase in the green fluorescence compared to normal cells) (Fig. 5D). In the other experiments with Rhodamine 123, decreasing fluorescent intensity in the PBA-ZnO-Q exposed cells compared to the control cells indicate that exposure to the nanohybrid can decrease the MMP, thereby induce mitochondrial dysfunction in a dose dependent manner (Fig. 5E). A combinatorial effect of PBA-ZnO-Q was observed compared to PBA-ZnO or free Q in the cells for both ROS and MMP. An increase in the level of intracellular ROS implies the occurrence of oxidative stress [57]. At the basal level, ROS is beneficial in promoting cell growth and differentiation. However, the accumulation of intracellular ROS beyond the threshold level primarily lead to irreversible impairment in different vital cellular functions, and further its excessive accumulation causes cell death [58]. Literature suggests that the occurrence of oxidative stress is associated with the downregulation of mitochondrial membrane protein, Bcl2 and simultaneous upregulation of Bax. Thereby, it causes pores in the mitochondrial membrane and results in the release of cytochrome C in the cytosol. It causes

depolarisation of the mitochondrial membrane, and these events are directly linked with the activation of the extrinsic and intrinsic pathway of apoptosis [59]. 3.7. Determination of quercetin accumulation in tumor tissues Upon administration of the nanohybrids, as described in the Section 2.2.9, it was observed that on all the cases for both time intervals, quercetin had been distributed in the tumor tissue. Results indicated that ZnO nanoparticle enhanced the tissue absorption of quercetin and functionalization of the nanoparticle with PBA is further beneficial in increasing the tissue absorption of quercetin. It was also observed that after 6 h of treatment the presence of quercetin was detected more in the tumor tissue compared to the tumor tissue dissected out after 24 h of treatment (Fig. 6A). Literature suggests that PBA functionalization increases the stability and tissue absorption of the nanoparticles [59]. The presence of more amount sialic acid receptor in the cancer cells compared to the normal cells can be attributed to the reason behind increased tissue absorption of the PBA functionalised ZnO nanoparticles [60].

Table 2 Time dependent change of hydrodynamic size and PDI of the nanocarrier. PBA-ZnO-Q

Average SD

Day 0

Day 7

Day 14

Size

PDI

Size

PDI

Size

PDI

252.65 66.110538

0.59825 0.161362

264.43333 64.503127

0.549 0.179853

273.9667 48.98381

0.603 0.127526

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Fig. 4. (A) Fluorescence microscopic image of PBA-ZnO-Q NPs obtained at 40× magnification. (B) Intracellular uptake of the ZnO and PBA-ZnO NPs showing FITC fluorescence in the MCF-7 cells, fluorescent micrographs obtained at 40× magnification, DAPI fluorescence indicates the nucleus of the cells. (C) FACS analysis of MCF-7 cells exposed to different nanoformulations. (D) Quantitative estimation of intracellular release of quercetin from PBA-ZnO-Q in MCF-7 and MCF-10a cells after 48 h of incubation. All values are expressed as mean ± SD.

nephrotoxicity and hepatotoxicity compared to the control mice. For hepatotoxicity, ALT and ALP levels indicate a sharp increase in the tumor control animals. Treatment with PBA-ZnO and PBA-ZnO-Q did not cause any added toxic effects in the tumor bearing mice (Fig. 7A and B). Similar results were observed when BUN and creatinine were measured in blood serum (Fig. 7C and D). Moreover, these nanoparticles were found to reduce tumor associated toxicity in liver and kidney. The effectiveness of free quercetin administration can be attributed to the prophylactic role of quercetin against cellular stress in liver and kidney. The amount of quercetin used for both free quercetin and PBA-ZnO-Q groups is similar. In the PBA-ZnO-Q treatment group, the accumulation of quercetin in liver and kidney is expected to be less due to the tumor-targeting property of the nanoparticle. On the other hand, in the free quercetin treatment group, quercetin accumulation in liver and kidney is expected to be higher, thereby imparting better beneficial role against hepatotoxicity and nephrotoxicity compared to animals treated with PBA-ZnO-Q. In the PBA-ZnO-Q administered animals, a reduction in the tumorinduced splenomegaly was observed compared to the untreated group (Fig. 7E). The reduction in splenomegaly in the tumor bearing animals can be attributed to the anti-inflammatory property of quercetin [61]. This results overall indicated that the dose used for free quercetin and different nanoparticles in the experimental protocol are physiologically relevant and do not have any adverse effect on the other vital organs of a living system.

3.8. Determination of in vivo antitumor activity In EAC solid tumor bearing Swiss albino mice, it has been observed that the newly synthesized PBA conjugated and quercetin loaded ZnO nanohybrid (PBA-ZnO-Q) showed enhanced antitumor activity (reduce tumor mass and volume) compared to only nanoparticles and free quercetin treated animals. From the curve of relative change in tumor volume among the several treatment groups, it is observed that the tumor size was grown continuously up to 14 days in the untreated tumor control group (Fig. 6B, C). Comparative statistical analysis between the animals treated with PBA-ZnO NPs and free quercetin showed a delayed increase in tumor volume over time. Intravenous administration of 33 mg/kg bodyweight of PBA-ZnO-Q is found to decrease the tumor volume compared to the tumor control animals, thereby exerting maximum tumor inhibition effect. In the animals treated with PBA-ZnO-Q, a significant combinatorial anticancer effect was observed compared to the PBA-ZnO NPs or free quercetin treated groups (Fig. 6B, C & D). These experimental data indicated that ZnO NPs enhances the antitumor efficacy of quercetin mainly by increasing its accumulation in the tumor tissue. We did not get any significant loss in body weight profile among the different experimental groups used in the study (Fig. 6E), which indicated that PBA-ZnO and PBA-ZnO-Q did not induce any systemic toxicity. 3.9. Determination of systemic toxicity following administration of nanoparticles in tumor bearing mice

4. Conclusion

Upon administration of nanoparticles, as described in the Section 2.2.10, different serum biomarkers were determined from the blood serum. It was observed that induction of EAC solid tumor causes

Overall, the results indicate the effectiveness of our synthesized ZnO NPs as a potential drug delivery system for loading and delivering 137

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Fig. 5. Analysis of cytotoxic potential of the nanoformulations. (A) MTT cell viability in MCF-7 cells, (B) bright field micrographs of MCF-7 cells obtained at 10× magnification. All values are expressed as mean ± SD. (C) MTT cell viability in MCF-10a cells. (D) Determination of intracellular ROS level using DCFDA staining by FACS analysis. (E) Determination of mitochondrial membrane potential using Rhodamine 123 staining by FACS analysis.

hydrophobic anticancer drugs. Conjugation with PBA is found to enhance its absorption in the tumor tissue through the interaction with the sialic acid (overexpressed in the cancer cells). Most interestingly, both the in vitro and in vivo results indicate that ZnO based nanoformulations can significantly enhance the anticancer effect of quercetin by increasing its bioavailability. Use of bioactive natural compounds (like quercetin) as anticancer drug candidate is advantageous than the conventionally used chemically synthesized drugs. These kinds

of molecules have differential cytotoxic effect in cancer cells and are nontoxic to the other vital organs [62,63]. Due to high metabolic activity, the oxidative load on the cancer cells is much higher than the normal cells. Therefore, cancer cells are generally more viable to external oxidative insults. Disruption of redox homeostasis in the cancer cells is regarded as the most convenient way to selectively kill the cancer cells without causing significant alteration in homeostasis of the other organs [54]. In this study, it was found that PBA-ZnO-Q can

Fig. 6. (A) Determination of tissue absorption of quercetin in different experimental animals. (B) Representative photographs of the tumor tissues dissected out from different experimental groups. (C) Relative change in tumor volumes expressed in cubic centimeter in the course of nanoparticle administration. (D) Tumor mass expressed in grams. (E) Relative change in body weight expressed in gram in the course of nanoparticle administration. All values are expressed as mean ± SD. 138

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Fig. 7. Analysis of systemic toxicity in different experimental animals. (A) ALT level. (B) ALP level. (C) BUN level. (D) Creatinine level. All values are expressed as mean ± SD. (E) Representative photographs of spleen dissected out from different experimental groups. “*” represents significant differences with tumor control animals.

induce profound cytotoxic effect via the combinatorial ROS enhancement effects of PBS-ZnO and free Q in cancer cells. Apart from its anticancer effect, PBA-ZnO-Q did not show any sign of systemic toxicity in tumor bearing mice and was found to reduce tumor associated toxicity in liver, kidney and spleen. Overall our study leads to the development of improved targeted therapy for the treatment of breast cancer which might also lead to clinical trials in the near future.

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