# Structural, Optical, Antibacterial, and Anticancer Properties of Cerium Oxide Nanoparticles Prepared by Green Synthesis UsingMorinda citrifolia Leaves Extract

Authors: Abozer Y. Elderdery; Badr Alzahrani; Abdulrahim A. Alabdulsalam; Siddiqa M. A. Hamza; Ahmed M. E. Elkhalifa; Abdulaziz H. Alhamidi; Fehaid Alanazi; A. Mohamedain; Suresh Kumar Subbiah; Pooi Ling Mok
Journal: Bioinorganic Chemistry and Applications
 (2022)

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## Abstract

Currently, new advancements in the area of nanotechnology opened up new prospects in the field of medicine that could provide us with a solution for numerous medical complications. Although a several varieties of nanoparticles is being explored to be used as nanomedicines, cerium oxide nanoparticles (CeO2 NPs) are the most attractive due to their biocompatibility and their switchable oxidation state (+3 and +4) or in other words the ability to act as prooxidant and antioxidant depending on the pH condition. Green synthesis of nanoparticles is preferred to make it more economical, eco-friendly, and less toxic. The aim of our study here is to formulate the CeO2 NPs (CeO2 NPs) using Morinda citrifolia (Noni) leaf extract and study its optical, structural, antibacterial, and anticancer abilities. Their optical and structural characterization was accomplished by employing X-ray diffractography (XRD), TEM, EDAX, FTIR, UV-vis, and photoluminescence assays. Our CeO2 NPs expressed strong antibacterial effects against Gram-positive S. aureus and S. pneumonia in addition to Gram-negative E. coli and K. pneumonia when compared with amoxicillin. The anticancer properties of the green synthesized CeO2 NPs against human acute lymphoblastic leukemia (ALL) MOLT-4 cells were further explored by the meticulous study of their ability to diminish cancer cell viability (cytotoxicity), accelerate apoptosis, escalate intracellular reactive oxygen species (ROS) accumulation, decline the mitochondria membrane potential (MMP) level, modify the cell adhesion, and shoot up the activation of proapoptotic markers, caspase−3, −8, and −9, in the tumor cells. Altogether, the outcomes demonstrated that our green synthesized CeO2 NPs are an excellent candidate for alternative cancer therapy.


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## Body

## 1. Introduction

Cancer is a burgeoning health ailment challenging the whole worldwide healthcare system [1, 2]. A rapid increase in the occurrence of cancer among the older population is observed recently [3]. ALL is a predominant pediatric blood cancer frequently diagnosed among children 2 to 5 years old [4]. Its pathogenesis is characterized by uncontrolled proliferation and malignant transformation of lymphoblasts present in the blood, bone marrow, and other extramedullary sites like the liver, spleen, etc. [5–7]. In well-developed countries, the prolonged chance of survival among ALL-diagnosed children is estimated to be more than 80% whereas it is substantially lower in the less developed countries [7, 8]. Contemporary ALL treatment incorporates various phases of chemotherapy including induction, consolidation, and post consolidation stage [9]. The first stage of induction involves the utilization of alternative protocols using corticosteroids, anthracycline, cyclophosphamide, cytarabine, vincristine, methotrexate, and daunorubicin [5, 7, 9]. This is followed by the consolidation and post consolidation or maintenance stage, which also involves several complicated protocols [5, 7, 9]. Altogether, chemotherapy can be described as a treatment involving high levels of toxicity and several formidable side effects [9, 10]. Despite all advanced treatment and enhanced management proceeding towards 90% of the curing rate of the disease, the healthcare system still endures significant problems of resistance to therapy and relapse of the malignancy [11, 12]. According to the studies among the ALL-diagnosed patients, more than 50% of adults and 20% of children relapse after a successful remission of cancer via chemotherapy [13, 14]. Knowing all the above facts, the researchers are now focused on the development of efficient and safer tools using new technologies to help patients diagnosed with ALL.Nanotechnology is one of the most interesting branches of science enjoying the limelight in recent decades. It has a wide spectrum of uses in diverse fields like imaging, commercial industries, electronics, and healthcare [15, 16]. In the healthcare system, nanotechnology is widely utilized in the formulation and delivery of novel medicament to treat or diagnose various diseases [15, 16]. Nanotechnology implicates the employment of nanoparticles (NPs) characterized as tiny particles of size ranging from 1 nm to 100 nm, possessing distinctive physicochemical properties that can be used in diverse areas of physics, chemistry, and biology [17]. Among a heterogeneous variety of NPs, cerium oxide (CeO2) is one of the widely utilized NPs due to its exclusive characteristic features of biocompatibility, stability, eco-friendliness, and eccentric surface chemistry [18, 19]. The lattice site of the CeO2 NPs or nanoceria is capable of producing more oxygen vacancies [20, 21] and this redox property can be significantly exploited in the treatment of various ailments related to oxidative stress. It is substantially employed in the production of sensors, catalysts, cells, drug-delivery agents, therapeutical agents, and antiparasitic creams [15, 18, 22]. Metals and metal oxides at the nanoscale are called nanostructured materials in science and technology. Furthermore, biocidal materials find use in a several range of fields, especially in the medical field. Due to their potential usage in drug-delivery, biosensing, and medical field, CeO2 NPs are explored in the field of nanomedicine. Additionally, the CeO2 NPs have low cost and feature low toxicity, are biocompatible, and are relatively stable [2]. As a result of CeO2 NPs, there are more oxygen vacancies at lattice sites [1]. In this way, the redox properties of CeO2 NPs may assist in curing oxidative stress mediated diseases (ROS essential for in vitro activity). The fabrication of CeO2 NPs is normally accomplished by numerous chemical and/or physical approaches [19, 23]; unfortunately, these methods pose various hazards to the ecosystem and biodiversity due to the usage of toxic reducing solvents in the process. Furthermore, the NPs acquired by these processes are also found to be unstable and toxic in nature, thus becoming less favorable [23, 24]. This problem is solved by the introduction of a new technique called green synthesis where natural resources like plants, microbes, or any kind of organic derivatives can be used to amalgamate NPs [25, 26]. Specifically, the process of photosynthesis of NPs involving plant extracts is considered an easier and safer method to produce nanostructures [27]. The phytochemicals like amines, phenols, enzymes, and ketones found in the plant extracts are presumed accountable for the stabilization and reduction of various salts into their corresponding NPs [28, 29]. Literature show that CeO2 NPs have excellent anticancer [30], antimicrobial [31], larvicidal [32], photocatalytic [33], and antioxidant [34] properties. Resistance to several commonly used antibiotics has been achieved by bacteria in recent years due to the expeditious evolution and adaptation of their genome [35, 36] making most of the available drugs useless in this stage. The biogenic nanoceria (CeO2 NPs) has proven to be very efficient in the treatment of such multiple drug-resistant bacteria and obstinate pathogenesis [29]. An amalgamation of CeO2 NPs with other organic and inorganic compounds has also proven promising to amplify the antimicrobial potentiality [37, 38]. Bio-assisted CeO2 NPs have demonstrated antifungal ability via mass gathering free radicals, which in turn disrupts the morphology and physiology of the fungal cells leading to their demise [31].Morinda citrifolia is a tree often recognized as Noni coming from the family Rubiaceae. Various parts of these plants have been consumed as food and used in indigenous medicine as anti-inflammatory, analgesic, antimicrobial, and anticancerous agents for over 2000 years [39]. Our present study particularly focuses on the photosynthesis of CeO2 NPs using the M. citrifolia and explores its structural, optical, antibacterial, and anticancer properties.


## 2. Experimental Methods

### 2.1. Leaf Extract Preparation

To use the leaves, ten grams ofMorinda citrifolia leaves were cleaned thrice with running water and deionized water. A 150 ml deionized water–filled 250 ml beaker containing 250 mlMorinda citrifolia leaves was then boiled at 100°C for 45 minutes to prepare the leaves to extract the solution. The light green color was produced by this Morinda citrifolia leaves extract solution. A clear solution was attained by filtering the leaf extract by Whatman No. 1 filter paper. For the preparation of CeO2 NPs, fresh Morinda citrifolia leaves extract was used.


### 2.2. Synthesis of CeO2 NPs

100 mL ofMorinda citrifolia leaf extract and 0.1 M of Ce(NO3)3·6H2O were mixed together using a magnetic stirrer for 6 hours under the constant stirring condition at 80°C. A from the reaction mixture, a precipitate with yellowish-brown color was developed. The reaction solution were centrifuged for 15 mins at 15,000 rpm. Then, the solid CeO2 were washed using deionized water and absolute ethanol. After calcination at 800°C for five hours, CeO2 nanopowder was formed.


### 2.3. Antibacterial Activity

Assays by agar diffusion were done with Gram-positive (G+)S.aureus and S.pneumonia, Gram-negative (G−) E.coli, and K.pneumonia. The strains were spread on the Petri plates prepared with a nutrient agar (NA) medium. The CeO2 NPs solution (1–2 mg/ml) was dispersed in 5% of sterilized dimethylsulfoxide in 40 μl, 50 μl, and 60 μl of wells, respectively. Following overnight incubation at 37°C, the inhibition zones around the wells were noted. As a positive control, we used standard amoxicillin (30 μg/ml), and a triplicate of each test was performed.


### 2.4. Characterization Analysis

To characterize the green CeO2 NPs synthesized by X’Pert PRO PANalytical, XRD is commonly used with masks from 2 mm to 20 mm, divergence slits varying from 1/2° to 1/32°, and nickel and copper filters. Analyzed by TEM using the Tecnai F20 model operating at 200 kV accelerating voltage, the chemical composition was determined using Carl Zeiss Ultra 55 FESEM and EDAX: Inca instruments. Using a Perkin-Elmer spectrometer, we measured the Fourier transform infrared spectra in the 400–4000 cm−1 range. A JASCO V-650 spectrophotometer was used to measure the UV-Visible absorption spectra. JASCO spectrofluorometer FP-8200 was used to measure photoluminescence spectra.


### 2.5. Chemicals and Reagents

The following materials were obtained from Corning (USA) and Sigma Aldrich (USA): fetal bovine serum (FBS), Dulbecco’s Modified Eagle Medium (DMEM), antibiotics, phosphate-buffered saline (PBS), ethidium bromide (EtBr), 3- (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), rhodamine-123 (RH-123) 2,7-dichlorofluorescein diacetate (DCFH-DA), dimethylsulfoxide (DMSO), and acridine orange (AO).


### 2.6. Cell Culture

The human ALL MOLT-4 cells were attained from ATCC, USA. The cells were grown in a DMEM medium complemented with antibiotics and 10% of heat-inactivated FBS and maintained in an incubator with 95% atmospheric air, 5% CO2, and 98% humidity at the temperature of 37°C.


### 2.7. MTT Cytotoxicity Assessment

The cytotoxic ability of CeO2 NPs prepared using M. citrifolia was assessed in agreement with Mosmann [40]. Initially, 96 wells were seeded with approximately 6 × 103 MOLT-4 cells and supplemented with several dosages of CeO2 NPs (10, 20, 30, 40, 50, and 60 μg/ml), meanwhile the control wells were deprived of the supplementation. After the incubation time of 24 hours, 20 μl of MTT stain was mixed. Subsequently, the microtiter plate was incubated at 37°C for 4 hours, and then the conceived crystals of formazan were liquefied with 150 μl of DMSO and the absorbance was evaluated colorimetrically at 570 nm using 620 nm as the reference wavelength. These readings were further used to estimate the cell viability in percentage by determining the 50% inhibitory concentration.


### 2.8. Apoptosis Assessment

AO/EtBr staining technique was employed to determine the apoptotic activity of human MOLT-4 cells administered with 30μg/ml and 40 μg/ml of CeO2 NPs fabricated using M. citrifolia in comparison with control MOLT-4 cells [41]. Primarily, the slides with the prepared samples were stained with AO/EtBr and a coverslip was placed immediately ensuring the proper spreading of the dye. Then incubated at 37°C for 5 minutes before examined under fluorescent microscope (40x magnification). The bright green fluorescent color emitted by the apoptotic cells was carefully counted and recorded.


### 2.9. Intracellular ROS Assessment

The DCFH-DA staining method was used to estimate the intracellular ROS accumulation [42]. The human ALL MOLT-4 cells administered with 30 μg/ml and 40 μg/ml of CeO2 NPs fabricated using M. citrifolia were collected and suspended in PBS solution of pH 7.4. Roughly 2 × 105 ml of this cell suspension was mixed with 10 μM DCFH-DA solution, and was allowed to incubate for 30 minutes at 37°C. Later, these cells were cleansed with PBS and fluorescence were determined spectrofluorimetrically by excitation at the wavelengths of 485 nm and 530 nm.


### 2.10. Mitochondria Membrane Potential (MMP) Assessment

The MMP of a cell is commonly estimated using rhodamine (Rh) 123 dye [43]. This procedure was executed using a 6-well plate seeded with human MOLT-4 cells administered with 30 μg/ml and 40 μg/ml of CeO2 NPs fabricated using M. citrifolia incubated for 24 hours. These cultures were then inoculated with Rh-123 dye and incubated at 37°C for another 30 minutes. The cells were then rinsed with PBS solution before observation under the fluorescence microscope with a blue filter (450–490 nm) and further analysis was done using ImageJ software.


### 2.11. Proapoptotic Marker (Caspase−3, −8, and −9) Evaluation

The sampling culture of human MOLT-4 cells (control) and cells supplemented with 30μg/ml and 40 μg/ml of CeO2 NPs fabricated using M. citrifolia were incubated for 24 hours and prepared to estimate the caspase activities following the manufacturer’s protocol of commercially available colorimetric protease assay kit (Thermo Scientific, USA). Each comes with a specified substrate like Ac-DEVD (acetyl-Asp-Glu-Val-Asp) for caspase-3, LEHD (Leu-Glu-His-Asp) for caspase-9, and IETD (Ile-Glu-Thr-Asp) for caspase-8. These substrates are initially tagged with the chromophore p-nitroanilide (pNA), which then are liberated by the caspase activity and are measured using a spectrophotometer at the wavelength of 405 nm [42].


### 2.12. Statistical Analysis

The outcomes were shown as mean ± SD for the triplicated trials. Statistical analysis was evaluated utilizing SPSS V20 software. The significance was computed employing one-way ANOVA accompanied by Duncan’s Multiple Range Test (DMRT) assessment. The differences in the means of experimental groups are considered as significant if thep<0.05.


## 2.1. Leaf Extract Preparation

To use the leaves, ten grams ofMorinda citrifolia leaves were cleaned thrice with running water and deionized water. A 150 ml deionized water–filled 250 ml beaker containing 250 mlMorinda citrifolia leaves was then boiled at 100°C for 45 minutes to prepare the leaves to extract the solution. The light green color was produced by this Morinda citrifolia leaves extract solution. A clear solution was attained by filtering the leaf extract by Whatman No. 1 filter paper. For the preparation of CeO2 NPs, fresh Morinda citrifolia leaves extract was used.


## 2.2. Synthesis of CeO2 NPs

100 mL ofMorinda citrifolia leaf extract and 0.1 M of Ce(NO3)3·6H2O were mixed together using a magnetic stirrer for 6 hours under the constant stirring condition at 80°C. A from the reaction mixture, a precipitate with yellowish-brown color was developed. The reaction solution were centrifuged for 15 mins at 15,000 rpm. Then, the solid CeO2 were washed using deionized water and absolute ethanol. After calcination at 800°C for five hours, CeO2 nanopowder was formed.


## 2.3. Antibacterial Activity

Assays by agar diffusion were done with Gram-positive (G+)S.aureus and S.pneumonia, Gram-negative (G−) E.coli, and K.pneumonia. The strains were spread on the Petri plates prepared with a nutrient agar (NA) medium. The CeO2 NPs solution (1–2 mg/ml) was dispersed in 5% of sterilized dimethylsulfoxide in 40 μl, 50 μl, and 60 μl of wells, respectively. Following overnight incubation at 37°C, the inhibition zones around the wells were noted. As a positive control, we used standard amoxicillin (30 μg/ml), and a triplicate of each test was performed.


## 2.4. Characterization Analysis

To characterize the green CeO2 NPs synthesized by X’Pert PRO PANalytical, XRD is commonly used with masks from 2 mm to 20 mm, divergence slits varying from 1/2° to 1/32°, and nickel and copper filters. Analyzed by TEM using the Tecnai F20 model operating at 200 kV accelerating voltage, the chemical composition was determined using Carl Zeiss Ultra 55 FESEM and EDAX: Inca instruments. Using a Perkin-Elmer spectrometer, we measured the Fourier transform infrared spectra in the 400–4000 cm−1 range. A JASCO V-650 spectrophotometer was used to measure the UV-Visible absorption spectra. JASCO spectrofluorometer FP-8200 was used to measure photoluminescence spectra.


## 2.5. Chemicals and Reagents

The following materials were obtained from Corning (USA) and Sigma Aldrich (USA): fetal bovine serum (FBS), Dulbecco’s Modified Eagle Medium (DMEM), antibiotics, phosphate-buffered saline (PBS), ethidium bromide (EtBr), 3- (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), rhodamine-123 (RH-123) 2,7-dichlorofluorescein diacetate (DCFH-DA), dimethylsulfoxide (DMSO), and acridine orange (AO).


## 2.6. Cell Culture

The human ALL MOLT-4 cells were attained from ATCC, USA. The cells were grown in a DMEM medium complemented with antibiotics and 10% of heat-inactivated FBS and maintained in an incubator with 95% atmospheric air, 5% CO2, and 98% humidity at the temperature of 37°C.


## 2.7. MTT Cytotoxicity Assessment

The cytotoxic ability of CeO2 NPs prepared using M. citrifolia was assessed in agreement with Mosmann [40]. Initially, 96 wells were seeded with approximately 6 × 103 MOLT-4 cells and supplemented with several dosages of CeO2 NPs (10, 20, 30, 40, 50, and 60 μg/ml), meanwhile the control wells were deprived of the supplementation. After the incubation time of 24 hours, 20 μl of MTT stain was mixed. Subsequently, the microtiter plate was incubated at 37°C for 4 hours, and then the conceived crystals of formazan were liquefied with 150 μl of DMSO and the absorbance was evaluated colorimetrically at 570 nm using 620 nm as the reference wavelength. These readings were further used to estimate the cell viability in percentage by determining the 50% inhibitory concentration.


## 2.8. Apoptosis Assessment

AO/EtBr staining technique was employed to determine the apoptotic activity of human MOLT-4 cells administered with 30μg/ml and 40 μg/ml of CeO2 NPs fabricated using M. citrifolia in comparison with control MOLT-4 cells [41]. Primarily, the slides with the prepared samples were stained with AO/EtBr and a coverslip was placed immediately ensuring the proper spreading of the dye. Then incubated at 37°C for 5 minutes before examined under fluorescent microscope (40x magnification). The bright green fluorescent color emitted by the apoptotic cells was carefully counted and recorded.


## 2.9. Intracellular ROS Assessment

The DCFH-DA staining method was used to estimate the intracellular ROS accumulation [42]. The human ALL MOLT-4 cells administered with 30 μg/ml and 40 μg/ml of CeO2 NPs fabricated using M. citrifolia were collected and suspended in PBS solution of pH 7.4. Roughly 2 × 105 ml of this cell suspension was mixed with 10 μM DCFH-DA solution, and was allowed to incubate for 30 minutes at 37°C. Later, these cells were cleansed with PBS and fluorescence were determined spectrofluorimetrically by excitation at the wavelengths of 485 nm and 530 nm.


## 2.10. Mitochondria Membrane Potential (MMP) Assessment

The MMP of a cell is commonly estimated using rhodamine (Rh) 123 dye [43]. This procedure was executed using a 6-well plate seeded with human MOLT-4 cells administered with 30 μg/ml and 40 μg/ml of CeO2 NPs fabricated using M. citrifolia incubated for 24 hours. These cultures were then inoculated with Rh-123 dye and incubated at 37°C for another 30 minutes. The cells were then rinsed with PBS solution before observation under the fluorescence microscope with a blue filter (450–490 nm) and further analysis was done using ImageJ software.


## 2.11. Proapoptotic Marker (Caspase−3, −8, and −9) Evaluation

The sampling culture of human MOLT-4 cells (control) and cells supplemented with 30μg/ml and 40 μg/ml of CeO2 NPs fabricated using M. citrifolia were incubated for 24 hours and prepared to estimate the caspase activities following the manufacturer’s protocol of commercially available colorimetric protease assay kit (Thermo Scientific, USA). Each comes with a specified substrate like Ac-DEVD (acetyl-Asp-Glu-Val-Asp) for caspase-3, LEHD (Leu-Glu-His-Asp) for caspase-9, and IETD (Ile-Glu-Thr-Asp) for caspase-8. These substrates are initially tagged with the chromophore p-nitroanilide (pNA), which then are liberated by the caspase activity and are measured using a spectrophotometer at the wavelength of 405 nm [42].


## 2.12. Statistical Analysis

The outcomes were shown as mean ± SD for the triplicated trials. Statistical analysis was evaluated utilizing SPSS V20 software. The significance was computed employing one-way ANOVA accompanied by Duncan’s Multiple Range Test (DMRT) assessment. The differences in the means of experimental groups are considered as significant if thep<0.05.


## 3. Results

### 3.1. UV-Vis Spectrum Analysis

Figure1(a) illustrates the UV-VIS absorption spectrum of CeO2 NPs. Photoexcitation of electrons from valence band to the conduction band accounts for the absorption peak observed for CeO2 NPs at 324 nm (Figure 1(a)). It can be written αhυ = A (hυ-Eg) n that is the relation between the absorption coefficient α and the incident photon energy hυ [14]. The bandgap of 2.65 eV for CeO2 NPs is revealed in Figure 1(b).Figure 1
Spectral analysis of CeO2 NPs. Uv-Vis spectrum of CeO2 NPs (a). The bandgap of CeO2 NPs (b). Photoluminescence spectra for CeO2 NPs at room temperature (c).
(a)(b)(c)


### 3.2. Photoluminescence (PL) Spectroscopic Analysis

According to Figure1(b), the CeO2 nanoparticle PL emission spectrum shows peaks at 362 nm, 370 nm, 394 nm, 415 nm, 436 nm, 460, and 493 nm, respectively. Based on the band-to-band recombination process, CeO2 NPs exhibit near-band edge emissions at 362–394 nm (362, 371, and 394), possibly caused by localized or free excitons [13]. It was suggested that the violet emission noticed at 415 nm is a result of the presence of defects between Ce 4f and O 2p valence bands [15]. Both blue emission peaks are located between 436 nm and 460 nm, and they are caused by abundant defects like dislocations that are needed for fast oxygen transport [16]. NPs made of CeO2 exhibit a blue-green emission at 493 nm because of the surface defect.


### 3.3. FTIR Analysis

FTIR spectra of the green synthesized CeO2 NPs are demonstrated in Figure 2. The O–H stretching were noted at 3375 cm−1 for CeO2 [11]. The symmetric and asymmetric C-H stretching was located at 2916 and 2853 cm−1. In the FTIR spectra, 1114 cm−1 was detected for the Ce–O–Ce stretching vibrations. This work found that the vibration frequencies of the Ce-O bands are 572 cm−1 for CeO2 and 415 cm−1 for CeO2 in the literature, respectively [12].Figure 2
FTIR transmittance vs. wavenumber chart of CeO2 NPs derived from infrared analysis.


### 3.4. Morphology and Chemical Composition

Figure3(a) reveals the TEM image of the fabricated CeO2 NPs. The CeO2 NPs formed spherical structures with uniform grain boundaries, and the average size of the CeO2 NPs is 40 nm. The nucleation reduction of nanoparticles, due to various phytochemicals, acts as reducing and capping agent. EDAX spectra of green formulated CeO2 NPs are revealed in Figure 3(b). The NPs’ atomic percentage was Ce at 20.72% and O at 79.28%, respectively.Figure 3
TEM micrographics of the CeO2 NPs: lower and higher magnification TEM image (a). Elements, weight %, and atomic % of the composition were attained by EDAX (b).
(a)(b)


### 3.5. XRD Analysis

The XRD results of green formulated CeO2 NPs are depicted in Figure 4. The different peaks were observed at angles (2θ) of 28.08, 32.6, 47.016, 55.89, 56.617, 69.00, and 76.2 corresponding to (111), (200), (220), (311), (222), (400), and (331) planes of the CeO2 NPs, structurally formed with the face-center cubic phase (JCPDS no: 34–0394).Figure 4
XRD pattern of CeO2 NPs.The following equation was utilized to assess the lattice constants “a” of the cubic structure of CeO2.(1)1d2=h2+k2+l2a2.The lattice constant “a” is attained by the relation a=d2h2+k2+l2. The lattice constant value “a” is 5.440 Å for CeO2 NPs. The crystallite size of the CeO2 is determined using Debye-Scherrer’s formula.Average crystallite sizeD=+0.9λ/βcosθ, where λ: wavelength of X-ray (1.5405 A), β: angular peak width at half maximum in radians, and θ: Bragg’s diffraction angle. D is calculated as 45 nm for CeO2 NPs. The lattice constant “a” is attained by the lattice constant value “a” is 5.440 Å for CeO2 NPs.


### 3.6. Antibacterial Activity

The antibacterial properties of CeO2 NPs were examined against Gram-positive (G+) S. aureus and S. pneumonia, and Gram-negative (G−) K. pneumonia and E. coli bacterial pathogens using different concentrations of CeO2 NPs (1, 1.5, and 2 mg/ml) and comparison was made with administration of 30 μg/ml amoxicillin alone. The results were shown in Figure 5(a). The inhibition zone formed after the incubation period was carefully measured, tabulated, studied, and presented as a bar diagram (Figure 5(b)). The observations made it clear that CeO2 NPs were far more efficient than conventional amoxicillin, and administration of higher concentration of CeO2 NPs showed relatively stronger bacterial growth inhibition when compared with lower concentration of CeO2 NPs. The antibacterial properties of CeO2 NPs generally depend on their size, surface area and topography. Furthermore, the electrostatic attraction of positively charged NPs and negatively charged bacterial cells could increase the ROS accumulation in bacterial cells, and finally lead to the growth inhibition and cell death [17, 18].Figure 5
Antibacterial activity of CeO2 NPs. NPs of CeO2 inhibited the growth of S. aureus, S. pneumonia, K. pneumonia, and E. coli (a). Antibacterial properties were assessed for CeO2 NPs by detecting the inhibition zones (b).
(a)(b)


### 3.7. Cytotoxicity of Green Synthesized CeO2 NPs

MTT assay was done to scrutinize the cytotoxic ability of green synthesized CeO2 NPs against MOLT-4 cells presented in Figure 6. The control MOLT-4 cells were observed to have a high percentage of viable cells which significantly declined in the cultures administered with the green synthesized CeO2 NPs in a concentration-dependent manner; i.e., the higher concentration caused higher cytotoxicity, reducing the number of viable cancer cells. It was observed that half-maximal inhibitory concentration (IC50-39.37 μg/ml) was achieved in 30 μg/ml and 40 μg/ml concentrations of CeO2 NPs doses; therefore, these cultures were subjected to further studies.Figure 6
CeO2 NP inhibited the MOLT-4 cell viability. Cells were administered with several dosages (0–60 μg/ml) of CeO2 NPs for 24 hours. Then cells were examined by MTT assay and the data are revealed as mean ± SD of three individual experiments.


### 3.8. Apoptosis by Green Synthesized CeO2 NPs

AO/EtBr staining revealed the ability of CeO2 NPs to instigate apoptosis in MOLT-4 cells (Figure 7(a)). The control sample with only MOLT-4 exhibited no apoptosis and showed just green fluorescence due to AO stain, whereas CeO2 NPs supplemented culture glowed intense orange fluorescence due to EtBr stain exhibiting apoptosis. Thus, both 30 μg/ml and 40 μg/ml of CeO2 NPs concentration succeeded in gushing the apoptosis of cancerous MOLT-4 cells proportional to their CeO2 NPs concentration (bar diagram Figure 7(b)).Figure 7
Effect of CeO2 NPs on the apoptosis in the MOLT-4 cells for 24 hours. AO/EtBr (1: 1) were utilized to stain the cells and examined under fluorescent microscopy. The green fluorescence were noticed in control cells, which reveals the absence of apoptosis. The 30 and 40 μg/ml of CeO2 NPs administered cells exhibited the yellow/orange fluorescence, which confirms the occurrence of apoptosis. (a) (A) Control (B) 30 μg/ml of CeO2 NPs and (C) 40 μg/ml of CeO2 NPs administered cells. (b) Arbitrary units (a.u.) of developed fluorescence. Values are revealed as mean ± SD of triplicates. ∗p<0.05 compared to the “control” group and ∗∗p<0.005 compared to the “control” group.
(a)(b)


### 3.9. ROS Estimation

DCFH-DA staining technique was employed to estimate the expression of ROS in untreated malignant MOLT-4 cells, and was compared with the MOLT-4 cells administered with 30μg/ml and 40 μg/ml of formulated CeO2 NPs. The intensity of the bright green fluorescence reveals the higher ROS accumulation (Figure 8(a)). The observation of the procedure depicted (bar diagram, Figure 8(b)) a high level of ROS in the cells of 40 μg/ml CeO2 NPs, which was slightly lower in the 30 μg CeO2 NPs sample and almost negligible in the control sample.Figure 8
Effect of CeO2 NPs on the intracellular ROS production in the MOLT-4 cells. The cells were examined by DCFH-DA to detect the ROS status and images were taken using fluorescent microscope. A dull green fluorescence were noted on the control cells, which revealed the poor ROS production. The CeO2 NPs (30 and 40 μg/ml) administered cells revealed the higher green fluorescence, which confirms the higher ROS production. (a) (A) Control (B) 30 μg/ml of CeO2NPs and (C) 40 μg/ml of CeO2 NPs administered cells. (b) Arbitrary units (a.u.) of developed fluorescence. Values are revealed as mean ± SD of triplicates. ∗p<0.05 compared to the “control” group and ∗∗p<0.005 compared to the “control” group.
(a)(b)


### 3.10. MMP Estimation

The Rh-123 staining procedure was performed to track the level of MMP in the MOLT-4 cells used as control and the MOLT-4 cells administered with 30μg/ml and 40 μg/ml of green synthesized CeO2 NPs. The results observed showed that the control cell culture emitted intense green fluorescence and the intensity diminished in the cells supplemented with 30 μg/ml and 40 μg/ml CeO2 NPs subsequently (Figure 9(a)). These observations support the ability of CeO2 NPs to reduce the level of MMP in blood cancer cells (bar diagram, Figure 9(b)).Figure 9
Effects of CeO2 NPs on the MMP status of MOLT-4 cells. CeO2 NPs administered cells revealed the reduced MMP status. The higher green fluorescence were noted on the control cells that demonstrate the higher MMP. The 30 and 40 μg/ml of CeO2 NPs administered cells revealed the dull green fluorescence that proves the decline in MMP. (a) (A) Control (B) 30 μg/ml of CeO2 NPs and (C) 40 μg/ml of CeO2 NPs administered cells. (b) Arbitrary units (a.u.) of developed fluorescence. Values are revealed as mean ± SD of triplicates. ∗p<0.05 compared to the “control” group and ∗∗p<0.005 compared to the “control” group.
(a)(b)


### 3.11. Expression of Caspase−3, −8, and −9

The expression of proapoptotic proteolytic caspase−3, −8, and −9 in the control, and 30μg/ml and 40 μg/ml of green synthesized CeO2 NPs administered MOLT-4 cells were estimated and illustrated in Figure 10. The outcomes revealed that the expression of caspase−3, −8, and −9 were higher in CeO2 NPs supplemented culture when compared with the untreated control, and hence it is conclusive that CeO2 NPs undeniably play a role in the upregulation of caspase-3, -8, and -9 expression and its efficiency to do so increases with its concentration (Figure 10).Figure 10
Effect of CeO2 NPs on the caspases activity. The administration with 30 μg/ml and 40 μg/ml of CeO2 NPs considerably elevated the caspase−3, −8, and −9 activities in the MOLT-4 cells. Values are revealed as mean ± SD of triplicates. ∗p<0.05 compared to the “control” group and ∗∗p<0.005 compared to the “control” group.
(a)(b)(c)


## 3.1. UV-Vis Spectrum Analysis

Figure1(a) illustrates the UV-VIS absorption spectrum of CeO2 NPs. Photoexcitation of electrons from valence band to the conduction band accounts for the absorption peak observed for CeO2 NPs at 324 nm (Figure 1(a)). It can be written αhυ = A (hυ-Eg) n that is the relation between the absorption coefficient α and the incident photon energy hυ [14]. The bandgap of 2.65 eV for CeO2 NPs is revealed in Figure 1(b).Figure 1
Spectral analysis of CeO2 NPs. Uv-Vis spectrum of CeO2 NPs (a). The bandgap of CeO2 NPs (b). Photoluminescence spectra for CeO2 NPs at room temperature (c).
(a)(b)(c)


## 3.2. Photoluminescence (PL) Spectroscopic Analysis

According to Figure1(b), the CeO2 nanoparticle PL emission spectrum shows peaks at 362 nm, 370 nm, 394 nm, 415 nm, 436 nm, 460, and 493 nm, respectively. Based on the band-to-band recombination process, CeO2 NPs exhibit near-band edge emissions at 362–394 nm (362, 371, and 394), possibly caused by localized or free excitons [13]. It was suggested that the violet emission noticed at 415 nm is a result of the presence of defects between Ce 4f and O 2p valence bands [15]. Both blue emission peaks are located between 436 nm and 460 nm, and they are caused by abundant defects like dislocations that are needed for fast oxygen transport [16]. NPs made of CeO2 exhibit a blue-green emission at 493 nm because of the surface defect.


## 3.3. FTIR Analysis

FTIR spectra of the green synthesized CeO2 NPs are demonstrated in Figure 2. The O–H stretching were noted at 3375 cm−1 for CeO2 [11]. The symmetric and asymmetric C-H stretching was located at 2916 and 2853 cm−1. In the FTIR spectra, 1114 cm−1 was detected for the Ce–O–Ce stretching vibrations. This work found that the vibration frequencies of the Ce-O bands are 572 cm−1 for CeO2 and 415 cm−1 for CeO2 in the literature, respectively [12].Figure 2
FTIR transmittance vs. wavenumber chart of CeO2 NPs derived from infrared analysis.


## 3.4. Morphology and Chemical Composition

Figure3(a) reveals the TEM image of the fabricated CeO2 NPs. The CeO2 NPs formed spherical structures with uniform grain boundaries, and the average size of the CeO2 NPs is 40 nm. The nucleation reduction of nanoparticles, due to various phytochemicals, acts as reducing and capping agent. EDAX spectra of green formulated CeO2 NPs are revealed in Figure 3(b). The NPs’ atomic percentage was Ce at 20.72% and O at 79.28%, respectively.Figure 3
TEM micrographics of the CeO2 NPs: lower and higher magnification TEM image (a). Elements, weight %, and atomic % of the composition were attained by EDAX (b).
(a)(b)


## 3.5. XRD Analysis

The XRD results of green formulated CeO2 NPs are depicted in Figure 4. The different peaks were observed at angles (2θ) of 28.08, 32.6, 47.016, 55.89, 56.617, 69.00, and 76.2 corresponding to (111), (200), (220), (311), (222), (400), and (331) planes of the CeO2 NPs, structurally formed with the face-center cubic phase (JCPDS no: 34–0394).Figure 4
XRD pattern of CeO2 NPs.The following equation was utilized to assess the lattice constants “a” of the cubic structure of CeO2.(1)1d2=h2+k2+l2a2.The lattice constant “a” is attained by the relation a=d2h2+k2+l2. The lattice constant value “a” is 5.440 Å for CeO2 NPs. The crystallite size of the CeO2 is determined using Debye-Scherrer’s formula.Average crystallite sizeD=+0.9λ/βcosθ, where λ: wavelength of X-ray (1.5405 A), β: angular peak width at half maximum in radians, and θ: Bragg’s diffraction angle. D is calculated as 45 nm for CeO2 NPs. The lattice constant “a” is attained by the lattice constant value “a” is 5.440 Å for CeO2 NPs.


## 3.6. Antibacterial Activity

The antibacterial properties of CeO2 NPs were examined against Gram-positive (G+) S. aureus and S. pneumonia, and Gram-negative (G−) K. pneumonia and E. coli bacterial pathogens using different concentrations of CeO2 NPs (1, 1.5, and 2 mg/ml) and comparison was made with administration of 30 μg/ml amoxicillin alone. The results were shown in Figure 5(a). The inhibition zone formed after the incubation period was carefully measured, tabulated, studied, and presented as a bar diagram (Figure 5(b)). The observations made it clear that CeO2 NPs were far more efficient than conventional amoxicillin, and administration of higher concentration of CeO2 NPs showed relatively stronger bacterial growth inhibition when compared with lower concentration of CeO2 NPs. The antibacterial properties of CeO2 NPs generally depend on their size, surface area and topography. Furthermore, the electrostatic attraction of positively charged NPs and negatively charged bacterial cells could increase the ROS accumulation in bacterial cells, and finally lead to the growth inhibition and cell death [17, 18].Figure 5
Antibacterial activity of CeO2 NPs. NPs of CeO2 inhibited the growth of S. aureus, S. pneumonia, K. pneumonia, and E. coli (a). Antibacterial properties were assessed for CeO2 NPs by detecting the inhibition zones (b).
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## 3.7. Cytotoxicity of Green Synthesized CeO2 NPs

MTT assay was done to scrutinize the cytotoxic ability of green synthesized CeO2 NPs against MOLT-4 cells presented in Figure 6. The control MOLT-4 cells were observed to have a high percentage of viable cells which significantly declined in the cultures administered with the green synthesized CeO2 NPs in a concentration-dependent manner; i.e., the higher concentration caused higher cytotoxicity, reducing the number of viable cancer cells. It was observed that half-maximal inhibitory concentration (IC50-39.37 μg/ml) was achieved in 30 μg/ml and 40 μg/ml concentrations of CeO2 NPs doses; therefore, these cultures were subjected to further studies.Figure 6
CeO2 NP inhibited the MOLT-4 cell viability. Cells were administered with several dosages (0–60 μg/ml) of CeO2 NPs for 24 hours. Then cells were examined by MTT assay and the data are revealed as mean ± SD of three individual experiments.


## 3.8. Apoptosis by Green Synthesized CeO2 NPs

AO/EtBr staining revealed the ability of CeO2 NPs to instigate apoptosis in MOLT-4 cells (Figure 7(a)). The control sample with only MOLT-4 exhibited no apoptosis and showed just green fluorescence due to AO stain, whereas CeO2 NPs supplemented culture glowed intense orange fluorescence due to EtBr stain exhibiting apoptosis. Thus, both 30 μg/ml and 40 μg/ml of CeO2 NPs concentration succeeded in gushing the apoptosis of cancerous MOLT-4 cells proportional to their CeO2 NPs concentration (bar diagram Figure 7(b)).Figure 7
Effect of CeO2 NPs on the apoptosis in the MOLT-4 cells for 24 hours. AO/EtBr (1: 1) were utilized to stain the cells and examined under fluorescent microscopy. The green fluorescence were noticed in control cells, which reveals the absence of apoptosis. The 30 and 40 μg/ml of CeO2 NPs administered cells exhibited the yellow/orange fluorescence, which confirms the occurrence of apoptosis. (a) (A) Control (B) 30 μg/ml of CeO2 NPs and (C) 40 μg/ml of CeO2 NPs administered cells. (b) Arbitrary units (a.u.) of developed fluorescence. Values are revealed as mean ± SD of triplicates. ∗p<0.05 compared to the “control” group and ∗∗p<0.005 compared to the “control” group.
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## 3.9. ROS Estimation

DCFH-DA staining technique was employed to estimate the expression of ROS in untreated malignant MOLT-4 cells, and was compared with the MOLT-4 cells administered with 30μg/ml and 40 μg/ml of formulated CeO2 NPs. The intensity of the bright green fluorescence reveals the higher ROS accumulation (Figure 8(a)). The observation of the procedure depicted (bar diagram, Figure 8(b)) a high level of ROS in the cells of 40 μg/ml CeO2 NPs, which was slightly lower in the 30 μg CeO2 NPs sample and almost negligible in the control sample.Figure 8
Effect of CeO2 NPs on the intracellular ROS production in the MOLT-4 cells. The cells were examined by DCFH-DA to detect the ROS status and images were taken using fluorescent microscope. A dull green fluorescence were noted on the control cells, which revealed the poor ROS production. The CeO2 NPs (30 and 40 μg/ml) administered cells revealed the higher green fluorescence, which confirms the higher ROS production. (a) (A) Control (B) 30 μg/ml of CeO2NPs and (C) 40 μg/ml of CeO2 NPs administered cells. (b) Arbitrary units (a.u.) of developed fluorescence. Values are revealed as mean ± SD of triplicates. ∗p<0.05 compared to the “control” group and ∗∗p<0.005 compared to the “control” group.
(a)(b)


## 3.10. MMP Estimation

The Rh-123 staining procedure was performed to track the level of MMP in the MOLT-4 cells used as control and the MOLT-4 cells administered with 30μg/ml and 40 μg/ml of green synthesized CeO2 NPs. The results observed showed that the control cell culture emitted intense green fluorescence and the intensity diminished in the cells supplemented with 30 μg/ml and 40 μg/ml CeO2 NPs subsequently (Figure 9(a)). These observations support the ability of CeO2 NPs to reduce the level of MMP in blood cancer cells (bar diagram, Figure 9(b)).Figure 9
Effects of CeO2 NPs on the MMP status of MOLT-4 cells. CeO2 NPs administered cells revealed the reduced MMP status. The higher green fluorescence were noted on the control cells that demonstrate the higher MMP. The 30 and 40 μg/ml of CeO2 NPs administered cells revealed the dull green fluorescence that proves the decline in MMP. (a) (A) Control (B) 30 μg/ml of CeO2 NPs and (C) 40 μg/ml of CeO2 NPs administered cells. (b) Arbitrary units (a.u.) of developed fluorescence. Values are revealed as mean ± SD of triplicates. ∗p<0.05 compared to the “control” group and ∗∗p<0.005 compared to the “control” group.
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## 3.11. Expression of Caspase−3, −8, and −9

The expression of proapoptotic proteolytic caspase−3, −8, and −9 in the control, and 30μg/ml and 40 μg/ml of green synthesized CeO2 NPs administered MOLT-4 cells were estimated and illustrated in Figure 10. The outcomes revealed that the expression of caspase−3, −8, and −9 were higher in CeO2 NPs supplemented culture when compared with the untreated control, and hence it is conclusive that CeO2 NPs undeniably play a role in the upregulation of caspase-3, -8, and -9 expression and its efficiency to do so increases with its concentration (Figure 10).Figure 10
Effect of CeO2 NPs on the caspases activity. The administration with 30 μg/ml and 40 μg/ml of CeO2 NPs considerably elevated the caspase−3, −8, and −9 activities in the MOLT-4 cells. Values are revealed as mean ± SD of triplicates. ∗p<0.05 compared to the “control” group and ∗∗p<0.005 compared to the “control” group.
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## 4. Discussion

The plant’s secondary metabolite compounds are an important source of oxidizing and capping agents for metal oxide nanoparticles [6, 8]. Plant extracts derived from Morinda citrifolia (Ce (NO3)3) have high van der wall forces, which results in the oxidization of Ce3+ to Ce4+. Morinda citrifolia L. plant has mono-ethoxyrubiadin, nordamnacanthal, quinoline, and rubiadin. CeO2 is oxidized to Ce (OH)4 by hydrolyzing it to OH−. Ce4+ slowly reacts with OH− to develop the Ce(OH)3 colloids (damnacanthol, digoxin, chrysophanol (1,8-dihydroxy-3-methyl anthraquinone), morindone-6-β-primeveroside, anthraquinones, and their glycosides, glucose (β-D-glucopyranose), indoles, purines, caprylic acid, flavones glycosides, fatty acids, alcohols (C5-9), caproic acid, flavonoids, and β-sitosterol). In the presence of this secondary metabolite, by hydrolyzing Ce4+ to OH− in the presence of this secondary metabolite, Ce4+ is converted to Ce (OH)4. Colloids of Ce(OH)4 are formed when Ce4+ reacts with OH+. It occurs when ions of a metal form complex compounds, producing nanosize particles of CeO2.The World Health Organization (WHO) published a report saying there are more than 10 million new cancer cases each year, as well as 7.9 million deaths due to cancer [44]. ALL is a form of blood cancer that can be defined as abnormal cell proliferation and differentiation of lymphoid progenitors [45], and it is extremely heart breaking to know that children are more susceptible to this disease in comparison with adults. Although a very high percentage of remission of ALL is achieved recently, the refractory relapse of ALL is still a great challenge to the current methods of treatment. In addition to this, a complication caused by the malignant cells showing resistance to these conventional therapies is also not to be neglected [46]. Chemotherapy is an extensively used standard cancer treatment; unfortunately, it is also extremely toxic in nature. Although chemotherapy includes a diverse number of mechanisms, its major task involves the indiscriminate killing of all rapidly growing cells which include both normal and tumor cells, thus accompanied by deleterious side effects [47]. Hence, now the invention of innovative cancer treatments has become a crucial problem faced by the whole world currently [48]. CeO2 NPs can be proposed as a solution for this problem as CeO2 NPs are extremely biocompatible, promptly eliminated from the body [49], and less toxic in nature [50]. In neutral pH, CeO2 NPs exhibit antioxidant abilities [51]. However, in an acidic tumoral environment, the NPs exhibit prooxidant abilities [18]. This peculiar ability of CeO2 NPs to switch antioxidant to prooxidant depending on the pH can be exploited to attack the cancer cell specifically and protect normal cells at the same time [50, 52].In our current investigation, CeO2 NPs were first formulated from the M. citrifolia leaves [42], and their structural and optical characteristics were studied which was following the prior studies [53, 54]. The X-ray diffraction showed that our CeO2 NPs were 40 nm in size and TEM revealed the morphology to be spherical average-sized, whereas EDAX assessment gave up the atomic percentage of Ce and O to be 20.72% and 79.28%, respectively. The UV spectrum assay provided the bandgap of CeO2 NPs as 2.65 eV. Spectroscopic photoluminescence assay provided evidence of defect states on a large scale within the O2 valency band [53] and defects responsible for quick oxygen transportation [55]. The FTIR assay proves that the phytochemicals and flavonoids occur in the leaves serve as the reducing and stabilizing factor during NPs synthesis and this could further enhance its antimicrobial ability [56]. The antibacterial assessment of green synthesized CeO2 NPs indicatedthat CeO2 NPs were relatively more efficient than the conventional antibiotic amoxicillin concerning both G+ and G− strains, which is in line with the previous studies conducted with Streptococcus mutants [57]. Generally, the ROS produced by NPs leads to the mechanical destruction of the bacterial cell membrane [58], which is believed to be responsible for the antibacterial efficacy of CeO2 NPs. Since the recent development, resistance toward NPs is almost impossible for bacteria as NPs attack many cellular pathways at the same time [59]. NPs can be a promising alternative for conventional treatments to combat antibiotic-resistant bacteria.The results of our MTT assay proved the viability of human (MOLT-4) cancer cells was severely declined by the treatment of formulated CeO2 NPs, and it was caused by the upstream event of cytotoxicity, triggered by the overproduction of ROS [60]. Hence, it is conclusive that the green synthesized CeO2 NPs have an excellent cytotoxic ability, which is in harmony with previous studies [61], making it one step closer to the prospect of being used in cancer therapy. It was also observed that the viability of cancer cells was reduced to half when treated with 30 μg/ml and 40 μg/ml and so these cultures were put through further examination. Apoptosis performs a key functions in the homeostasis of an organism to get rid of the unwanted cells [62], inadequate or complete omission of apoptosis will be the principle feature of tumorigenicity, and hence encouraging apoptosis becomes a momentous goal for cancer treatment [63]. Our test results exhibited an exemplary apoptotic potency of green synthesized CeO2 NPs in both 30 μg/ml and 40 μg/ml doses, which is similar to the former studies [64], again adding up to the anticancerous ability of our green synthesized CeO2 NPs.Excess of ROS is a well-known factor contributing to severe damage to DNA and disturbance of the cell cycle, hence causing apoptosis of the cell [65]. Estimation of ROS intracellular accumulation in untreated human ALL MOLT-4 cells (control) and MOLT-4 cells treated with 30 μg/ml and 40 μg/ml of CeO2 NPs fabricated using M. citrifolia was executed in our current study, which gave the conclusion that CeO2 NPs induced expeditious production of ROS in the cancer cell, and this is in harmony with previous studies [65] and hence supports the CeO2 NPs as the anticancerous agent. MMP is vital to sustaining the mitochondrial membrane, and dropping of MMP level is the primary stage during apoptosis of the cell culture which leads to the deposition of ROS [66]. In conformity with the prior investigation [67], our outcomes demonstrate a considerable diminution of MMP in the malignant cells due to the supplementation of 30 μg/ml and 40 μg/ml of CeO2 NPs synthesized using M. citrifolia.During the metastasis stage of cancer, some malignant cells from the primary tumor disseminate and travel through the circulatory and lymphatic systems and take over new organs [68, 69]. In harmony with the past investigation by [70], our current study also demonstrated the upregulation of proapoptotic markers, namely, caspase-3, -8, and -9 in the ALL MOLT-4 cancer cells, induced by the supplementation of 30 μg/ml and 40 μg/ml CeO2 NPs prepared using leaf extract of M. citrifolia. Here, caspase-8 and caspase-9 are known to be the initiator caspases which subsequently activate caspase-3 known to be the executioner caspase which then instigates the destruction of vital structural proteins and activates a few enzymes that finally cause DNA fragmentation and membrane disruption, which ultimately leads to apoptosis [71]. Hence, the CeO2 NPs fabricated utilizing M. citrifolia again prove themselves to be worthy as anticancer therapeutic agents.


## 5. Conclusion

In our study, the CeO2 NPs were successfully synthesized using M. citrifolia, and their structural and optical characters were carefully studied, after which their antibacterial and anticancer abilities were tested. The results proved that our green synthesized CeO2 NPs revealed a considerable antibacterial effect against the tested strains. Concerning cancer, evidence strongly suggests that our green synthesized CeO2 NPs could be a potential candidate to treat cancer as it shows full potency to be much safer and more efficient and economical, and most importantly it is a target specific cancer treatment.



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*Source: 6835625-2022-09-28.xml*