Apoptosis-Mediated Anticancer Activity of Zinc Oxide Nanoparticles Derived and Characterized from Halophila beccarii

introduction: 1.INTRODUCTION Nanotechnology is one of the most emerging trends which has brought prodigious growth in recent years, which aims to synthesize materials in nano scale [1] and proved to be a blessing in therapeutic field as it opens new prospects for the treatment of various disorders. Although a variety of physical and chemical techniques are used to build nanoparticles, the green approach found to be more dependable due to its significant implications. The conventional chemical mode is more laborious, over-priced and make use of various chemical compounds for reducing, stabilizing and capping which end up with toxic by-products. Plants, Fungi, bacteria and algae are the prolific resources for bio-fabrication and chemical profiles especially from different plants is a growing trend which is considered much more simple, non-toxic and effective than other sources. Because of their unique biological characteristics, oxide nanomaterials prepared with metals are a major concern. Of these, zinc oxide nanoparticles (ZnONPs) have drawn a lot of attention due to their numerous biomedical uses, including anti-inflammatory, wound-healing, anti-cancerous, anti-oxidant, and photochemical properties [2]. When compared with terrestrial plants, marine plants are remarkable source of medicinal properties as they are mostly grown in extreme conditions like pressure, salinity and temperature [3]. Marine plants, including seagrasses, have evolved as an appealing for the bioinspiration of nanoparticles due to their ample bioactive compounds and distinctive environmental adaptations. Seagrasses are marine angiosperms and the only flowering plants that grow in marine environment. Around 60 species of marine seagrass are divided into four families–Cymodoceaceae, Posidoneaceae, Zosteraceae, and hydrocharitaceae. Previous literature reported that there is an inadequate exploration regarding Halophila beccarii which belongs to hydrocharitaceae family, in contrast to experimental research on other seagrass species, including Enhalus acoroides , Thalassia hemprichii, Cymodocea serrulata, Halophila ovalis, and Halophila decipens [4]. Keeping in view the importance of seagrasses, Halophila beccarii was chosen for developing ZnONPs and investigate their effects on the apoptosis and cell division of triple negative breast cancer cells, BT-549. Apoptosis plays a significant part in regression of tumor cells. Which is initiated through mitochondrial / intrinsic or extrinsic /death receptor pathways. Both pathways lead to activation of caspases which are essential in dismantling the cell by cytoplasmic shrinkage, condensation of the nuclear cells and membrane blebbing leading to cell death. This research aims to explore the functional role of Halophila beccarii derived ZnONPs (Hb-ZnONPs) in the induction of apoptosis. materials and methods: 2. Materials and Methods 2.1 Fabrication of Zinc oxide nanoparticles The Seagrass, H. beccarii collected from Bay of Bengal was subjected to aqueous extraction by the method standardized in our laboratory [5]. The ZnO NPs were synthesized through seagrass induced process by adding 2mg of aqueous extract of H.beccarii to 100 ml of 0.02 M Zinc-acetate dihydrate [6]. Alkaline conditions with pH at 12 were maintained by 1M NaOH and stirring was continued for 4h to obtain the precipitate. Further, the white precipitate was subjected to ultrasonication for 15min and centrifugation for 5min at 7500g in order to collect Hb-ZnONPs. The nano particles were stabilized by calcination at 100ºC for 4 h. 2.2 Characterization of ZnO nano particles Ultraviolet -visible Spectrophotometer (UV-1601, SHIMADZU) was utilized initially to confirm the nanoparticle formation based on the absorption maxima [7]. Using an FTIR spectrophotometer (BRUKER / ALPHA-T), the different functional groups linked to the synthesis of Hb-ZnONPs were identified in the 400 cm-1 to 4000 cm-1 range [8]. Scherrer's formula was utilized in X-ray diffraction (Malvern PANalytical-AERIES) pattern to comprehend the crystalline nature and behavior of the reduced Hb-ZnONPs [9]. Further the morphology and aggregations of Hb-ZnONPs was assessed through scanning electron microscopy (JSM-IT500-JEOL) [10]. Energy Dispersive X-Ray (EDX) analysis was used to determine the elemental composition [11]. The magnitude and stability of the Hb-ZnONPs was measured by using a Dynamic light scattering instrument (HORIBA SZ-100) [12, 13] and the surface topology was captured with Atomic force microscopy (A100-AFM)[14]. 2.3 Propagation of Cell lines Triple negative breast cancer BT-549 cells and Raw 264.7 cells (macrophage like cells) were purchased from National centre for cell sciences (NCCS), Pune. They were maintained in Dulbecco Modified Eagle Medium comprising 10% heat -inactivated fetal bovine serum (FBS) at 37o C in a 5% CO2 incubator. The cells were allowed to grow until they reach 85% confluency. 2.4 Cytotoxicity analysis The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was used to study the cell proliferation through oxidative phosphorylation by measuring the intensity of the formazan crystals at 570 nm [15]. Both Raw 264.7 and BT-549 cells at a population of 4×103 were cultured in 96 well plate for 12-24 h under 5% of CO2 in a humidified chamber. Various concentrations of aqueous extract (100 µg, 200 µg, 300 µg, 400 µg, 500 µg) and Hb-ZnONPs (5 µg, 10 µg, 20 µg, 40 µg, 80 µg) were added to both Raw 264.7 and BT-549 cells in triplicates and left it for incubation for 24 h. After incubation, the cells were treated with MTT (5 mg/ml) after complete aspiration of spent medium and incubated for four hours. The formazan crystals formed after incubation were dissolved in 100 µl of dimethyl sulfoxide for 5 min and absorbance was measured at 570 nm (SpectraMax M5 – Molecular Devices). Data was analyzed using Graph-pad prism 1010 (316) and the probability impact was calculated using one way ANOVA. 2.5 Apoptosis detection using flow cytometry Annexin V-FITC and propidium iodide method was adopted for the quantitative measurement of apoptotic cells [16]. BT-549 cell lines at 2X104cells/ml were cultured in 12 well plates in the presence of aqueous extract (100µg/ml) and two concentrations of Hb-ZnONPs (5µg/ml and 10µg/ml) for 12 h. Following a 12 h incubation period, the cells were rinsed and reconstituted in 50 µl of binding buffer containing 05 µl of Annexin V-FITC. They were then incubated for 15 minutes at 4ºC in a dark environment. After incubation, the cells in each well were stained with 5µl of propidium iodide and incubated for 5min Finally, the cells were transferred to cytometry tubes for quantitative analysis using flow cytometry (Sysmax C1/VSC/NSLPOR/005). results: 3.Results 3.1 Synthesis and Characterization of ZnO nanoparticles The reaction between phytochemicals of H. beccarii and Zinc acetate under alkaline conditions results in white precipitate supports seagrass mediated synthesis of Hb-ZnONPs (Fig.1). 3.2 Functional features of ZnONPs derived from H.beccarii The fabrication of Hb-ZnONPs was confirmed based on the distinct peak centered around 367 nm (Fig. 2a) with a band gap energy 336eV. FT-IR spectra serve as a molecular fingerprint to determine the functional groups of the seagrass chemical profile intricated in the reduction and capping of Hb-ZnONPs. The intense broad spectrum at 3424 cm-1 in aqueous extract and 3417 cm-1 in Hb-ZnONPs represent the O-H stretching. The slight shift from 3424 cm-1 to 3417 cm-1 suggests an interaction between the O-H groups and ZnO. The signatures at 2930 cm-1, 1633 cm-1, 1414 cm-1, 1075 cm-1 in aqueous extract correspond to the C-H and CH2 stretch in aliphatic hydrocarbons, C=O in amide groups or C=C stretching in aromatic groups, C-O stretching vibration in phenolic compounds respectively. The spectra at 478 cm-1 represents metal-oxygen stretching vibrations. The absorption of infrared light at 2922 cm-1, 1565 cm-1, 1412 cm-1 & 1052 cm-1 were observed in Hb-ZnONPs. The spectra shifted from 1633 cm-1 in aqueous extract towards a lower wave number (1565 cm-1) in Hb-ZnONPs suggest that the carbonyl or aromatic groups are involved in reduction and capping of the Hb-ZnONPs. The molecular signature at 491 cm-1 reflects the formation of Zinc oxide (Fig.2b). The mean size of Hb-ZnONPs was 35 nm (Fig. 2c) and further the zeta potential is important parameter for measuring the dispersion stability and electrostatic potential of Hb-ZnONPs was found to be -447mV (Fig. 2d). 3.3 Composition and morphological properties of Hb-ZnONPs SEM micrograph of ZnO nanoparticles indicates hexagonal shape with varied sizes between 20 and 80 nm (Fig. 3a). Majority of the Hb-ZnONPs were found to be with 36 nm size, which is in accordance with the AFM analysis ,where the calcinated Hb-ZnONPs were in hexagonal shape with low level of agglomeration. EDX profile demonstrated strong emission spectra of Zn at ~1keV and 87keV and minor peak of carbon at 03keV and oxygen 05kev (Fig-3b). which accounts for 62.3% Zn, 25.79% oxygen and 11.9 discussion: 4.Discussion Plants have been extensively exploited for the fabrication of metal oxide nanoparticles, including gold, silver, and zinc oxide. However, there are limited reports on the use of seagrasses in synthesizing zinc oxide nanoparticles. The appearance of a white coloration, resulting from the reduction of zinc acetate precursor by seagrass metabolites, is considered to be an initial indicator. Several techniques were used to specify nanoparticles and speculate their utmost properties including size, shape, surface charge and elemental composition of the nanoparticles. UV- Spectroscopy provided insights about the optical behaviour and material properties of Hb-ZnONPs, which exhibited a band gap energy of 337eV with an absorption peak at 367 nm. The results are in line with earlier observations, from the ZnONPs with characteristic absorption peak at 350nm synthesized using Cymodocea serrulate, a seagrass. The band gap energy of Hb-ZnONPs is consistent with that of ZnONPs reported from the leaf extract of Sonneratia alba [17] and Helichrysum chymosum [18]. FTIR results were utilized to consider the reactive groups of phytochemical molecules responsible for the reduction and stabilization of Hb-ZnONPs. The peaks at 2930 cm⁻¹, 1633 cm⁻¹, 1414 cm⁻¹, and 1075 cm⁻¹ in the aqueous extract correspond to C-H and CH₂ stretching in aliphatic hydrocarbons, C=O or C=C stretching in amide or aryl or cyclic groups, and carbonyl (C-O) in phenolic compounds respectively and are good players in the reduction of Hb-ZnONPs. The peak at 478 cm-1represents the stretching vibrational mode of ZnONPs [17]. The peaks at 2922 cm-1, 1565 cm-1, 1412 cm-1 & 1052 cm-1were observed in ZnONPs [19]. The stretching fingers of 656-432 cm-1 represents the metal oxide properties of nanoparticles [20]. Dynamic light scattering analysis revealed the average size of Hb-ZnONPs as 35 nm, while the zeta potential, indicating dispersion stability and electrostatic potential, was measured at -44.7 mV. The magnitude of zeta potential smaller than -30 mV and greater than +30 mV form stable suspension and prevent aggregation [21]. SEM, XRD and AFM results confirm the formation of crystalline and hexagonal particles with size approximately 36 nm, making them highly versatile for various applications [22] and the data confirms the consistency and reliability of the experimental results [23]. The prepared ZnO NPs contain 13- atoms distributed with 12-bonds among which 3-bonds are polyhedral. The wurtzite structure of ZnO NPs is central for their unique physical and chemical properties and the hexagonal arrangement influences their optical and surface behaviour [24]. EDX demonstrating the pure composition of particles with 62.3% Zn, 25.79% oxygen and 11.9 conclusion: 5.Conclusion Gaining knowledge about apoptosis opens the door to tumor-specific apoptosis treatment. This study demonstrated the synthesis of Hb-ZnONPs from seagrass and its characterization techniques manifest the presence of hexagonal and crystalline nano size particles. Further analysis, confirmed that the extracts from H beccarii and Hb-ZnONPs have the potential for inducing apoptosis and build immune competence in the host .These findings indicate that ZnO nanoparticles derived from H beccarii may effectively combat breast cancer by inducing apoptosis, highlighting their potential as multifunctional tools in cancer therapeutics