Kei-apple-mediated NiO nanoparticles and biological studies: anti-inflammatory and cytotoxicity study against HeLa and HEK 293 cell lines

Nickel oxide (NiO) nanoparticles are gaining popularity in multiple fields owing to their useful properties. The application in biomedicine has been further enhanced by combining them with plant extracts that possess unique biological properties. In this report, NiO nanoparticles were synthesized by mediating the reaction process with the leaf extracts of Kei-apple (Dovyallis Caffra), a local fruit tree found in South Africa. Various characterization techniques such as x-ray diffraction (XRD), V–Vis spectroscopy (UV–Vis), transmission electron microscope (TEM), Scanning electron microscope (SEM), and Energy dispersive x-ray analysis (EDX) were employed to establish the different physicochemical properties of the prepared NiO material. The XRD report obtained confirmed the formation of NiO nanoparticles with an average crystallite size of 9 mm. Furthermore, the microscopic techniques showed that the material possessed a spherical-shaped structure, with diameter sizes smaller than 27.18 nm (average size of about 11 nm). The purity of the material was shown in the EDX analysis, in which the primary composition was only the elemental constituents of the NiO nanoparticles. The nanoparticles exhibited good cytotoxicity comparable to the 5-Fluorouracil in both the Human embryo kidney (HEK 293) and Human cervical cancer (HeLa) cell lines, with a half maximal inhibitory concentration (IC50) value of 0.00015 and 13.8 μg ml−1, respectively. The anti-inflammatory study, on the other hand, exhibited a weak anti-inflammatory effect in the used Bovine serum albumin denaturation assay. The finding here thus suggests that the Kei-apple mediated NiO nanoparticles can be safely used in different fields without causing any appreciable harm to the human body due to the specificity to the cancerous cell line and the observed weak viability in the used non-cancerous embryonic kidney cells.


Introduction
Nanotechnology plays a crucial role in material science by providing new ways to manipulate and engineer materials at the nanoscale, leading to the development of advanced materials with unprecedented properties and functionalities [1]. These advancements have opened new opportunities for a wide range of applications in various fields, leading to significant developments in material science and technology [2]. Its global significance in biomedical sciences, cosmetics, energy science, environmental science, food science, and drug-gene delivery has been attributed to the minute size and high surface-to-volume ratio, which result in distinct chemical and physical properties from their bulk counterparts [3][4][5]. Consequently, nanomaterials-based products have thus garnered lots of attention such that the industry has been estimated to be about $55 billion by 2022 with an annual growth rate of 20% [6].
The role of metal-based nanoparticles in the field of nanotechnology cannot be farfetched due to their unique properties which have resulted in their wide applications in numerous areas of life [7]. In medicine, for Any further distribution of this work must maintain attribution to the author(s) and the title of the work, journal citation and DOI.
instance, metal nanoparticles have provided novel approaches for disease diagnostics and treatments which were initially thought to be impossible due to size limitations [3]. Hence, several properties such as ease of preparation, stability, ease of control in engineering size, shape, porosity, optical properties, and cellular penetration properties have made them highly desirable in various fields, especially medicine [3]. This has thus brought about increased attention and wide preparations using various means and technology. Metal oxide nanoparticles are one of the most prepared and used classes of metal-based nanoparticles due to their ease of preparation (which could be achieved by simply using plant extracts) and their outstanding properties [8]. They possess distinctive characteristics with far-reaching implications in the fields of science and technology including a high surface-to-volume ratio, extensive surface area, and widespread availability on Earth [9]. Other unique properties which made them applicable to a field like biomedical sciences include, the ease of incorporation into hydrophilic and hydrophobic systems and functionalization by various molecules due to their negatively charged surfaces [10]. Hence, they are currently mostly used in sensors, energy storage devices, biomedicine, catalysis, Magnetic Resonance Imaging (MRI), etc [11]. Moreover, their ease of preparation and environmental friendliness via facile approaches such as the use of plant extract makes them highly sort after amongst researchers [12].
Many synthetic methods have been used to prepare metal oxide nanoparticles and this has been well documented in literature [13]. However, due to the environmental concern because of the use of harmful reagents and by-products, green facile methods such as those involving the use of plant extract, which is costeffective, have provided a channel that is environmentally safe and more biocompatible for biological research [14]. To simply put, these synthetic approaches enable the convenient, swift, eco-friendly, and biocompatible production of various highly sort oxides [13]. One notable example of metal oxide that has caught the attention of many researchers is nickel oxide (NiO) nanoparticles due to their unique physical and chemical properties such as chemical stability, electro-catalysis, superconductivity, and electron transfer potentials which make them attractive for a variety of applications in electronics, energy storage, catalysis, and biomedicine [15]. Furthermore, Nickel oxide nanoparticles possess a wide band gap (3.7-4.0 eV) with an intrinsic p-type semiconducting property originating from the positive charge compensation at the thermodynamically favored Ni 2+ vacancies [16]. This has led to their wide studies in the area of energy storage and supercapacitors, anode material in electrochromic devices, photocatalysis for water purification, chemical process catalysis, and sensing [13]. Nickel oxide has been reported to possess unique properties such as effective hole-transport properties, high work function, and electron-blocking characteristics, alongside good thermal stability [13,16]. Generally, Ni compounds, according to the International Agency for Research on Cancer (IARC Monographs on the Evaluation of Carcinogenic Risks to Human, Volume 49, 1990), have been classified as class I carcinogenic material, which means they are relatively safe and possess lower toxicity [17]. Biologically, they have been reported to possess anti-inflammatory properties and also exhibit cytotoxic properties against cancer cell lines [11]. Mariam et al, have reported the exertion of cytotoxic properties by NiO and Ni nanoparticles against the human colorectal adenocarcinoma cell line (HT29) [18]. Furthermore, in their study, Ada et al reported a 20% induction of apoptosis and neurosis in the HeLa cells following the screening with NiO nanoparticles after a 16 h incubation period using double staining immunostaining assay [19]. Other biological properties of NiO nanoparticles that have been documented include anti-Alzheimer's, antioxidant, anti-leishmanial, and antimicrobial, Protein kinase (PK) inhibition, Alpha-amylase inhibition potential [11,15]. Seeing the biological potentials of this material we thus herein examined the use of Kei-apple leaves, which have been reported to possess numerous phytochemicals and in turn several medicinal properties, as a mediating agent for the synthesis of NiO nanoparticles. This material is then screened for cytotoxicity against cancer and human cell lines of human cervical cancer (HeLa) and normal human embryo kidney (HEK 293) cell lines using the MTT assay approach. Furthermore, the anti-inflammatory properties using Bovine serum albumin denaturation assay was also examined, seeing that most cytotoxic compound always possess a measure of anti-inflammatory potentials.

Collection, identification and preparation of kei-apple leaves
Fresh leaves of the Kei apple tree, scientifically known as Dovyalis caffra, were gathered from the prickly branches within the North-West University Garden in Mafikeng. After proper identification, the leaves underwent a thorough cleansing process with tap water and double-distilled water to eliminate any undesired substances. Subsequently, they were air-dried in the laboratory for a period of three weeks and finely powdered. Approximately 100 g of the obtained powder was combined with 1 l of deionized water and subjected to heating at 70°C for a duration of 2 h. The resulting mixture was then filtered through Whatman filter paper, which has a pore size capable of retaining particles with a diameter of 11 μm.

Preparation of kei-apple-mediated NiO nanoparticles
Subsequently, a modified version of the literature procedure was employed for the synthesis of NiO nanoparticles [20]. In order to maintain an alkaline environment, a small amount of NaOH was introduced into a solution containing 55 ml of Kei apple leaf extracts. Following this, 155 ml of 1 mM NiCl 2 ⋅6H 2 O was incorporated into the mixture and agitated for a duration of 1 h at 75°C. A noticeable colour transformation from dark to pale green occurred, indicating the formation of NiO nanoparticles. The obtained mixture was then subjected to centrifugation at 10,000 rpm for 15 min, followed by rinsing with a mixture of deionized water and ethanol to isolate the particles. After drying this material in an oven for 12 h, the particles were subjected to calcination in a muffle furnace at 410°C for 2 h, resulting in the formation of brown material.

Synthesis
Generally, plant extract-based nanoparticle synthesis proceeds in three stages: activation, growth, and termination [21]. During the activation phase, phytochemicals containing -OH groups in the plant extracts reduce metal ions from the precursor salt to a zero-valent state, leading to metal atom nucleation [22]. As the biological reduction process continues, separated metal atoms start to combine, resulting in the growth phase [21]. This phase leads to increased thermodynamic stability and the accumulation of the nanoparticles, which affects their morphology. The nanoparticles finally reach their maximum activity level, and plant metabolites cap their consistent morphology [21]. The diverse constituents of phytochemicals in the plant extracts can influence the resulting morphology of nanoparticles. Thus, the various components of the plants can affect the shape and biological usefulness of the prepared material [23]. A schematic representation of the synthesis and the mechanistic process is presented in figure 1.

2.3.
Characterization of the nanoparticles 2.3.1. X-ray powder diffraction (XRD) was carried to determine the phase and the crystallite size of the material using a Bruker D8-Advance x-ray diffractometer in locked coupled mode with Cu-Kα radiation (λ = 1.5406 Å) at a 40 kV tube voltage and 40 mA tube current. The diffraction pattern was recorded over the 2θ range of 35-80°, with a position-sensitive Lynx-Eye detector employed, and the data were collected at a speed of 0.5 s s −1 te −1 p −1 , corresponding to 92 s/step for a scintillation counter.

UV-Vis spectroscopy
The absorption spectrum was collected from Perkin Elmer Lambda 20UV-vis spectrophotometer at room temperature in the wavelength range of 250-700 nm. Transmission and Scanning electron microscopy and x-ray spectroscopy (EDS). The microscopic imaging of the material was performed using a ThermoFisher Scientific FEI Quanta 250 field emission gun scanning electron microscope operating at an accelerating voltage of 15 kV. For energy-dispersive x-ray spectroscopy (EDS) line analysis, the Oxford Inca software was employed.

Cytotoxicity study
The in vitro anticancer studies were conducted on the immortalized Human cervical carcinoma (HeLa) and human embryonic kidney (HEK 293) cells using standard procedures [24]. Upon procuring both cell lines from the ATCC, USA, these cells were cultured in EMEM medium with fetal bovine serum, 10%, streptomycin, 100 μg ml −1 and penicillin, 100 μg ml −1 , in a 25 cm 2 tissue culture flasks. To investigate the viability of the cells, the MTT assay was performed in a 96-well plate which already contain 2.5×10 2 cells/well in 100 μl EMEM cells. After incubating the cells at 38°C overnight, the medium was replaced. The prepared sample at different concertation of 10, 25, 50, and 100 μg ml −1 was immediately added to this mixture and then incubated at 37°C for 48 h, followed by the MTT assay. Treated and untreated cells + DMSO were used as control 1 and control 2, respectively. Also, the currently available anticancer drug of 5-Fluorouracil was used as the standard to compare the activity of the NiO nanoparticles. The medium in the assay was then replaced using 10% MTT reagent which was then followed by incubation for 4 h at 37°C. Formazan crystals were dissolved in 100 μl of DMSO, and the absorbance of the mixture in the 96 well plate at 570 nm was recorded with DMSO act as a blank. This was repeated three times.

Anti-inflammatory study
The Bovine serum albumin denaturation assay was used to investigate the anti-inflammatory potentials of NiO nanoparticles in this study, with the procedure modified from previously studies [25]. In this experimental procedure, varying concentrations of NiO nanoparticles or diclofenac at 50, 25, 12.5, 6.25, and 3.12 μM were mixed with 0.5 ml of egg albumin and 2.5 ml of phosphate-buffered saline solution (pH 6.4) in 2 ml volumes. The obtained mixtures were incubated for 15 min at 38°C and then boiled for 10 min at 70°C. After cooling to 25°C, 250 μl of the mixtures were put into a 96-well microplate. Their absorbances at 655 nm were measured using a microplate reader (680-BIORAD, USA) for the samples and diclofenac, with diclofenac serving as the standard drug. The was carried out thrice, and the measure of the percentage inhibition of protein denaturation was calculated using equation (1).
NiO Ct V NiO = the absorbance of NiO and diclofenac; V ct = absorbance of control.

Results and discussions
3.1. X-ray diffraction (XRD) analysis X-ray diffraction (XRD) study was used to ascertain the crystallinity and crystalline phase of the prepared NiO nanoparticles. The XRD peak pattern for the NiO nanoparticles is presented in figure 2, which showed that the crystal structure was face-cantered cubic NiO.  using Debye-Scherrer formula [12] and presented in table 1. This is well within the size range (9.8 nm) reported in literature on the facile synthesis of NiO at similar calcination temperature [12].

Optical properties
The optical absorption spectra of Kei-apple-mediated NiO nanoparticles were measured at room temperature in the wavelength range of 250-700 nm, as depicted in figure 3. The absorption spectrum was collected from Perkin Elmer Lambda 20UV-vis spectrophotometer. Prior to recording the UV-vis spectra, the samples were uniformly dispersed in distilled methanol and subjected to ultrasonication for 15-20 min. The optical absorption spectra of the NiO nanoparticles showed an absorption edge ranging from 300 nm to 450 nm, with the peak absorption occurring at approximately 369 nm. The peak absorption of the NiO nanoparticles exhibited a blue shift. This means that the shift of the absorption peak towards shorter wavelengths in the absorption spectrum and may be due to the surface plasmon resonance resulting in surface defects or crystallite size and quantum confinement of particles [27]. This effect arises from the quantum mechanical behaviour of electrons and results in changes to the energy levels and bandgap of the nanoparticles. Thus, this observed absorption band is associated with the electronic transition from the valence band to the conduction band in the NiO semiconductor [28]. The absorption band reported here is within the range in literature for other plantmediated NiO nanoparticles [11,27].

Morphology of Kei-apple mediated NiO nanoparticles
The morphology of the Kei-apple leaf-mediated NiO nanoparticles was studied using SEM and TEM, and the micrographs were obtained, as shown in figure 4. The particles were agglomerated and possessed a somewhat spherical shape which tends towards an irregular polyhedron morphology, as shown in figures 4(a)-(d), respectively. This is similar to the report made by Ascencio et al for NiO nanoparticles calcined at the temperature range of 300 to 600°C [29]. At low magnification, as shown in figure 3(d), the diameter size of the prepared nanoparticle was estimated using the ImageJ software and the size distribution histogram. The diameters ranged between 4.34 and 27.18 nm, with an average size of 11.38 nm for the nanoparticles for a count  of 50 particles, which agrees with the crystallite size estimated with XRD and other literature reports [29]. As already established in literature, the agglomeration observed is an indication of magnetic interaction and polymeric adherence among the particles [30]. Kei-apple leaf extracts thus showed a promising potential to effectively mediate the synthesis of NiO nanoparticles. Although most of the morphology reported for NiO nanoparticles in literature are spherical in nature [6,11,15], other morphologies which are polyhedral in nature have been reported for plant-mediated approaches. For instance, using V amygdalina leaf extracts, an octahedral-shaped morphology has been reported by Habtemariam and Omar [31]. Furthermore, the constituting elements of the nanoparticles were examined using energy-dispersive x-ray spectroscopy (EDS), which quantified the elemental constituent by showing only O and Ni peaks without any appearance of any other impurity peaks safe for carbon due to the used tape for sample support during analysis ( figure 4).

Cytotoxicity study
The MTT assay method was used to evaluate the cytotoxic effect of the prepared Kei-apple mediated NiO nanoparticles on the cellular mitochondrial metabolism using human cervical cancer (HeLa) and Human embryo kidney (HEK 293) cell lines. Although many reports have been made on the Cytotoxicity study of NiO nanoparticles, no report was found, at the time of the drafting of this manuscript, for the cytotoxicity study of NiO on both Hela and HEK 293 cell lines. Statistical data were collected as means ± standard deviations of three replicates and were processed in Excel, as shown in table 2. Results obtained, alongside the estimated minimum inhibitory concentration (IC 50 ) from this study, are summarized and presented as a Histogram in figure 5. The nanoparticles showed a concentration-dependent profile, as seen in figure 5, similar to most metal-based nanoparticles and compared favourably well with the standard 5-Flurouracil (5-FU) [6,24,27,32]. For the synthesized NiO nanoparticles, the lowest cell viability of about 13.44 and 4.92% was found at the highest concentration used in both HEK 293 and HeLa cells, respectively. However, the standard drug 5-Flurouracil (5-FU) possessed cell viability of 13 and 32% at the same concentration for the respective cell lines. Moreover, the estimated IC 50 value obtained for the NiO nanoparticles was 13.8 and 0.00015 μg ml −1 for HEK 293 and HeLa cell lines, respectively, while the standard drugs were estimated to be 6.05 and 17.48 μg ml −1 in the same cell lines. This finding shows that the Kei-apple mediated NiO nanoparticles have a good cytotoxic property, which is exceptionally better in HeLa cell lines than the used standard 5-Flurouracil. The Kei-apple mediated NiO nanoparticles had a weaker viability on HEK 293 cells compared to the effect of the standard drug and similar to the previous report in literature [27]. This thus suggests the safe usage of the NiO nanoparticle for different applications, such as in water treatment seeing that it is specific for only cancerous cells. The result obtained here agrees with other reports in literature [27,33]. It has been noted that the cytotoxicity of most metal-based nanoparticles is inversely proportional to the size of the nanoparticles. In this report, it was noted that nanoparticles were predominantly spherical and quasi-spherical shapes with cytotoxicity at a median dose of 1-20 μg ml −1 [33]. These reasons, amongst many others, may have accounted for the outstanding cytotoxicity displayed in this report. Few reports have been made on the cytotoxicity of NiO. Cambre et al, evaluated and compared the cytotoxicity of NiO and Ni(OH) nanoparticles against human bronchoalveolar carcinoma (A549) and human hepatocellular carcinoma (HepG2) cell lines [6]. In this report, a cell line-specific cytotoxicity in which both nanoparticles were toxic to A549 cells but relatively non-toxic to HepG2 cells [6]. In another study in which Geranium wallichianumas plant extract was used as a mediating agent, the obtained IC 50 value of 37.84 μgml −1 was reported after screening against HepG2 cancer cells [15]. These literature reports thus corroborate our present study and show the potential of NiO as an antiproliferation agent against cancer cell lines.

Anti-inflammatory study
The relationship between cancer and inflammation was first suggested by Rudolf Virchow in the mid-19th century that inflammation may be linked to cancer, as cancer seemed to arise in areas with chronic inflammation, and tumour biopsies showed an abundance of inflammatory cells [34]. Nowadays, scientists recognize that inflammation can play a role in the development of cancer. Also, although there are limited studies on the relationship between cytotoxicity and anti-inflammatory properties, it is already established that inflammation can also be a response to cellular damage caused by cytotoxic agents. Therefore, it is possible that compounds with both cytotoxic and anti-inflammatory properties may be more effective in reducing inflammation caused by cytotoxicity [34]. Hence, the advantage of a compound possessing both cytotoxic and anti-inflammatory properties is that it can potentially target cancer cells while reducing inflammation caused by the cytotoxic effects [35]. In this study, therefore, the anti-inflammatory properties of the Kei apple NiO nanoparticles and the obtained data, and the estimated minimum inhibitory concentration IC 50 value was obtained and compared with the standard anti-inflammatory drug called diclofenac. Statistical data were   collected as means ± standard deviations of three replicates and were processed in Excel, as shown in table 3. These results are presented as a histogram in figure 6. A dose-dependent profile was observed for the used concentration range. The estimated IC 50 value for the nanoparticles was 9.34 ± 0.010 μg ml −1 while the standard drug was 2.94 ± 0.014 μg ml −1 . This data thus show that the NiO nanoparticles compare favourably with diclofenac and possess the potential to inhibit inflammations. And as expected, most cytotoxic compounds are expected to exhibit some level of anti-inflammatory properties which this finding adequately corroborates [36,37]. No known reports exist for the anti-inflammatory studies of NiO nanoparticles using the Bovine serum albumin denaturation assay approach as of the time of this report.

4. Conclusion
NiO nanoparticles were successfully synthesized by mediating with Kei-apple leaf extracts. The formation and characteristics of the NiO nanoparticles were qualitatively confirmed by XRD and different microscopic techniques. The distinct peaks in the XRD pattern revealed the crystallinity of these NiO nanoparticles, and the EDS successfully confirmed the elemental constituents of the prepared material. Furthermore, the microscopic techniques of TEM and SEM revealed a spherical morphology with an average diameter size of 11 nm, which agrees with the estimated crystallite size obtained in the XRD (9 nm). The results of the cytotoxicity screen against human cervical cancer (HeLa) and human embryonic kidney (HEK 293) cell lines suggest that the prepared nanoparticles were specific only against the HeLa cancer cell line but had a weak effect on the noncancerous kidney cell line in comparison to the standard drug. Moreover, the cytotoxic activity of the HeLa cells, when compared with the standard anticancer drug 5-Fluorouracil, showed far superior activity with an IC 50 of 0.00015 μg ml −1 . Additionally, the anti-inflammatory study showed that the prepared NiO nanoparticles possessed weaker activity compared to the standard diclofenac. Since the goal of the current material is biological in nature, there is thus a need to carry out stability studies in other to establish the shelf life during the period of storage so as to guarantee its quality, effectiveness and safety. The study reported here suggests that the material may be safe for different applications, such as water treatment, considering it is only specific to cancer cells.