Green-Synthesis of MgO and ZrO 2 Nanocomposite: Physicochemical Properties and Anti- Plasmodial Activity in a Mouse Model

Green-Synthesis of MgO and ZrO 2 Nanocomposite: Physicochemical Properties and Anti- Plasmodial Activity in a Mouse Model | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Green-Synthesis of MgO and ZrO 2 Nanocomposite: Physicochemical Properties and Anti- Plasmodial Activity in a Mouse Model Augustine Innalegwu Daniel, Sarah Udenyi Onogwu, Theresa Yebo Gara, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5662380/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Malaria remains a life-threatening disease, particularly in Sub-Saharan Africa, with the emergence of Plasmodium-resistant parasites necessitating new chemotherapeutic drugs. This study evaluated the anti-plasmodial potential of phyto-mediated MgO@ZrO2 nanocomposites in Plasmodium berghei -infected mice. Green-synthesized nanomaterials were characterized using various analytical techniques. In the suppressive test, 25 mice were infected with 0.2 mL of blood containing ~ 1x10⁷ parasites and treated with 50, 100, and 200 mg/kg bwt of nanoparticles for 4 days. In the curative test, 25 infected mice were divided into 5 groups: groups I-III received 50, 100, and 200 mg/kg bwt nanoparticles; group IV received 1.4 mg/kg bwt artemether/lumefantrine; group V received saline. Bodyweight, packed cell volume, and survival time were monitored. Scanning and transmission microscopy revealed agglomerated spherical nanoparticles (42.71 ± 11.60, 60.22 ± 6.54, and 39.25 ± 9.80 nm). XRD showed MgO-NPs, ZrO2-NPs, and MgO/ZrO2-NPs with varied lattice patterns. Acute toxicity tests showed LD 50 > 2000 mg/kg bwt. The nanoparticles suppressed parasites dose-dependently, with MgO-NPs, ZrO 2 -NPs, and MgO/ZrO 2 -NPs achieving 66.79%, 34.72%, and 41.02% suppression, respectively, at 200 mg/kg bwt. In the curative test, MgO-NPs > MgO/ZrO2-NPs > ZrO2-NPs showed significant inhibition (p < 0.05) compared to controls. These nanomaterials may serve as leads for antimalarial drug development. Antimalaria Green synthesis Acute toxicity Plasmodium berghei Nanotechnology Malaria Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Malaria is an acute illness caused by Plasmodium parasites, which is spread through the bites of infected female Anopheles mosquitoes (Steketee et al., 2021 ). There are 5 parasite species that cause malaria namely;- Plasmodium falciparum, Plasmodium malariae, Plasmodium ovale, Plasmodium knowlesi and Plasmodium vivax with P. falciparum and P. vivax and pose the greatest threat to human (Kwansa-Bentum et al., 2019 ; Udayabhanu et al., 2018 ). Plasmodium falciparum is the deadliest malaria parasite and the most prevalent on the African continent (Steketee et al., 2021 ). Nigeria, Democratic Republic of Congo, Uganda, Mozambique, Angola, and Burkina Faso account for over 90% of malaria cases in Africa constituting 55% of the global cases (Jagannathan & Kakuru, 2022 ). Also, according to WHO 2020 report, over 627, 000 deaths occurred globally as a result of malaria out of 241 million malaria related cases with 77% of this death involving children less than 5 years of age (Jagannathan & Kakuru, 2022 ). Despite efforts by the WHO and other international organisation to eradicate malaria in the world, the disease has continued to be a leading cause of death in Africa particularly Sub-Saharan Africa where access to medical facilities and medications still remain a big challenge (Okaiyeto et al., 2021 ). In 2018, the Commonwealth Leaders agreed to reduce the global cases of malaria by half on or before 2023. Unfortunately, the vision did not materialize due to the outbreak of COVID-19 pandemic that disrupted global economic growth and distort the program leading increased malaria cases and mortality rates. This among other factors clearly shows that the Commonwealth did not achieve its goal (Steketee et al., 2021 ). Furthermore, the evolution and spread of parasites resistant to the current available antimalarial drugs is one of the major challenges for effective global malaria control (GMC) (Kojom Foko et al., 2019 ). Recently, Artemisinin-based combination therapy (ACT) which is currently the most successful treatment available played a crucial role in GMC achievement (Kojom Foko et al., 2019 ). However, the advent of artemisinin-resistant P. falciparum strains, primarily recorded in Western Cambodia, Greater Mekong Subregion (GMS) and Southern China poses another threat to these artemisinin-based combinations (Souleymane et al., 2017 ). The spread of parasites that are resistant to artemisinin (ART) and/or to associated drugs in ACT is a major setback in the treatment of malaria patients and control strategies. Studies have shown that ACT-resistant parasites are responsible for higher failure rate from ACT treatment over the years (Baker et al., 2022 ; Haldar et al., 2018 ; Roux et al., 2021 ). Also, the spread of parasite resistant strain from Asia to Africa due to the resistance to antimalaria drugs such as chloroquine and sulfadoxine-pyrimethamine have also frustrated the efforts of malaria eradication around the globe(Su et al., 2019 ). Although. there have been many concerns about the potential emergence of k13 mutations linked to artemisinin resistance in Africa, the mutations that have been observed thus far are uncommon and unrelated to k13 polymorphisms that have been linked to decrease susceptibility in Asia (Djaman et al., 2017 ; Kamau et al., 2015 ; Menard et al., 2016 ; Murugan et al., 2017 ). Therefore, the future of malaria control and global elimination strategies would rely largely on cutting edge research and development of novel chemotherapeutic agents to the next generation of antimalarial drugs (Tse et al., 2019 ). There are different strategies and methods available for developing novel anti-malarial drugs, some of which have been derived from biological materials such as plants, animals and microorganisms (Kojom Foko et al., 2019 ). Recently, nanotechnology which is an emerging field of science with wide applications in diverse field of human endeavours have offered a new approach to the development of novel therapeutic drugs for the management of diseases (Okaiyeto et al., 2021 ). Different studies have shown that biological methods of synthesis of nanomaterials is a promising alternative to the conventional chemical methods (Avitabile et al., 2020 ; Kumaresan et al., 2018 ; Younis et al., 2021 ). Studying the physical and chemical properties of any nanomaterials for biomedical applications is essential because it provide the connection between their biological activity with specific parameters such as composition, size, shape, and capping (Avitabile et al., 2020 ). Also, size as well as surface properties of nanomaterials are an important criterion to determine their pharmacokinetic, bioavailability and biological activity (Younis et al., 2021 ). Due to the growing number of reports on the development of drug resistance to the primary treatments used to treat malaria, it is necessary to find new alternatives for the management of this disease (Busari et al., 2017 ). The potential of nanomaterials to provide controlled-release of active materials/ingredients is advantageous to address recrudescence, which is frequently encountered during artemisinin-based therapy (Pawar & Shende, 2020 ). The use of nanomaterials has been reported to enhance treatment results and efficacies due to their unique properties such as large surface area and morphology, sizes distribution, catalytic properties, high conductivity and stability and the ability to manipulate them to carry out a desired function in the biological system such as functionalization for active targeting of cells or organs (Varadharaj et al., 2020 ). Metallic nanoparticles are useful and safe in nature, with many applications in biomedical and agricultural research, electronics, cosmetics, food, and environmental remediation (Ahmed et al., 2016 ; Khandel et al., 2018 ). The biological uses of metallic nanoparticles such as zinc oxide, iron oxide, silver and titanium nanoparticles have been used as antibacterial (Patra & Baek, 2017 ; Patra & Baek, 2016 ), antifungal (Arciniegas-Grijalba et al., 2017 ; Mallmann et al., 2015 ), and antivirus (Broglie et al., 2015 ; Narasimha, 2013 ). In vivo anti-plasmodial study of MgO-NPs and ZrO 2 -NPs are scanty in literature. This is the first study to explore the anti-plasmodial potential of MgO/ZrO 2 -NPs in Plasmodium berghei infected mice. Other metallic nanoparticles such as silver and zinc oxide nanoparticles have been used in vitro for their anti-plasmodial potential. In a study by Najoom et al. ( 2021 ), ZnO nanoparticles significantly inhibit plasmodium parasites with lower doses while Hawadak et al. ( 2022 ) shows that silver nanoparticles inhibit plasmodium parasites with less haemolysis of the red blood cells. Therefore, the aim of this study is to evaluate the anti-plasmodial potentials of phyto-mediated magnesium and zirconium oxide nanocomposite against chloroquine sensitive NK-65 Plasmodium berghei infected albino mice. Materials and Methods Materials Fresh leaves of Eucalyptus camaldulensis were collected from Bosso Campus of Federal University of Technology, Minna, Niger State, Nigeria. The leaves were washed with clean water and air dried at the Laboratory of Biochemistry Department for 2 weeks to a constant dried weight. Zirconium tetrachloride octahydrate (ZrCl4.8H 2 O) ≥ 99.5%, Magnesium sulfate heptahydrate (MgSO 4 .7H 2 O) ≥ 98% and sodium hydroxide ≥ 97% were used for the synthesis of magnesium oxide, zirconium oxide and magnesium/zirconium oxide nanoparticles. Methods Preparation of plant extracts The dried leaves material was blended into fine powder using kitchen blender and (20 g) of the powder was extracted with 400 mL of distilled water at 45 ℃ for 30 minutes using magnetic stirrer to obtain a concentration of 50 mg/mL. The extract was filtered using muslin clothe and centrifuged to obtain a fine filtrate which was preserved in the refrigerator at 4 ℃. Synthesis of magnesium oxide nanoparticles (MgO-NPs) and zirconium oxide nanoparticles (ZrO-NPs) The synthesis of magnesium or zirconium oxide nanoparticles was carried out following the reported protocol of Khan et al. ( 2020 ) and Chau et al. ( 2023 ) with slight modifications. Exactly 20 mL of the plant extract and 100 mL of 0.2 mM solution of either magnesium sulphate or zirconium tetrachloride was stirred continuously on a magnetic stirrer for 2 hours at 60 ℃. The pH of the mixture was adjusted to 9 using 0.2 M NaOH and stirred further for another 1 h. The synthesis of the nanoparticles was confirmed by a colour change from light green to milky colour for MgO-NPs and light green to yellow for ZrO 2 -NPs. The solution was immediately centrifuged to collect the nanoparticles washed severally with deionized water to remove any trace of impurities. The nanoparticles were oven dried at 60 ℃ to a powdered form and calcined at 500 ℃ for 3 hours. Synthesis of magnesium oxide/zirconium oxide nanocomposite (MgO/ZrO-NPs) For the synthesis of MgO/ZrO 2 -NPs, 50 mL each of 0.2 mM solution of MgSO 4 and ZrCl 4 .8H 2 O solution were combined and stirred continuously for 30 minutes this was followed by the addition of 20 mL of the plant extract before stirring for 3 h. The pH of the solution was adjusted to 9 using 0.2 M NaOH and stirred further for another 1 hour. The synthesis of the nanoparticle was confirmed by colour change from light green to grey black. The solution was immediately centrifuged, washed severally with deionized water to obtain the synthesized nanoparticles in pellet form. The pellet was oven dried overnight at 60 ℃ and calcined at 500 ℃ for 3 hours. Characterization of the nanoparticles The synthesized nanoparticles were preliminarily characterized using scanning and transition electron microscope (SEM and TEM) (Zeiss Aunga) to determine the size and morphology of the nanoparticles. Powder X-ray diffractometer (Bruker d8) was used to acquire the X-ray diffraction (XRD) patterns of the synthesized nanoparticles and to determine the crystallinity of the nanoparticles using the Derby Sherer equation: \(\:L=\:\frac{K\lambda\:}{FWHM\:X\:Cos\theta\:}\) where L is the crystallite size, K is the constant, which is equivalent to 0.94, λ = 1.54178 and θ is the peak position in radian. Experimental Animals A total of seventy-five albino mice weighing between 18–25 g of both sexes were used for this study. The mice were obtained from the animal house of the Veterinary Department of the University of Jos, Nigeria. The animals were acclimatized and housed for a period of 21 days in standard cages and fed with standard pelleted feed (Guinea feed) and water ad libitum . Ethical approval for the animal studies was obtained from the Directorate of Research, Innovation and Development (Animal Ethics Committee), Federal University of Technology, Minna, Nigeria. Acute Toxicity Testing of the nanoparticles Oral acute toxicity test of the nanoparticles was carried following the standard guidelines of the Organisation for Economic Co-operation and Development (OECD) as described by Misganaw et al. ( 2020 ). For a sighting study, a single dose of 2000 mg/kg bodyweight (bwt) each of MgO-NPs, ZrO 2 -NPs and MgO/ZrO 2 -NPs were separately administered to a mouse by oral gavage. Since no mortality was recorded within 24 h, an additional four mice each were used and administered the same dose of the nanoparticles and distilled water was administered to the normal control. The mice were monitored continuously every 30 minutes for 4 h and then daily for the next 14 days for the general signs and symptoms of toxicity. Plasmodium berghei parasites A chloroquine sensitive strain of Plasmodium berghei (NK-69) was obtained from the National Institute of Medical Research (NIMER), Yaba Lagos, Nigeria and was maintained by sub-passage in mice. Parasite inoculation The parasites were inoculated intraperitoneally into the mouse with 0.2 mL of infected blood containing about 1 x 10 7 P. berghei parasitized erythrocytes. The inoculum consists of 5 x 10 7 P. berghei erythrocytes per mL. This was prepared by determining both the percentage parasitaemia and the erythrocytes count of the donor mouse under light microscope and diluting the blood with isotonic normal saline in ratio indicated by both determinations (Kumatia et al., 2021 ). Drug administration Artemether/lumefantrine (Art/Lum) was used as the positive control in this study. The drug, MgO-NPs, ZrO 2 -NPs and MgO/ZrO 2 -NPs used for the anti-plasmodial study were administered orally using a stainless metallic feeding cannula. In vivo anti-plasmodial activity of the nanoparticles Evaluation of Peters’4-day suppressive test of the nanoparticles This test was used to evaluate the schizontocidal activity of the nanoparticles and artesunate against early P. berghei infection in mice. A total of 25 Swiss albino mice were used in a Peters’ 4-day suppressive test. On the first day, the mice were infected with the parasite and randomly divided into 5 groups consisting of 5 mice each. These were orally administered with different doses of the nanoparticles (50, 100, and 200 mg/kg bwt) and Art/Lum (1.4 mg/kg bwt) while distilled water was administered to the negative control group. On the fifth day, thin blood film was made from tail blood of each mouse. The film was stained with Giemsa stain to reveal parasitized erythrocytes out of 500 in a random field of the microscope (Kumatia et al., 2021 ). The average percentage suppression of parasitaemia was calculated in comparison with the controls as follows: Evaluation of curative activities of the nanoparticles (Rane’s test) This was used to evaluate the schizontocidal activity of the nanoparticles, and artesunate in established infection. The study was carried out according to the method described by Ryley and Peters ( 1970 ). Plasmodium berghei was injected intraperitoneally into another 25 mice on the first day. After 72 h, the mice were divided randomly into five groups of 5 mice each. Different doses of the nanoparticles (50, 100 and 200 mg/kgbwt) were orally administered respectively to mice in groups I-III, 1.4 mg/kg/bwt of Art/Lum was administered to group IV (positive control) and group V was given distilled water (negative control). The nanoparticles and drug were administered once daily for 7 days. Giemsa-stained thin smears were prepared from tail blood samples collected every two days of treatment to monitor parasitaemia level. The bodyweight changes and packed cell volume (PCV) of the animals were also monitored every 2 days for the entire treatment period. Data Analysis Data were analysed using GraphPad Prism for windows version 9.0.0(121). Results for the study parameters are expressed as mean ± standard error of the mean (SEM). One-way ANOVA followed by Duncan post-hoc test for multiple comparisons was used to compare results among groups and the results were considered significant at 95% confidence level when the p-value was < 0.05. Results and Discussion Characterization of phyto-mediated nanoparticles The synthesis of MgO-NPs, ZrO 2 -NPs and MgO/ZrO 2 -NPs was conducted via green route. Scanning and transmission electron microscopy (SEM and TEM) analysis of the spherical shape MgO-NPs shows that the nanomaterials were agglomeration and monodispersed with distinct particles sizes (Fig. 1 a and b) while the oval shape ZrO 2 -NPs shows the agglomeration of the nanomaterials with the particle’s grains compacted (Fig. 1 c and d). In MgO/ZrO 2 -NPs, there was a mixture of spherical and oval shape nanomaterials which are compacted and agglomerated with no distinct separation between the grains of the nanomaterials (Fig. 1 d and e). The agglomeration of the nanomaterials might be due the electrostatic or van der Waal force of attraction resulting from the metallic elements used for the synthesis. This attraction was even more evident in the nanocomposite (Fig. 1 c) creating a hollow pore at the core of the nanomaterials. According to Arsuaga et al. ( 2013 ), agglomeration of nanomaterials due to attractive van der Waals forces can give rise to in homogeneities and defects in their morphology. Particle size analysis shows that the nanomaterials are made of particles of varied sizes with the nanocomposites (MgO/ZrO 2 -NPs) having the smallest particle size followed closely by MgO-NPs and then by ZrO 2 -NPs (Fig. 2 a-c). Particle sizes analysis of the nanoparticles shows that MgO-NPs contains particles ranging between 20–80 nm with an average particle size of 42.71 ± 11.60 nm (Fig. 2 a), ZrO 2 -NPs contains particles ranging between 30–100 nm with an average particle size of 60.22 ± 6.54 nm (Fig. 2 b) while MgO/ZrO 2 -NPs contains particles ranging from 20–70 nm with an average particle size of 39.25 ± 9.80 nm (Fig. 2 c). The particle size of MgO-NPs and ZrO 2 -NPs reported in this study is higher than 11.68 and 5 nm particle size reported by Ammulu et al. ( 2021 ) and Kumaresan et al. ( 2018 ) respectively for MgO-NPs and ZrO 2 -NPs. The differences in the particle sizes can be attributed to the concentration of the metallic salts and capping agents used and the synthesis condition (Tran et al., 2022 ). Also, it has been reported that the particle size of ZrO 2 -NPs ranges between 5–150 nm but particle size less than 50 nm are usually obtained when chemical or biological synthesis methods is used (Tran et al., 2022 ). Finally, Amrulloh et al. ( 2021 ) reported a particle size range between 60–100 nm for MgO-NPs synthesized using Mangifera indica bark. Elemental compositions of the nanomaterials show the presence of the constituent elements (Mg, Zr, and O) in different proportions. In MgO-NPs, there was 41.4% O and 50.69% Mg while ZrO 2 -NPs contain 51.4% O and 43.35% of the Zr metal. For MgO/ZrO 2 -NPs, the nanomaterials contain 20.51%, 33.89% and 39.34% of Mg, Zr and O respectively (Table 1 ). All the nanomaterials contain small amount of C which may be contributed by the plant materials used as a reducing or capping agent during the synthesis. The presence of carbon in the nanomaterials may be due to the bioactive metabolites of E. camaldulensis which play a dual role in both reducing and stabilizing the nanomaterials Abdel-Aziz et al. ( 2020 ). According to Shayoub et al. (2015) and Ghareeb et al. ( 2018 ), leaf extract of E. camaldulensis contains phytochemicals such as flavonoids and phenols which has been reported to be a capping and reducing agents for the synthesis of nanoparticles. Table 1 Elemental composition of the metallic nanoparticles synthesized using extract of E. camaldulensis Elements MgO-NPs ZrO 2 -NPs MgO/ZrO 2 -NP C 7.91 5.25 6.26 O 41.4 51.4 39.34 Mg 50.69 - 20.51 Zr - 43.35 33.89 Total 100.00 100.00 100.00 X-ray diffraction analysis of the nanoparticles shows the appearance of different diffraction peaks in all the nanomaterials with different fragmentation patterns and distinct phases (Fig. 3 ). In MgO-NPs, the periclase and lattice face-centered cubic nanomaterial shows the appearance of peaks at position 36.94°, 42.92°, 62.30°, 74.69° and 78.63° with a fragmentation pattern of (111), (200), (220), (311) and (222) respectively and a crystallite size of 11.69 ± 0.23 nm. Monoclinic tetragonal ZrO 2 -NPs shows the presence of similar peaks with MgO/ZrO 2 -NPs at position 30.22°, 34.57°, 50.22° and 59.28° with a fragmentation pattern of (101), (002), (112) and (103) with a crystallite size of 4.16 ± 0.82 and 6.07 ± 0.51 nm for ZrO 2 -NPs and MgO/ZrO 2 -NPs respectively. There was an emergence of unidentified peak at position 50.22° and 60.00° in MgO-NPs which becomes prominent in MgO/ZrO 2 -NPs while peaks at position 74.69° and 78.63° which are prominent in MgO-NPs where absent in MgO/ZrO 2 -NPs (Fig. 3 ). Studies have shown that increase crystallite size increases with increasing temperature and reaction time (Hassanzadeh-Tabrizi, 2023 ; Upadhyay et al., 2016 ). From this study, a temperature of 60 ℃ and 3 hrs reaction time was chosen based on the reported optimal condition for the synthesis of these nanomaterials in literature (Mourdikoudis et al., 2018 ). Crystallite size is one of the important parameters that influence physical properties of nanomaterials. Crystallite size is inversely proportional to the surface area of material (Upadhyay et al., 2016 ). Therefore, the synthesized nanomaterials shows that ZrO 2 -NPs have more surface area followed by MgO/ZrO 2 -NPs and MgO-NPs. Finally, the diffraction peaks pattern is similar to those identified by Younis et al. ( 2021 ) who synthesized MgO-NPs using Rosa floribunda charisma extract and Bishwokarma et al. ( 2021 ) for ZrO 2 -NPs synthesized using Curcuma longa extract. Acute oral toxicity test of the nanoparticles Acute oral toxicity test shows that the nanoparticles (MgO-NPs, ZrO 2 -NPs, and MgO/ZrO 2 -NPs) did not caused any gross behavioural changes and mortality within 24 h as well as in the following days, indicating that the LD 50 values of the nanoparticles is greater than 2000 mg/kg in mice as per OECD 423 guidelines. Acute toxicity study of the nanomaterials indicates that they are safe in the mice at a dose of 2000 mg/kg bodyweight in compliance with the Organization for Economic Cooperation and Development (OECD) guidance 423 document on acute toxicity (Bedi & Krishan, 2020 ). The mice show no loss of appetite and weakness in the first hour of administration of the nanoparticles with normal breathing, eye colour, skin, and furs position throughout the study period. Therefore, 50, 100 and 200 mg/kg bodyweight doses of the nanomaterials were selected for anti-plasmodial study. Anti-plasmodial activity of the nanoparticles Peter’s 4 days suppressive study of the nanoparticles shows a dose-dependent suppression of the parasites (Table 2 ). MgO-NPs at 50, 100 and 200 mg/kg bwt significantly inhibited 38.54 ± 3.91, 55.34 ± 2.60 and 66.79 ± 2.05% of the plasmodium parasites. The level of parasite inhibition by ZrO 2− NPs and MgO/ZrO 2 -NPs was not significantly difference (p > 0.05) at 50 and 100 mg/kg bwt doses but at 200 mg/kg bwt, MgO/ZrO 2 -NPs show a slightly higher inhibition of the parasites compared to ZrO 2 -NPs (Table 2 ). This result was further confirmed using the Rane’s curative test. The nanoparticles show a significant dose-dependent clearance of the parasites from the erythrocytes of the mice which was comparable to the standard drug (artesunate) used as positive control. The observed anti-plasmodial activity of the nanoparticles can be attributed to their particle and crystallite sizes. Smaller particle size will have a have larger surface area and will have more penetration into the cells resulting in more efficient interaction with the parasites. Also, the high surface-volume ratio of this smaller nanoparticles will lead to a significant release of reactive oxygen species that can alter the cellular structure of the parasite (Hanna et al., 2023 ). Finally, the crystallite sizes of the nanoparticles may lead to higher surface energy which can enhance the interaction between the nanoparticles and the biological membrane of the parasite resulting in a better anti-plasmodial effect (Parthiban et al., 2019 ). Table 2 Percentage parasite suppression by metallic nanoparticles synthesized using extract of E. camaldulensis % Suppression Nanoparticles 50 mg/kg bwt 100 mg/kg bwt 200 mg/kg bwt MgO-NPs 38.54 ± 3.91 b 55.34 ± 2.60 b 66.79 ± 2.05 c ZrO 2 -NPs 29.76 ± 5.97 a 31.21 ± 3.28 a 34.72 ± 3.33 a Mg/Zr-NPs 29.76 ± 3.23 a 31.86 ± 2.36 a 41.023.44 b Values are expressed in mean ± standard error of mean of 5 replicates. Values with the same superscript on the same column have no significance difference at p < 0.05. Effect of the nanoparticle’s treatment on the packed cell volume (PCV), bodyweight changes and mean survival time of the mice Treatment of the mice with different doses (50, 100 and 200 mg/kg bwt) of the nanoparticles shows a significant change (p < 0.05) in the PCV of the mice compared to group treated with 1.4 mg/kg bwt of artesunate (Fig. 5 ). The negative control group show a significant change (p < 0.01) in the bodyweight of the mice with a significant drop on the 8th days of the study (Fig. 5 ). Furthermore, the nanomaterials had no significant effect on the bodyweight of the mice throughout the period of treatment (Fig. 6 ). There was no significant change in the bodyweight of mice treated with the different doses of the nanoparticles between the first 3 days (Fig. 6 ) however, on the 8th day, there was a significant change in the bodyweight of the mice particularly the negative control group which shows a significant drop in the bodyweight of the animals. The different doses of the nanoparticles were able to averagely maintain the bodyweight of the mice throughout the treatment period compared with the positive control group. Treatment with different doses of the nanoparticles was also able to extend the survival time of the mice in a dose-dependent manner compared to the negative control group which have a mean survival time below 10 days (Fig. 7 ). The negative control group which was infected with the plasmodium parasite but not treated show a significant decrease (p < 0.05) in their bodyweight because of the parasites. During malaria infection, loss of appetite is preliminary symptoms which affect the feeding behaviour of the patient. From the acute toxicity study, the nanomaterials had no effect on the feeding behaviour of the mice hence maintenance of their bodyweight during the treatment period. Finally, treatment with different doses of the nanoparticles was found to extend the survival time of the mice compared with the negative control group (Fig. 7 ). This is an indication that the nanoparticle was able to reduce the virulence of the parasite and hence prevent the mortality of the mice (Fig. 7 ). In the negative control group, the mice were able to withstand the virulence of the parasite for about 8–9 days and majority of the mice died after 8 days because of the increase in the number of parasites in their erythrocytes. Conclusion The result from this study shows the successful synthesis of MgO-NPs, ZrO 2 -NPs and MgO/ZrO 2 -NPs using leaf extract of E. camaldulensis. Anti-plasmodial activity of the nanoparticles at different doses shows a significant inhibition of the parasites in a dose-dependent manner with moderate effect on the PCV and bodyweight changes of the mice. The treatment also shows a significant increase in the survival time of the P. berghei infected mice compared with the negative control mice with shorter survival time. This study therefore shows that the synthesis nanoparticles maybe a good candidate compound for the formulation of chemotherapeutic drug to control malaria parasites infections. Declarations Authors contribution Augustine Innalegwu Daniel, Jimoh Oladejo Tijani, Marshal Keyster, Ashwil Klein and Alechine Emmanuel Ameh contributed to project conceptualization, design, supervision, and administration. Sarah Udenyi Onogwu, Theresa Yebo Gara, Amuda Oladunni and Samson Oselusi contributed to writing, editing, analysis, and Manuscript draft. Hazeezah Mustapha Garba, Samad Hussein, Aminat Oluwatoyin Salaudeen contributed to writing, review and editing. Augustine Innalegwu Daniel carried out the data curation and validation. Funding This research did not receive funding from any source. Data availability The data and results generated in the current study are available from the corresponding author on reasonable request. Conflict of interest The authors have no competing interests. Ethical approval This study was approved by the Research Ethics Committee, Federal University of Technology, Minna, Niger State, Nigeria. Assigned number: 000079. Consent to participate The authors have given their consent to participate in the manuscript. Consent for publication The authors have given their consent to publish the manuscript. References Abdel-Aziz, M. M., Emam, T. M., & Elsherbiny, E. A. (2020). Bioactivity of magnesium oxide nanoparticles synthesized from cell filtrate of endobacterium Burkholderia rinojensis against Fusarium oxysporum. Materials Science and Engineering: C , 109 , 110617. Ahmed, S., Ahmad, M., Swami, B. L., & Ikram, S. (2016). A review on plants extract mediated synthesis of silver nanoparticles for antimicrobial applications: a green expertise. 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Influence of crystallite size on the magnetic properties of Fe3O4 nanoparticles. Journal of Alloys and Compounds , 678 , 478-485. Varadharaj, V., Ramaswamy, A., Sakthivel, R., Subbaiya, R., Barabadi, H., Chandrasekaran, M., & Saravanan, M. (2020). Correction to: Antidiabetic and Antioxidant Activity of Green Synthesized Starch Nanoparticles: An In Vitro Study. Journal of Cluster Science , 31 (6), 1267-1267. Younis, I. Y., El-Hawary, S. S., Eldahshan, O. A., Abdel-Aziz, M. M., & Ali, Z. Y. (2021). Green synthesis of magnesium nanoparticles mediated from Rosa floribunda charisma extract and its antioxidant, antiaging and antibiofilm activities. Scientific Reports , 11 (1), 16868. Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5662380","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":391477156,"identity":"d6388a53-37fb-45d7-bf20-f1fa53cabed0","order_by":0,"name":"Augustine Innalegwu Daniel","email":"","orcid":"","institution":"University of the Western Cape","correspondingAuthor":false,"prefix":"","firstName":"Augustine","middleName":"Innalegwu","lastName":"Daniel","suffix":""},{"id":391477157,"identity":"0669e5a1-fde5-4b82-ab7d-4f1fbd6ab761","order_by":1,"name":"Sarah Udenyi Onogwu","email":"","orcid":"","institution":"Joseph Sarwuan Tarka 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13:53:47","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5662380/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5662380/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":71884178,"identity":"0f2c7c51-d85c-4fe4-b5ce-281a0822a38d","added_by":"auto","created_at":"2024-12-19 12:00:17","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":469337,"visible":true,"origin":"","legend":"\u003cp\u003eSEM and TEM images of metallic (a and b) MgO-NPs, (c and d) ZrO\u003csub\u003e2\u003c/sub\u003e-NPs and (d and e) MgO/ZrO\u003csub\u003e2\u003c/sub\u003e-NPs respectively synthesized using extract of E. camaldulensis. The SEM image of MgO/ZrO\u003csub\u003e2\u003c/sub\u003e-NPs shows a hollow pore at the centre of the nanomaterials which can be attributed to the attraction force between the metallic Mg and Zr present in the nanomaterials.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-5662380/v1/243aaba9d030f40a8222bd36.png"},{"id":71884179,"identity":"3ce05bf7-b8dd-4b76-9504-fa13061e817b","added_by":"auto","created_at":"2024-12-19 12:00:17","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":265587,"visible":true,"origin":"","legend":"\u003cp\u003eParticle size distribution of (a) MgO-NPs, (b) ZrO\u003csub\u003e2\u003c/sub\u003e-NPs and (c) MgO/ZrO\u003csub\u003e2\u003c/sub\u003e-NPs synthesized using extract of E. camaldulensis\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-5662380/v1/236839eaf6f76e1621cdcdff.png"},{"id":71882866,"identity":"07bfc84d-627c-42b9-9a6a-f231d2b34c62","added_by":"auto","created_at":"2024-12-19 11:52:17","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":189370,"visible":true,"origin":"","legend":"\u003cp\u003eX-ray diffraction pattern of MgO-NPs, ZrO\u003csub\u003e2\u003c/sub\u003e-NPs and MgO/ZrO\u003csub\u003e2\u003c/sub\u003e-NPs synthesized using extract of E. camaldulensis\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-5662380/v1/8934723cbf2ed40c6191ca6b.png"},{"id":71882874,"identity":"66ff3ab0-f568-4fa8-a34d-b84ad0916957","added_by":"auto","created_at":"2024-12-19 11:52:17","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":191881,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of phyto-mediated metallic nanoparticles on the mean parasitaemia level of mice infected with Plasmodium berghei parasites and treated with 50, 100 and 200 mg/kg bwt of (a) MgO-NPs, (b) ZrO\u003csub\u003e2\u003c/sub\u003e-NPs and (c) MgO/ZrO\u003csub\u003e2\u003c/sub\u003e-NPs. The control group was administered Art/Lum (1.4 mg/kg bwt) while negative control group consist of mice infected with the parasite and left untreated throughout the period of the study.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-5662380/v1/bd75c8cfb71a6991a5b7a376.png"},{"id":71882870,"identity":"b9eb5c50-4943-4b7a-96f4-a0e1f22f73be","added_by":"auto","created_at":"2024-12-19 11:52:17","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":205052,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of phyto-mediated metallic nanoparticles on packed cell volume (PCV) of P. berghei infected mice. Mice were treated with 50, 100 and 200 mg/kg bwt of (a) MgO-NPs, (b) ZrO\u003csub\u003e2\u003c/sub\u003e-NPs and (c) MgO/ZrO\u003csub\u003e2\u003c/sub\u003e-NPs for a period of 7 days. The control group was administered Art/Lum (1.4 mg/kg bwt) while negative control group consist of mice infected with the parasite and left untreated throughout the period of the study. Violin with * shows statistically significant difference at p\u0026lt;0.05 while violin with ** were statistically significant at p\u0026lt;0.01.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-5662380/v1/a3a6fd1bc62ae1d7c2c95923.png"},{"id":71885746,"identity":"cab67c32-00a4-44bd-ad98-b0617dddd477","added_by":"auto","created_at":"2024-12-19 12:16:17","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":190909,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of phyto-mediated metallic nanoparticles on the mean bodyweight of \u003cem\u003eP. berghei \u003c/em\u003einfected mice. Mice were treated with 50, 100 and 200 mg/kg bwt of (a) MgO-NPs, (b) ZrO\u003csub\u003e2\u003c/sub\u003e-NPs and (c) MgO/ZrO\u003csub\u003e2\u003c/sub\u003e-NPs for a period of 7 days. The control group was administered Art/Lum (1.4 mg/kg bwt) while negative control group consist of mice infected with plasmodium parasite and left untreated throughout the period of the study. Bars with ** shows statistically significant difference at p\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-5662380/v1/227414a3380af668a59ff5bf.png"},{"id":71882869,"identity":"815a2be5-4239-4d04-beaf-0181fcdfff15","added_by":"auto","created_at":"2024-12-19 11:52:17","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":124634,"visible":true,"origin":"","legend":"\u003cp\u003eMean survival time of mice treated with different doses (50, 100 and 200 mg/kg bwt) of MgO-NPs, ZrO\u003csub\u003e2\u003c/sub\u003e-NPs, MgO/ZrO\u003csub\u003e2\u003c/sub\u003e-NPs and Art/Lum (1.4 mg/kg bwt). Negative control group consist of mice infected with the parasite and left untreated throughout the period of the study Bars with * are significantly different at p\u0026lt;0.05 while bars with ** are significantly different at p\u0026lt;0.01.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-5662380/v1/0f90e5e377d75d4830f014c1.png"},{"id":72414065,"identity":"c2c3af44-2f0a-4449-9738-2512c76152ea","added_by":"auto","created_at":"2024-12-26 19:16:32","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2415965,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5662380/v1/579e100b-88d7-483c-a0b8-17b8e2c5ca8b.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Green-Synthesis of MgO and ZrO 2 Nanocomposite: Physicochemical Properties and Anti- Plasmodial Activity in a Mouse Model","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMalaria is an acute illness caused by \u003cem\u003ePlasmodium\u003c/em\u003e parasites, which is spread through the bites of infected female \u003cem\u003eAnopheles\u003c/em\u003e mosquitoes (Steketee et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). There are 5 parasite species that cause malaria namely;- \u003cem\u003ePlasmodium falciparum, Plasmodium malariae, Plasmodium ovale, Plasmodium knowlesi\u003c/em\u003e and \u003cem\u003ePlasmodium vivax\u003c/em\u003e with \u003cem\u003eP. falciparum\u003c/em\u003e and \u003cem\u003eP. vivax\u003c/em\u003e and pose the greatest threat to human (Kwansa-Bentum et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Udayabhanu et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). \u003cem\u003ePlasmodium falciparum\u003c/em\u003e is the deadliest malaria parasite and the most prevalent on the African continent (Steketee et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Nigeria, Democratic Republic of Congo, Uganda, Mozambique, Angola, and Burkina Faso account for over 90% of malaria cases in Africa constituting 55% of the global cases (Jagannathan \u0026amp; Kakuru, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Also, according to WHO 2020 report, over 627, 000 deaths occurred globally as a result of malaria out of 241\u0026nbsp;million malaria related cases with 77% of this death involving children less than 5 years of age (Jagannathan \u0026amp; Kakuru, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eDespite efforts by the WHO and other international organisation to eradicate malaria in the world, the disease has continued to be a leading cause of death in Africa particularly Sub-Saharan Africa where access to medical facilities and medications still remain a big challenge (Okaiyeto et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In 2018, the Commonwealth Leaders agreed to reduce the global cases of malaria by half on or before 2023. Unfortunately, the vision did not materialize due to the outbreak of COVID-19 pandemic that disrupted global economic growth and distort the program leading increased malaria cases and mortality rates. This among other factors clearly shows that the Commonwealth did not achieve its goal (Steketee et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFurthermore, the evolution and spread of parasites resistant to the current available antimalarial drugs is one of the major challenges for effective global malaria control (GMC) (Kojom Foko et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Recently, Artemisinin-based combination therapy (ACT) which is currently the most successful treatment available played a crucial role in GMC achievement (Kojom Foko et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). However, the advent of artemisinin-resistant \u003cem\u003eP. falciparum\u003c/em\u003e strains, primarily recorded in Western Cambodia, Greater Mekong Subregion (GMS) and Southern China poses another threat to these artemisinin-based combinations (Souleymane et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The spread of parasites that are resistant to artemisinin (ART) and/or to associated drugs in ACT is a major setback in the treatment of malaria patients and control strategies. Studies have shown that ACT-resistant parasites are responsible for higher failure rate from ACT treatment over the years (Baker et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Haldar et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Roux et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Also, the spread of parasite resistant strain from Asia to Africa due to the resistance to antimalaria drugs such as chloroquine and sulfadoxine-pyrimethamine have also frustrated the efforts of malaria eradication around the globe(Su et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Although. there have been many concerns about the potential emergence of \u003cem\u003ek13\u003c/em\u003e mutations linked to artemisinin resistance in Africa, the mutations that have been observed thus far are uncommon and unrelated to \u003cem\u003ek13\u003c/em\u003e polymorphisms that have been linked to decrease susceptibility in Asia (Djaman et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Kamau et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Menard et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Murugan et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Therefore, the future of malaria control and global elimination strategies would rely largely on cutting edge research and development of novel chemotherapeutic agents to the next generation of antimalarial drugs (Tse et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThere are different strategies and methods available for developing novel anti-malarial drugs, some of which have been derived from biological materials such as plants, animals and microorganisms (Kojom Foko et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Recently, nanotechnology which is an emerging field of science with wide applications in diverse field of human endeavours have offered a new approach to the development of novel therapeutic drugs for the management of diseases (Okaiyeto et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Different studies have shown that biological methods of synthesis of nanomaterials is a promising alternative to the conventional chemical methods (Avitabile et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Kumaresan et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Younis et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Studying the physical and chemical properties of any nanomaterials for biomedical applications is essential because it provide the connection between their biological activity with specific parameters such as composition, size, shape, and capping (Avitabile et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Also, size as well as surface properties of nanomaterials are an important criterion to determine their pharmacokinetic, bioavailability and biological activity (Younis et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Due to the growing number of reports on the development of drug resistance to the primary treatments used to treat malaria, it is necessary to find new alternatives for the management of this disease (Busari et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The potential of nanomaterials to provide controlled-release of active materials/ingredients is advantageous to address recrudescence, which is frequently encountered during artemisinin-based therapy (Pawar \u0026amp; Shende, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The use of nanomaterials has been reported to enhance treatment results and efficacies due to their unique properties such as large surface area and morphology, sizes distribution, catalytic properties, high conductivity and stability and the ability to manipulate them to carry out a desired function in the biological system such as functionalization for active targeting of cells or organs (Varadharaj et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Metallic nanoparticles are useful and safe in nature, with many applications in biomedical and agricultural research, electronics, cosmetics, food, and environmental remediation (Ahmed et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Khandel et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The biological uses of metallic nanoparticles such as zinc oxide, iron oxide, silver and titanium nanoparticles have been used as antibacterial (Patra \u0026amp; Baek, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Patra \u0026amp; Baek, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), antifungal (Arciniegas-Grijalba et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Mallmann et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), and antivirus (Broglie et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Narasimha, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). \u003cem\u003eIn vivo\u003c/em\u003e anti-plasmodial study of MgO-NPs and ZrO\u003csub\u003e2\u003c/sub\u003e-NPs are scanty in literature. This is the first study to explore the anti-plasmodial potential of MgO/ZrO\u003csub\u003e2\u003c/sub\u003e-NPs in \u003cem\u003ePlasmodium berghei\u003c/em\u003e infected mice. Other metallic nanoparticles such as silver and zinc oxide nanoparticles have been used \u003cem\u003ein vitro\u003c/em\u003e for their anti-plasmodial potential. In a study by Najoom et al. (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), ZnO nanoparticles significantly inhibit plasmodium parasites with lower doses while Hawadak et al. (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) shows that silver nanoparticles inhibit plasmodium parasites with less haemolysis of the red blood cells. Therefore, the aim of this study is to evaluate the anti-plasmodial potentials of phyto-mediated magnesium and zirconium oxide nanocomposite against chloroquine sensitive NK-65 \u003cem\u003ePlasmodium berghei\u003c/em\u003e infected albino mice.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003eMaterials\u003c/h2\u003e\n \u003cp\u003eFresh leaves of \u003cem\u003eEucalyptus camaldulensis\u003c/em\u003e were collected from Bosso Campus of Federal University of Technology, Minna, Niger State, Nigeria. The leaves were washed with clean water and air dried at the Laboratory of Biochemistry Department for 2 weeks to a constant dried weight. Zirconium tetrachloride octahydrate (ZrCl4.8H\u003csub\u003e2\u003c/sub\u003eO)\u0026thinsp;\u0026ge;\u0026thinsp;99.5%, Magnesium sulfate heptahydrate (MgSO\u003csub\u003e4\u003c/sub\u003e.7H\u003csub\u003e2\u003c/sub\u003eO)\u0026thinsp;\u0026ge;\u0026thinsp;98% and sodium hydroxide\u0026thinsp;\u0026ge;\u0026thinsp;97% were used for the synthesis of magnesium oxide, zirconium oxide and magnesium/zirconium oxide nanoparticles.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eMethods\u003c/h3\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n \u003ch2\u003ePreparation of plant extracts\u003c/h2\u003e\n \u003cp\u003eThe dried leaves material was blended into fine powder using kitchen blender and (20 g) of the powder was extracted with 400 mL of distilled water at 45 ℃ for 30 minutes using magnetic stirrer to obtain a concentration of 50 mg/mL. The extract was filtered using muslin clothe and centrifuged to obtain a fine filtrate which was preserved in the refrigerator at 4 ℃.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eSynthesis of magnesium oxide nanoparticles (MgO-NPs) and zirconium oxide nanoparticles (ZrO-NPs)\u003c/h3\u003e\n\u003cp\u003eThe synthesis of magnesium or zirconium oxide nanoparticles was carried out following the reported protocol of Khan et al. (\u003cspan class=\"CitationRef\"\u003e2020\u003c/span\u003e) and Chau et al. (\u003cspan class=\"CitationRef\"\u003e2023\u003c/span\u003e) with slight modifications. Exactly 20 mL of the plant extract and 100 mL of 0.2 mM solution of either magnesium sulphate or zirconium tetrachloride was stirred continuously on a magnetic stirrer for 2 hours at 60 ℃. The pH of the mixture was adjusted to 9 using 0.2 M NaOH and stirred further for another 1 h. The synthesis of the nanoparticles was confirmed by a colour change from light green to milky colour for MgO-NPs and light green to yellow for ZrO\u003csub\u003e2\u003c/sub\u003e-NPs. The solution was immediately centrifuged to collect the nanoparticles washed severally with deionized water to remove any trace of impurities. The nanoparticles were oven dried at 60 ℃ to a powdered form and calcined at 500 ℃ for 3 hours.\u003c/p\u003e\n\u003ch3\u003eSynthesis of magnesium oxide/zirconium oxide nanocomposite (MgO/ZrO-NPs)\u003c/h3\u003e\n\u003cp\u003eFor the synthesis of MgO/ZrO\u003csub\u003e2\u003c/sub\u003e-NPs, 50 mL each of 0.2 mM solution of MgSO\u003csub\u003e4\u003c/sub\u003e and ZrCl\u003csub\u003e4\u003c/sub\u003e.8H\u003csub\u003e2\u003c/sub\u003eO solution were combined and stirred continuously for 30 minutes this was followed by the addition of 20 mL of the plant extract before stirring for 3 h. The pH of the solution was adjusted to 9 using 0.2 M NaOH and stirred further for another 1 hour. The synthesis of the nanoparticle was confirmed by colour change from light green to grey black. The solution was immediately centrifuged, washed severally with deionized water to obtain the synthesized nanoparticles in pellet form. The pellet was oven dried overnight at 60 ℃ and calcined at 500 ℃ for 3 hours.\u003c/p\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003eCharacterization of the nanoparticles\u003c/h2\u003e\n \u003cp\u003eThe synthesized nanoparticles were preliminarily characterized using scanning and transition electron microscope (SEM and TEM) (Zeiss Aunga) to determine the size and morphology of the nanoparticles. Powder X-ray diffractometer (Bruker d8) was used to acquire the X-ray diffraction (XRD) patterns of the synthesized nanoparticles and to determine the crystallinity of the nanoparticles using the Derby Sherer equation:\u003c/p\u003e\n \u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u0026nbsp;\u003cspan class=\"mathinline\"\u003e\\(\\:L=\\:\\frac{K\\lambda\\:}{FWHM\\:X\\:Cos\\theta\\:}\\)\u003c/span\u003e\u0026nbsp;\u003c/span\u003e where L is the crystallite size, K is the constant, which is equivalent to 0.94, \u0026lambda;\u0026thinsp;=\u0026thinsp;1.54178 and \u0026theta; is the peak position in radian.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eExperimental Animals\u003c/h3\u003e\n\u003cp\u003eA total of seventy-five albino mice weighing between 18\u0026ndash;25 g of both sexes were used for this study. The mice were obtained from the animal house of the Veterinary Department of the University of Jos, Nigeria. The animals were acclimatized and housed for a period of 21 days in standard cages and fed with standard pelleted feed (Guinea feed) and water \u003cem\u003ead libitum\u003c/em\u003e. Ethical approval for the animal studies was obtained from the Directorate of Research, Innovation and Development (Animal Ethics Committee), Federal University of Technology, Minna, Nigeria.\u003c/p\u003e\n\u003ch3\u003eAcute Toxicity Testing of the nanoparticles\u003c/h3\u003e\n\u003cp\u003eOral acute toxicity test of the nanoparticles was carried following the standard guidelines of the Organisation for Economic Co-operation and Development (OECD) as described by Misganaw et al. (\u003cspan class=\"CitationRef\"\u003e2020\u003c/span\u003e). For a sighting study, a single dose of 2000 mg/kg bodyweight (bwt) each of MgO-NPs, ZrO\u003csub\u003e2\u003c/sub\u003e-NPs and MgO/ZrO\u003csub\u003e2\u003c/sub\u003e-NPs were separately administered to a mouse by oral gavage. Since no mortality was recorded within 24 h, an additional four mice each were used and administered the same dose of the nanoparticles and distilled water was administered to the normal control. The mice were monitored continuously every 30 minutes for 4 h and then daily for the next 14 days for the general signs and symptoms of toxicity.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePlasmodium berghei\u003c/strong\u003e \u003cstrong\u003eparasites\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA chloroquine sensitive strain of \u003cem\u003ePlasmodium berghei\u003c/em\u003e (NK-69) was obtained from the National Institute of Medical Research (NIMER), Yaba Lagos, Nigeria and was maintained by sub-passage in mice.\u003c/p\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003eParasite inoculation\u003c/h2\u003e\n \u003cp\u003eThe parasites were inoculated intraperitoneally into the mouse with 0.2 mL of infected blood containing about 1 x 10\u003csup\u003e7\u003c/sup\u003e \u003cem\u003eP. berghei\u003c/em\u003e parasitized erythrocytes. The inoculum consists of 5 x 10\u003csup\u003e7\u003c/sup\u003e \u003cem\u003eP. berghei\u003c/em\u003e erythrocytes per mL. This was prepared by determining both the percentage parasitaemia and the erythrocytes count of the donor mouse under light microscope and diluting the blood with isotonic normal saline in ratio indicated by both determinations (Kumatia et al., \u003cspan class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003ch2\u003eDrug administration\u003c/h2\u003e\n \u003cp\u003eArtemether/lumefantrine (Art/Lum) was used as the positive control in this study. The drug, MgO-NPs, ZrO\u003csub\u003e2\u003c/sub\u003e-NPs and MgO/ZrO\u003csub\u003e2\u003c/sub\u003e-NPs used for the anti-plasmodial study were administered orally using a stainless metallic feeding cannula.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eIn vivo\u003c/strong\u003e \u003cstrong\u003eanti-plasmodial activity of the nanoparticles\u003c/strong\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n \u003ch2\u003eEvaluation of Peters\u0026rsquo;4-day suppressive test of the nanoparticles\u003c/h2\u003e\n \u003cp\u003eThis test was used to evaluate the schizontocidal activity of the nanoparticles and artesunate against early \u003cem\u003eP. berghei\u003c/em\u003e infection in mice. A total of 25 Swiss albino mice were used in a Peters\u0026rsquo; 4-day suppressive test. On the first day, the mice were infected with the parasite and randomly divided into 5 groups consisting of 5 mice each. These were orally administered with different doses of the nanoparticles (50, 100, and 200 mg/kg bwt) and Art/Lum (1.4 mg/kg bwt) while distilled water was administered to the negative control group. On the fifth day, thin blood film was made from tail blood of each mouse. The film was stained with Giemsa stain to reveal parasitized erythrocytes out of 500 in a random field of the microscope (Kumatia et al., \u003cspan class=\"CitationRef\"\u003e2021\u003c/span\u003e). The average percentage suppression of parasitaemia was calculated in comparison with the controls as follows:\u003c/p\u003e\n \u003cp\u003e\u003cimg src=\"https://myfiles.space/user_files/122228_c8a1650c59388082/122228_custom_files/img1734608305.png\"\u003e\u003cbr\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n \u003ch2\u003eEvaluation of curative activities of the nanoparticles (Rane\u0026rsquo;s test)\u003c/h2\u003e\n \u003cp\u003eThis was used to evaluate the schizontocidal activity of the nanoparticles, and artesunate in established infection. The study was carried out according to the method described by Ryley and Peters (\u003cspan class=\"CitationRef\"\u003e1970\u003c/span\u003e). \u003cem\u003ePlasmodium berghei\u003c/em\u003e was injected intraperitoneally into another 25 mice on the first day. After 72 h, the mice were divided randomly into five groups of 5 mice each. Different doses of the nanoparticles (50, 100 and 200 mg/kgbwt) were orally administered respectively to mice in groups I-III, 1.4 mg/kg/bwt of Art/Lum was administered to group IV (positive control) and group V was given distilled water (negative control). The nanoparticles and drug were administered once daily for 7 days. Giemsa-stained thin smears were prepared from tail blood samples collected every two days of treatment to monitor parasitaemia level. The bodyweight changes and packed cell volume (PCV) of the animals were also monitored every 2 days for the entire treatment period.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n \u003ch2\u003eData Analysis\u003c/h2\u003e\n \u003cp\u003eData were analysed using GraphPad Prism for windows version 9.0.0(121). Results for the study parameters are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM). One-way ANOVA followed by Duncan post-hoc test for multiple comparisons was used to compare results among groups and the results were considered significant at 95% confidence level when the p-value was \u0026lt;\u0026thinsp;0.05.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Results and Discussion","content":"\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eCharacterization of phyto-mediated nanoparticles\u003c/h2\u003e \u003cp\u003eThe synthesis of MgO-NPs, ZrO\u003csub\u003e2\u003c/sub\u003e-NPs and MgO/ZrO\u003csub\u003e2\u003c/sub\u003e-NPs was conducted via green route. Scanning and transmission electron microscopy (SEM and TEM) analysis of the spherical shape MgO-NPs shows that the nanomaterials were agglomeration and monodispersed with distinct particles sizes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea and b) while the oval shape ZrO\u003csub\u003e2\u003c/sub\u003e-NPs shows the agglomeration of the nanomaterials with the particle\u0026rsquo;s grains compacted (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec and d). In MgO/ZrO\u003csub\u003e2\u003c/sub\u003e-NPs, there was a mixture of spherical and oval shape nanomaterials which are compacted and agglomerated with no distinct separation between the grains of the nanomaterials (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed and e). The agglomeration of the nanomaterials might be due the electrostatic or van der Waal force of attraction resulting from the metallic elements used for the synthesis. This attraction was even more evident in the nanocomposite (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec) creating a hollow pore at the core of the nanomaterials. According to Arsuaga et al. (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), agglomeration of nanomaterials due to attractive van der Waals forces can give rise to in homogeneities and defects in their morphology. Particle size analysis shows that the nanomaterials are made of particles of varied sizes with the nanocomposites (MgO/ZrO\u003csub\u003e2\u003c/sub\u003e-NPs) having the smallest particle size followed closely by MgO-NPs and then by ZrO\u003csub\u003e2\u003c/sub\u003e-NPs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea-c). Particle sizes analysis of the nanoparticles shows that MgO-NPs contains particles ranging between 20\u0026ndash;80 nm with an average particle size of 42.71\u0026thinsp;\u0026plusmn;\u0026thinsp;11.60 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea), ZrO\u003csub\u003e2\u003c/sub\u003e-NPs contains particles ranging between 30\u0026ndash;100 nm with an average particle size of 60.22\u0026thinsp;\u0026plusmn;\u0026thinsp;6.54 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb) while MgO/ZrO\u003csub\u003e2\u003c/sub\u003e-NPs contains particles ranging from 20\u0026ndash;70 nm with an average particle size of 39.25\u0026thinsp;\u0026plusmn;\u0026thinsp;9.80 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). The particle size of MgO-NPs and ZrO\u003csub\u003e2\u003c/sub\u003e-NPs reported in this study is higher than 11.68 and 5 nm particle size reported by Ammulu et al. (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) and Kumaresan et al. (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) respectively for MgO-NPs and ZrO\u003csub\u003e2\u003c/sub\u003e-NPs. The differences in the particle sizes can be attributed to the concentration of the metallic salts and capping agents used and the synthesis condition (Tran et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Also, it has been reported that the particle size of ZrO\u003csub\u003e2\u003c/sub\u003e-NPs ranges between 5\u0026ndash;150 nm but particle size less than 50 nm are usually obtained when chemical or biological synthesis methods is used (Tran et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Finally, Amrulloh et al. (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) reported a particle size range between 60\u0026ndash;100 nm for MgO-NPs synthesized using \u003cem\u003eMangifera indica\u003c/em\u003e bark.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eElemental compositions of the nanomaterials show the presence of the constituent elements (Mg, Zr, and O) in different proportions. In MgO-NPs, there was 41.4% O and 50.69% Mg while ZrO\u003csub\u003e2\u003c/sub\u003e-NPs contain 51.4% O and 43.35% of the Zr metal. For MgO/ZrO\u003csub\u003e2\u003c/sub\u003e-NPs, the nanomaterials contain 20.51%, 33.89% and 39.34% of Mg, Zr and O respectively (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). All the nanomaterials contain small amount of C which may be contributed by the plant materials used as a reducing or capping agent during the synthesis. The presence of carbon in the nanomaterials may be due to the bioactive metabolites of \u003cem\u003eE. camaldulensis\u003c/em\u003e which play a dual role in both reducing and stabilizing the nanomaterials Abdel-Aziz et al. (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). According to Shayoub et al. (2015) and Ghareeb et al. (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), leaf extract of \u003cem\u003eE. camaldulensis\u003c/em\u003e contains phytochemicals such as flavonoids and phenols which has been reported to be a capping and reducing agents for the synthesis of nanoparticles.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eElemental composition of the metallic nanoparticles synthesized using extract of \u003cem\u003eE. camaldulensis\u003c/em\u003e\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eElements\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMgO-NPs\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eZrO\u003csub\u003e2\u003c/sub\u003e-NPs\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMgO/ZrO\u003csub\u003e2\u003c/sub\u003e-NP\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7.91\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e6.26\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eO\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e41.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e51.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e39.34\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMg\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e50.69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e20.51\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eZr\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e43.35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e33.89\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e100.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e100.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e100.00\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eX-ray diffraction analysis of the nanoparticles shows the appearance of different diffraction peaks in all the nanomaterials with different fragmentation patterns and distinct phases (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). In MgO-NPs, the periclase and lattice face-centered cubic nanomaterial shows the appearance of peaks at position 36.94\u0026deg;, 42.92\u0026deg;, 62.30\u0026deg;, 74.69\u0026deg; and 78.63\u0026deg; with a fragmentation pattern of (111), (200), (220), (311) and (222) respectively and a crystallite size of 11.69\u0026thinsp;\u0026plusmn;\u0026thinsp;0.23 nm. Monoclinic tetragonal ZrO\u003csub\u003e2\u003c/sub\u003e-NPs shows the presence of similar peaks with MgO/ZrO\u003csub\u003e2\u003c/sub\u003e-NPs at position 30.22\u0026deg;, 34.57\u0026deg;, 50.22\u0026deg; and 59.28\u0026deg; with a fragmentation pattern of (101), (002), (112) and (103) with a crystallite size of 4.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.82 and 6.07\u0026thinsp;\u0026plusmn;\u0026thinsp;0.51 nm for ZrO\u003csub\u003e2\u003c/sub\u003e-NPs and MgO/ZrO\u003csub\u003e2\u003c/sub\u003e-NPs respectively. There was an emergence of unidentified peak at position 50.22\u0026deg; and 60.00\u0026deg; in MgO-NPs which becomes prominent in MgO/ZrO\u003csub\u003e2\u003c/sub\u003e-NPs while peaks at position 74.69\u0026deg; and 78.63\u0026deg; which are prominent in MgO-NPs where absent in MgO/ZrO\u003csub\u003e2\u003c/sub\u003e-NPs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Studies have shown that increase crystallite size increases with increasing temperature and reaction time (Hassanzadeh-Tabrizi, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Upadhyay et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). From this study, a temperature of 60 ℃ and 3 hrs reaction time was chosen based on the reported optimal condition for the synthesis of these nanomaterials in literature (Mourdikoudis et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Crystallite size is one of the important parameters that influence physical properties of nanomaterials. Crystallite size is inversely proportional to the surface area of material (Upadhyay et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Therefore, the synthesized nanomaterials shows that ZrO\u003csub\u003e2\u003c/sub\u003e-NPs have more surface area followed by MgO/ZrO\u003csub\u003e2\u003c/sub\u003e-NPs and MgO-NPs. Finally, the diffraction peaks pattern is similar to those identified by Younis et al. (\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) who synthesized MgO-NPs using \u003cem\u003eRosa floribunda\u003c/em\u003e charisma extract and Bishwokarma et al. (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) for ZrO\u003csub\u003e2\u003c/sub\u003e-NPs synthesized using \u003cem\u003eCurcuma longa\u003c/em\u003e extract.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eAcute oral toxicity test of the nanoparticles\u003c/h2\u003e \u003cp\u003eAcute oral toxicity test shows that the nanoparticles (MgO-NPs, ZrO\u003csub\u003e2\u003c/sub\u003e-NPs, and MgO/ZrO\u003csub\u003e2\u003c/sub\u003e-NPs) did not caused any gross behavioural changes and mortality within 24 h as well as in the following days, indicating that the LD\u003csub\u003e50\u003c/sub\u003e values of the nanoparticles is greater than 2000 mg/kg in mice as per OECD 423 guidelines. Acute toxicity study of the nanomaterials indicates that they are safe in the mice at a dose of 2000 mg/kg bodyweight in compliance with the Organization for Economic Cooperation and Development (OECD) guidance 423 document on acute toxicity (Bedi \u0026amp; Krishan, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The mice show no loss of appetite and weakness in the first hour of administration of the nanoparticles with normal breathing, eye colour, skin, and furs position throughout the study period. Therefore, 50, 100 and 200 mg/kg bodyweight doses of the nanomaterials were selected for anti-plasmodial study.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eAnti-plasmodial activity of the nanoparticles\u003c/h2\u003e \u003cp\u003ePeter\u0026rsquo;s 4 days suppressive study of the nanoparticles shows a dose-dependent suppression of the parasites (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). MgO-NPs at 50, 100 and 200 mg/kg bwt significantly inhibited 38.54\u0026thinsp;\u0026plusmn;\u0026thinsp;3.91, 55.34\u0026thinsp;\u0026plusmn;\u0026thinsp;2.60 and 66.79\u0026thinsp;\u0026plusmn;\u0026thinsp;2.05% of the plasmodium parasites. The level of parasite inhibition by ZrO\u003csub\u003e2\u0026minus;\u003c/sub\u003eNPs and MgO/ZrO\u003csub\u003e2\u003c/sub\u003e-NPs was not significantly difference (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05) at 50 and 100 mg/kg bwt doses but at 200 mg/kg bwt, MgO/ZrO\u003csub\u003e2\u003c/sub\u003e-NPs show a slightly higher inhibition of the parasites compared to ZrO\u003csub\u003e2\u003c/sub\u003e-NPs (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). This result was further confirmed using the Rane\u0026rsquo;s curative test. The nanoparticles show a significant dose-dependent clearance of the parasites from the erythrocytes of the mice which was comparable to the standard drug (artesunate) used as positive control. The observed anti-plasmodial activity of the nanoparticles can be attributed to their particle and crystallite sizes. Smaller particle size will have a have larger surface area and will have more penetration into the cells resulting in more efficient interaction with the parasites. Also, the high surface-volume ratio of this smaller nanoparticles will lead to a significant release of reactive oxygen species that can alter the cellular structure of the parasite (Hanna et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Finally, the crystallite sizes of the nanoparticles may lead to higher surface energy which can enhance the interaction between the nanoparticles and the biological membrane of the parasite resulting in a better anti-plasmodial effect (Parthiban et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePercentage parasite suppression by metallic nanoparticles synthesized using extract \u003cem\u003eof E. camaldulensis\u003c/em\u003e\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e \u003cp\u003e% Suppression\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNanoparticles\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e50 mg/kg bwt\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e100 mg/kg bwt\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e200 mg/kg bwt\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMgO-NPs\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e38.54\u0026thinsp;\u0026plusmn;\u0026thinsp;3.91\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e55.34\u0026thinsp;\u0026plusmn;\u0026thinsp;2.60\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e66.79\u0026thinsp;\u0026plusmn;\u0026thinsp;2.05\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eZrO\u003csub\u003e2\u003c/sub\u003e-NPs\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e29.76\u0026thinsp;\u0026plusmn;\u0026thinsp;5.97\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e31.21\u0026thinsp;\u0026plusmn;\u0026thinsp;3.28\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e34.72\u0026thinsp;\u0026plusmn;\u0026thinsp;3.33\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMg/Zr-NPs\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e29.76\u0026thinsp;\u0026plusmn;\u0026thinsp;3.23\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e31.86\u0026thinsp;\u0026plusmn;\u0026thinsp;2.36\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e41.023.44\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eValues are expressed in mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of mean of 5 replicates. Values with the same superscript on the same column have no significance difference at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eEffect of the nanoparticle\u0026rsquo;s treatment on the packed cell volume (PCV), bodyweight changes and mean survival time of the mice\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTreatment of the mice with different doses (50, 100 and 200 mg/kg bwt) of the nanoparticles shows a significant change (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in the PCV of the mice compared to group treated with 1.4 mg/kg bwt of artesunate (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The negative control group show a significant change (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) in the bodyweight of the mice with a significant drop on the 8th days of the study (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Furthermore, the nanomaterials had no significant effect on the bodyweight of the mice throughout the period of treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). There was no significant change in the bodyweight of mice treated with the different doses of the nanoparticles between the first 3 days (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e) however, on the 8th day, there was a significant change in the bodyweight of the mice particularly the negative control group which shows a significant drop in the bodyweight of the animals. The different doses of the nanoparticles were able to averagely maintain the bodyweight of the mice throughout the treatment period compared with the positive control group. Treatment with different doses of the nanoparticles was also able to extend the survival time of the mice in a dose-dependent manner compared to the negative control group which have a mean survival time below 10 days (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). The negative control group which was infected with the plasmodium parasite but not treated show a significant decrease (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in their bodyweight because of the parasites. During malaria infection, loss of appetite is preliminary symptoms which affect the feeding behaviour of the patient. From the acute toxicity study, the nanomaterials had no effect on the feeding behaviour of the mice hence maintenance of their bodyweight during the treatment period. Finally, treatment with different doses of the nanoparticles was found to extend the survival time of the mice compared with the negative control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). This is an indication that the nanoparticle was able to reduce the virulence of the parasite and hence prevent the mortality of the mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). In the negative control group, the mice were able to withstand the virulence of the parasite for about 8\u0026ndash;9 days and majority of the mice died after 8 days because of the increase in the number of parasites in their erythrocytes.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe result from this study shows the successful synthesis of MgO-NPs, ZrO\u003csub\u003e2\u003c/sub\u003e-NPs and MgO/ZrO\u003csub\u003e2\u003c/sub\u003e-NPs using leaf extract of \u003cem\u003eE. camaldulensis.\u003c/em\u003e Anti-plasmodial activity of the nanoparticles at different doses shows a significant inhibition of the parasites in a dose-dependent manner with moderate effect on the PCV and bodyweight changes of the mice. The treatment also shows a significant increase in the survival time of the \u003cem\u003eP. berghei\u003c/em\u003e infected mice compared with the negative control mice with shorter survival time. This study therefore shows that the synthesis nanoparticles maybe a good candidate compound for the formulation of chemotherapeutic drug to control malaria parasites infections.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthors contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAugustine Innalegwu Daniel, Jimoh Oladejo Tijani, Marshal Keyster, Ashwil Klein and Alechine Emmanuel Ameh contributed to project conceptualization, design, supervision, and administration. Sarah Udenyi Onogwu, Theresa Yebo Gara, Amuda Oladunni and Samson Oselusi contributed to writing, editing, analysis, and Manuscript draft. Hazeezah Mustapha Garba, Samad Hussein, Aminat Oluwatoyin Salaudeen contributed to writing, review and editing. Augustine Innalegwu Daniel carried out the data curation and validation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis research did not receive funding from any source.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data and results generated in the current study are available from the corresponding author on reasonable request.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e The authors have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval\u003c/strong\u003e This study was approved by the Research Ethics Committee, Federal University of Technology, Minna, Niger State, Nigeria. Assigned number: 000079.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate\u003c/strong\u003e The authors have given their consent to participate in the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e The authors have given their consent to publish the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAbdel-Aziz, M. M., Emam, T. M., \u0026amp; Elsherbiny, E. A. (2020). Bioactivity of magnesium oxide nanoparticles synthesized from cell filtrate of endobacterium Burkholderia rinojensis against Fusarium oxysporum. \u003cem\u003eMaterials Science and Engineering: C\u003c/em\u003e,\u003cem\u003e 109\u003c/em\u003e, 110617. \u003c/li\u003e\n\u003cli\u003eAhmed, S., Ahmad, M., Swami, B. L., \u0026amp; Ikram, S. (2016). 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M., \u0026amp; Ali, Z. Y. (2021). Green synthesis of magnesium nanoparticles mediated from Rosa floribunda charisma extract and its antioxidant, antiaging and antibiofilm activities. \u003cem\u003eScientific Reports\u003c/em\u003e,\u003cem\u003e 11\u003c/em\u003e(1), 16868.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Antimalaria, Green synthesis, Acute toxicity, Plasmodium berghei, Nanotechnology, Malaria","lastPublishedDoi":"10.21203/rs.3.rs-5662380/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5662380/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMalaria remains a life-threatening disease, particularly in Sub-Saharan Africa, with the emergence of Plasmodium-resistant parasites necessitating new chemotherapeutic drugs. This study evaluated the anti-plasmodial potential of phyto-mediated MgO@ZrO2 nanocomposites in \u003cem\u003ePlasmodium berghei\u003c/em\u003e-infected mice. Green-synthesized nanomaterials were characterized using various analytical techniques. In the suppressive test, 25 mice were infected with 0.2 mL of blood containing\u0026thinsp;~\u0026thinsp;1x10⁷ parasites and treated with 50, 100, and 200 mg/kg bwt of nanoparticles for 4 days. In the curative test, 25 infected mice were divided into 5 groups: groups I-III received 50, 100, and 200 mg/kg bwt nanoparticles; group IV received 1.4 mg/kg bwt artemether/lumefantrine; group V received saline. Bodyweight, packed cell volume, and survival time were monitored. Scanning and transmission microscopy revealed agglomerated spherical nanoparticles (42.71\u0026thinsp;\u0026plusmn;\u0026thinsp;11.60, 60.22\u0026thinsp;\u0026plusmn;\u0026thinsp;6.54, and 39.25\u0026thinsp;\u0026plusmn;\u0026thinsp;9.80 nm). XRD showed MgO-NPs, ZrO2-NPs, and MgO/ZrO2-NPs with varied lattice patterns. Acute toxicity tests showed LD\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;\u0026gt;\u0026thinsp;2000 mg/kg bwt. The nanoparticles suppressed parasites dose-dependently, with MgO-NPs, ZrO\u003csub\u003e2\u003c/sub\u003e-NPs, and MgO/ZrO\u003csub\u003e2\u003c/sub\u003e-NPs achieving 66.79%, 34.72%, and 41.02% suppression, respectively, at 200 mg/kg bwt. In the curative test, MgO-NPs\u0026thinsp;\u0026gt;\u0026thinsp;MgO/ZrO2-NPs\u0026thinsp;\u0026gt;\u0026thinsp;ZrO2-NPs showed significant inhibition (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) compared to controls. These nanomaterials may serve as leads for antimalarial drug development.\u003c/p\u003e","manuscriptTitle":"Green-Synthesis of MgO and ZrO 2 Nanocomposite: Physicochemical Properties and Anti- Plasmodial Activity in a Mouse Model","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-12-19 11:52:12","doi":"10.21203/rs.3.rs-5662380/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"cd7608e2-1564-4f09-8083-4d166b87d5e6","owner":[],"postedDate":"December 19th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-01-13T04:23:14+00:00","versionOfRecord":[],"versionCreatedAt":"2024-12-19 11:52:12","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5662380","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5662380","identity":"rs-5662380","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}