Luteolin-Fabricated ZnO Nanostructures Showed PLK-1 Mediated Anti-Breast Cancer Activity

The present work describes a facile and convenient procedure for synthesizing zinc oxide nanoparticles using luteolin isolated from Eclipta alba plant (L-ZnONPs) at room temperature. The formation of as-grown L-ZnONPs was confirmed by X-ray diffraction analysis (XRD), scanning electron microscopy (SEM), transmission electron microscopy (TEM), high-resolution transmission electron microscopy (HR-TEM), and selected area electron diffraction (SAED). The Wurtzite structure of ZnO was observed by its hexagonal phases in diffraction patterns. The SEM images revealed the different sizes and morphologies of L-ZnONPs, with diameters between 12 and 25 nm. The HR-TEM result showed that the inter-planar distance between two lattice fringes was 0.262 nm, which coincides with the d-spacing of (002) and (101) lattice planes of the as-obtained material. The anticancer activity of L-ZnONPs against the breast cancer cell line MCF-7 was greater as compared to that of luteolin or ZnO alone. The mechanistic evaluation of such an activity carried out using in silico methods suggested that the anti-breast cancer activity of L-ZnONPs was mediated by polo-like kinase 1 (PLK1) proteins.


Introduction
Recent advances in the field of nanoscience and nanotechnology, with a particular aptitude for the preparation of highly ordered nanoparticulates of all types of morphologies, have led to the development of novel materials at the nanoscale level. The large realm in the field of nanoscience lies in the fact that nanoparticles deliver desirable properties and have wide applications in highly functional and effective therapeutic, catalytic, sensing, and photoelectronic devices [1][2][3][4][5][6]. Among metal oxide nanoparticles, zinc oxide is interesting due to its enormous range of applications in various areas such as the medical, optical, magnetic, and gas sensing fields. In addition to these properties, the ZnO nanostructure demonstrates high catalytic efficiency and strong adsorption ability, and is used routinely in the manufacture of sunscreens [7], in ceramics and rubber processing, in wastewater treatment, and as an antimicrobial agent [8,9].
The development of various processes for the synthesis of nano-and micro-scaled inorganic materials has contributed to an understanding of a relatively new and largely unexplored area of research based on the biosynthesis of nanomaterials. Plant extracts possessing metal-interacting multi-functional groups such as hydroxyl, carboxyl, and heteroaromatic rings offer an excellent opportunity to develop eco-friendly and cost-effective nanostructures that exhibit enhanced biological significance [10][11][12]. Biological applications of green synthesized ZnONPs have gained ample interest in the present scientific scenario [13,14]. Although there are many reports explaining the extraction and isolation of phytomolecules from Eclipta alba (E. alba) and their biological potencies [15][16][17][18], no work has been reported on the use of these phytomolecules in capping the metal nanoparticles. In this study, we aimed to synthesize ZnO nanoparticles using a phytomolecule, luteolin, isolated from E. alba. The above zinc oxide nanoparticles synthesized using luteolin isolated from Eclipta alba (L-ZnONPs) were further screened for their tumoricidal efficacy.
Furthermore, L-ZnONPs have shown very effective inhibition of several genes involved in breast cancer signaling pathways. Moreover, L-ZnONP-protein interactions have been analyzed using in silico molecular docking approaches. Molecular interaction studies reveal the ability of the ligand/small molecule to bind to the specific protein by forming hydrophobic and non-hydrophobic bonds, thus modifying its expressions and functions.
Meanwhile, zinc oxide (ZnO) nanoparticles, which represent a versatile drug delivery tool, have recently been reported to possess significant tumoricidal activity via ROS generation or the caspase-8 and p53 pathway [51][52][53][54]. However, a better understanding of the mechanistic mode and the resultant cellular consequences is essential. Although the metal oxide has been considered by the US FDA to be a "generally recognized as safe" (GRAS) substance [55], this categorization typically applies to substances that are larger than a micron. Hence, it may be deemed necessary to evaluate the cytotoxicity of the same in both in vitro and in vivo systems.

Materials and Methods
The precursors, anhydrous zinc acetate (Zn(Oac) 2 ) and pristine ZnONPs, were obtained from S.D. Fine Chemicals Ltd. (Mumbai, India), while ethanol and acetone were purchased from Merck Chemical Suppliers (Pune, India). Deionized water collected from an ELGA RO water purifier was used throughout the experiments (Elga Veolia, Lane End, UK). Powder XRD values were recorded on Bruker X-ray diffractometer with a scan range of 20-80 • at a 2 • /min scan rate using Cu Kα (1.5406 Å) radiation (Bruker, Karlsruhe, Germany). Scanning electron microscopy (SEM) and X-ray mapping images were recorded on a Zeiss microscope (Carl Zeiss, White Plains, NY, USA). Transmission electron microscopy (TEM) images and SAED patterns were recorded on a JEOL 2100F FEG apparatus operating at 200 kV after casting a drop of L-ZnONP for dispersion in ethanol over a Cu grid (Jeol, Akishima, Tokyo, Japan). The 1 H-NMR spectrum was recorded on a Bruker AC (300 MHz, Yokohama, Japan) spectrometer using tetramethylsilane (TMS) as an internal standard in DMSO-d 6 solvent. Chemical shifts (δ) are expressed in ppm. Mass spectra were recorded on a Waters SYNAPT G2 mass spectrometer (Malvern, UK) using electrospray ionization (ESI-TOF) operating at an ionization potential of 70 eV.

Plant Material Collection and Extraction of Eclipta alba Phytochemicals
Whole plants of E. alba were collected from Srirangapatna, Karnataka, India, (Geographical coordinates: 12.4237 • N, 76.6829 • E) from May to September 2019. Plant identification was unambiguously performed, and a voucher specimen (No. FLSDWH201) was deposited at the herbarium at the Department of Water and Health, JSS Academy of Higher Education and Research (Mysuru, India). The samples were shade-dried, homogenized using a mixer, and subjected to extraction using various solvents. Fifty grams of the coarse powder of the plant were subjected to hot solvent extraction using methanol (99%). The resulting filtrate was concentrated under a vacuum using a rotary evaporator (Rotavapor R-200, Buchi, Geneva, Switzerland), and the yield of methanol extract was recorded. The extract was further subjected to phytochemical screening in order to evaluate the phyto-constituents based on standard protocols.

Preparation of L-ZnONPs
The synthesis of L-ZnONPs was carried out according to the procedure mentioned previously [56]. An aqueous solution of Zn(Oac) 2 (0.115 g in 10 mL) and luteolin (0.069 g in 10 mL water) was mixed and stirred for 3 h at room temperature (a pH of 8.5 was maintained during synthesis using sodium bicarbonate). The white precipitate formed was filtered off using Whatmann No.1 filter (Analytics, Mumbai, India) paper and washed with ethanol (×5 times) in order to remove the adhered impurities. The above sample was then subjected to calcination in a preheated furnace at 400 • C for 3 h and used for further studies. We could reproduce the experimental results through the aforementioned synthetic procedure. The formation of L-ZnONPs was via 2,3-dihydoxyl groups present in the luteolin molecule, which were oxidized by the reducing the zinc ions to ZnONPs with their electron-donating abilities. The as-obtained luteolin-decorated ZnO nanostructures are depicted in Figure 2. The FT-IR and UV-Visible spectra of the as-obtained L-ZnONPs are depicted in Figures S3 and S4.

Measurement of Cell Viability Using MTT Assay
The MTT assay was performed as previously described by Denizot and Lang (1986) [57]. After 24 h, the treated cells were fixed using MTT reagent (5 mg/mL) in each well; cells were incubated at 37 • C for 1 h and centrifuged at 3000 rpm for 5 min. Plates were removed from centrifuge and the excess dye was washed, with 100 µL of DMSO added to solubilize the crystal. Optical density (OD) was taken at 570 nm, and percentage of inhibition was calculated using the formula mentioned below. The observations were represented graphi-cally. Statistical one-way ANOVA analysis followed by Tukey's test were conducted using the Prism 8 statistical analysis tool (GraphPad Software, San Diego, CA, USA).

% Inhibition = [(OD of control -OD of sample)/OD of control] × 100
(1) 2.6. In Silico Anticancer Study 2.6.1. Ligand Optimization Using Bioinformatics Software Chemsketch 12.0 software was used to sketch the two-dimensional structure of the L-ZnONPs. After sketching, the structure was cleaned and the explicit hydrogens were added and saved in a file in .cml format for further use. For the molecular docking purpose we required the .pdb format file of the ligand, and hence the 2D .cml format file of the L-ZnONPs was converted to 3D .pdb format file by generating the 3D coordinates using

Protein Modeling, Validation, and Preparation Using Bioinformatics Software and Tools
Based on earlier reports, in this study six proteins (human polo-like kinase 1 (PDB Id: 1Q40), human protein kinase C (PDB Id: 2FK9), human HER2 kinase domain (PDB Id: 3PP0), human EGFR/HER3 kinase (PDB Id: 4RIW), human ataxia telangiectasia-mutated and Rad3-related (PDB Id: 5YZ0), and human vaccinia-related kinase 1 (PDB Id: 2LAV)), were considered for in silico validation of the mechanistic interactions responsible for L-ZnONP activity against cancer cells.
The 3D structures (.pdb format) of the above proteins were taken from the Protein Data Bank database (PDB) (https://www.rcsb.org/, (accessed on 28 November 2000)) depending on their resolution values (≥2Å) [60]. The downloaded .pdb format files of all the proteins were visualized using Chimera v1.3.7 software [61] (https://www.cgl.ucsf.edu/chimera/ download.html, (accessed on 18 December 2020)) to edit the protein by deleting water and the other non-standard amino acids present with the protein (Figure 4A-F). The structures of the edited proteins were validated by the Ramachandran plot using RAMPAGE online tool in order to learn the number of residues in the favored and allowed regions [62]. The protein structure with~96% of residues in the favored region and~2 residues in the allowed region was selected for the molecular docking interaction purpose.

Binding Site Residues
The active site pocket residues or the binding site residues where the ligand interacted with the protein molecule of all the selected proteins were obtained using the Galaxy web online tool http://galaxy.seoklab.org/cgi-bin/submit.cgi?type=REFINE, (accessed on 11 October 2020) [63].

Molecular Interaction Studies
The validated proteins were now fit for the study of molecular interactions with the optimized ligand using the freely available and user-friendly PyRx 0.7 molecular docking software [64]. PyRx follows three main steps.
Step one is to load the respective protein and L-ZnONP ligand to make the macromolecules and ligands to generate the .pdbqt files that have all of the required structural parameters for docking purposes. The second step is to select the binding site amino acid residues and build a grid box around the selected residues. In the final step, the docking process gets started by considering the genetic algorithm.

Statistical Analysis
The results of anticancer activity are calculated as mean ± SE of three independent experiments. One-way analysis of variance (ANOVA) followed by Tukey's multiple comparisons were carried out using the GraphPad Prism 8.0 statistical analysis software.

XRD Studies
The diffraction pattern was in accordance with the common ZnO hexagonal phase, i.e., the Wurtzite structure (JCPDS 36-1451) as manifested in Figure 5

SEM Analysis
The morphology of L-ZnONPs under study was mainly composed of nanospheres and nanosheets with an average size ranging between 12 and 25 nm ( Figure 6). It can be clearly seen from this Figure that the morphology is comprised of dense cloud with randomly oriented, overlapping nanosheets and cluttered nanospheres.

TEM Analysis
The size and morphology of the as-obtained L-ZnONPs was further confirmed by TEM studies. As shown in Figure 7a, the TEM analysis of L-ZnONPs confirms that the particles reported here were almost hexagonal with particle size of approximately 17 nm, which is consistent with the observed morphology in SEM investigations. In addition, we can also observe spherical and rod-shaped nanostructures with some agglomerations of larger and smaller particles. The crystallinity results obtained by XRD analysis were further supported by HRTEM studies. The diffraction lattice fringes (Figure 7b) in the obtained L-ZnONPs show the d-spacing between two lattice fringes. In our case, the inter-planar distance between the two fringes was 0.262 nm, which corresponds with the d-spacing of the (002) crystal plane of ZnO [12].

L-ZnONPs Showed Greater Cytotoxicity in the MCF-7 Cell Line
All the treatment groups (luteolin, ZnO, and L-ZnONPs) showed dose-dependent cytotoxicity in MCF-7. However, the cytotoxicity in the treatment group containing the nanoparticle-coated luteolin was significantly greater than that of the individual treatments of the compound and ZnO. The cells were treated in hypoxic conditions for 24 h, with differential concentrations of the test samples ranging from 2.5 to 40 µM. While the highest concentration of luteolin showed cell growth inhibition of~52%, the MCF-7 cell growth in the ZnO nanoparticle-treated group was inhibited by up to~25% with a similar dosage ( Figure 8).
Surprisingly, the anti-breast cancer potential against the MCF-7 cells was evidently greater with L-ZnONP treatment at a 40-µM concentration, with the number of viable cells reduced to a paltry 15%. Luteolin has been reported to have an IC 50 value of about 43 µM for the breast cancer cell line MCF-7 [35]. Furthermore, the nanoparticle delivery of luteolin was found to reduce its IC 50 value in both in vitro and in vivo models [65], thereby suggesting that ZnO nanoparticle-guided delivery of luteolin improves its antitumorigenic activity.

In Silico Protein Validation
In this study, all the selected proteins showed over 96% of residues in the favored region and more than 2% of residues in the allowed region; thus, the RAMPAGE results were significant for considering proteins for further molecular interactions (Table 1).

Molecular Docking Studies
After the docking process, the six docked poses of the L-ZnONP system against the particular protein were obtained based on the increasing value of the binding affinity. The pose with the least binding affinity was selected and its .pdb format file was saved.
The docked ligand result file with the protein .pdb was visualized using Pymol 1.4.1 software https://pymol.org/2/, (accessed on 25 January 2021) to check for bonded and non-bonded interactions between the ligand and the protein [66].

Interaction of L-ZnONPs with Proteins
Among the six selected proteins, L-ZnONPs showed significant interactions with 1Q4O, 3PP0, and 2LAV by forming 11, 6, and 5 hydrogen bonds with values of −9.7, −8.3 and −10.1 for binding affinity, respectively ( Figures 9A, 10-13 and 14B). The other three proteins 4RIW, 2FK9, and 5YZ0 showed comparatively less i.e., 3 (−5.2), 2 (−7.6) and no (−7.5) hydrogen bonding with the L-ZnONPs, respectively. The best docked poses of the ligand with the selected six proteins were found to have conserved salt bridges with large numbers of bonded and non-bonded interactions ( Figure S5).
Based on the above results, it was hypothesized that the L-ZnONPs inhibited the MCF-7 cell proliferation by the means of molecular interactions involving the human polo-like kinase 1 (PLK1) protein [34,42].

Conclusions
In the present study, we obtained zinc oxide nanoparticles with a convenient green approach using a phyto-molecule, luteolin, isolated from Eclipta alba. The analysis of the as-obtained L-ZnONPs by electron microscope studies revealed that the prepared material assumed a hexagonal shape with particle size of approximately 17 nm. The d-spacing between two lattice fringes was shown to be 0.262 nm, corresponding with the d-spacing of the (002) crystal plane of ZnO. The luteolin-capped ZnONPs showed better tumoricidal behavior as compared to the two other components when tested individually. Based on in silico observations it may be hypothesized that the MCF-7 cytotoxicity of L-ZnONPs occurs via the involvement of the PLK1 proteins.

Conflicts of Interest:
There are no potential conflict of interest between the authors in publishing this work.