2.1 Reagents and chemicals

CeNPs were synthesized in the Chemistry Laboratory, at the Federal University of Paraíba, Brasil. Cerium (IV) sulfate (p.a., purity ≥ 98%) and sodium hydroxide (Vetec™ reagent grade, purity ≥ 97%) were purchased from Sigma-Aldrich (Merck, Merck KGaA, Darmstadt, Germany). All chemicals used were of analytical grade and used as received without further purification. Dimethylsulfoxide was purchased from Mallinckrodt Chemicals® (Phillipsburg, NJ, USA). Sodium thiopental (Thiopentax®) was purchased from Cristália (Itapira, SP, Brazil), and heparin (Parinex®) from Hipolabor (Sabará, MG, Brazil). Kits for biochemical and hematological analysis were purchased from LABTEST® (Lagoa Santa, MG, Brazil). Sabouraud dextrose broth and agar were purchased from HiMedia, India. LIVE/DEAD BacLight™ Viability Kit was purchased from Invitrogen™ (Molecular Probes Inc., OR, USA). Universal pH-indicator strips were purchased from Merck Quimical, São Paulo, Brazil. All other reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA).

2.2 Synthesis and characterization of cerium oxide nanoparticles

The synthesis of CeNP was performed by the microwave hydrothermal method (Soren et al., 2015), starting from 0.5 mmol of cerium (IV) sulfate in 100 ml of distilled water. Then, 5 mol/L NaOH solution was added drop wise under mild stirring until the pH of solution becomes 14. The reaction system was heated and treated in microwave at 150 °C for 5 min.

The material (after drying) was referred for characterization where its structural properties (Khan et al., 2019) were evaluated by X-ray diffraction (XRD); Stephen Brunauer, Paul Hugh Emmett and Edward Teller (BET) method (Brunauer et al., 1938; density of 7.2 g/ cm³ for the cerium oxide sample), high resolution scanning electron microscopy (SEM), transmission electron microscopy (TEM) and X-ray energy dispersion analysis spectroscopy (EDS). The optical proprieties of CeNP was evaluate using spectra in the UV–Vis in the range of wavelengths between 190 and 900 nm.

The average CeNP crystallite size ( $P_{\mathit{BET}}$ ) was also calculated using the following formula (Eq. (1); Raghupathi et al., 2011).

The average crystallite of the sample too was calculated by using the Debye-Scherrer formula (Eq. (2)):

The zeta potential was measured using a Zetatrac instrument (Microtrac, USA). The CeNPs were resuspended in distilled-deionized water (pH 7.2). The analysis was performed in triplicate. Measurements were essentially as described elsewhere (Pelletier et al., 2010).

2.3 Minimum inhibitory concentration and minimum fungicidal concentration

In order to investigate the minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of CeNPs, the following C. albicans clinical (LM), and American Type Culture Collection (ATCC) strains were used: ATCC 1106, ATCC 76485, ATCC 76645, ATCC 60193, ATCC 26645, LM 62, LM 92, LM 157, LM 38, LM 227 and LM 65 (1–5 × 10⁶ UFC/ mL). They were cultivated on Sabouraud Dextrose Broth (SDB).

CeNP resuspended in DMSO (dimethylsulfoxide) was prepared in concentrations of 150 mg/mL, along with a positive control (Miconazole Nitrate). The concentration of DMSO did not exceed 0.4% in the assays.

MIC of the suspension was assessed by microdilution method. Six rows of wells were prepared with final concentrations from 0.01 to 75 mg/mL for CeNP, and from 5 to 400 µg/mL for Miconazole Nitrate. All three remaining rows of wells were filled with the oxidation–reduction indicator resazurin.

Subsequently, the concentration corresponding to the minimum inhibitory and the two concentrations immediately more concentrated were cultivated on Sabouraud dextrose agar plates. MFC was determined as the lowest concentration showing no visible colony formation after incubation for 24 h at 37 °C.

2.4 Growth kinetic study

Three C. albicans strains (ATCC 1106, ATCC 76485 and LM 38) were selected for viability in experimental fluorescence assays (Jin et al., 2005). In summary, the fluorescence assay distinguishes between live and dead fungi. A standard curve was constructed using different proportions of viable (green fluorescence) and non-viable (red fluorescence) fungi cells. The curve was constructed with concentrations of 0, 20, 50, 80, and 100% live fungi using the LIVE/DEAD BacLight™ Viability Kit. A standardized concentration of 0.5 McFarland scale (10⁶ CFU/mL) from the fungi cells was prepared with saline solution. The growth of the fungal strain was evaluated at 1, 4, 8, and 10 h; and measured for optical density at 600 nm by spectrophotometer (Fluostar Optima - BMG Labtech, Germany). The tests were carried out in triplicate and repeated three times on different days.

2.5 Analysis of the substrate pH

A special care was dedicated to measure the influence of the interaction between nanoparticles and microorganisms studied on the pH of cultivation medium. Then, biotic and abiotic environment was established. Evaluation of change in the pH of the medium on CeNP contact with C. albicans was performed with universal pH 0–14 tape. The ATCC 60193 strain was randomly selected, grown in SDB for 48 h at 37° C and standardized (1–5 × 10⁶ CFU/mL). The CeNP suspension 5 mg/mL received more SDB and fungal inoculum suspension to obtain a final 3.17 mg/mL (MIC selected strain) in 100 uL. Controls were SDB + fungi and SDB + CeNP suspension at 3.17 mg/mL. The test was performed in quadruplicate on three different days.

2.6 DPPH radical scavenging activity

The free radical scavenging capacities of the CeNP were tested by their ability to bleach the stable 1,1-diphenyl-2-picrylhydrazyl hydrate (DPPH) radical. The reaction mixture contained 30 μM DPPH and different concentrations of CeNP (1, 5, 10, 15, 20 µg/mL) in 1 ml methanol. After 90 min of incubation at room temperature, the radical scavenging potential of the antioxidant was measured by % color inhibition the absorbance was taken at 517 nm (Soren et al., 2015). The inhibition rate of the DPPH free radical was calculated as follows (Eq. (3)):

Ascorbic acid was used as standard. The tests were carried out in triplicate and repeated three times on different days.

2.7 Animals, housing, diet, and water

The animals were obtained from the Dr. Thomas George Bioterium (Research Institute in Drugs and Medicines/Federal University of Paraíba, Brazil). In the acute oral toxicity study, 8–12 weeks old Swiss female mice (Mus musculus), in the weight range from 28.0 to 32.0 g on the first day of treatment, were acclimatized for 7 days before initiation of dosing.

Animals were housed in standard polypropylene cages with a controlled 12 h light/dark cycle. Temperature was maintained at 19–25 °C with a relative humidity of 30–55%. Potable water and standardized diet were provided ad libitum.

Experimental protocols and procedures were approved by the local animal ethics committee (protocol number 016/2016) which follows the international principles in ethics for animal experimentation.

2.8 Experimental design-acute toxicity on mice

The acute oral toxicity of CeNP was assessed in accordance with the Organization for Economic Cooperation and Development (OECD) guideline 423 (OECD, 2001). Briefly, CeNP (300 mg/kg) was administered orally in a single dose by gavage to three mice. After 14 days of observation, and accounting of the number of live and dead animals, three animals were dosed sequentially with the same dose. The study continued by testing two more groups of three animals with 2000 mg/kg of CeNP.

CeNP was resuspended in 5% (v/v) Tween-80 in saline. For each assay with a dose of CeNP, a group control (n = 3) administered with vehicle alone (5% (v/v) Tween 80 in saline) was used. After the administration of CeNP, the animals were reviewed cage-side for signs of toxicity, morbidity and mortality at the following time points: 15 min, 30 min, 1 h, 4 h and once a day for 14 days. Histopathological examination was performed for all living mice on day 15. Mice were observed for behavioral changes related to central nervous system (stimulant and depressant activity) and autonomic nervous system, among other abnormal behaviors (Almeida et al., 1999). Food and water consumption were measured daily and body weight of each mouse was taken at the initiation and the end of treatment (day 14).

At the end of the study, blood samples for hematology and biochemistry evaluation were collected from the retroorbital plexus under light anesthesia induced by sodium thiopental intraperitoneal administration (40 mg/kg) and heparin was used as an anticoagulant. Food was withheld for approximately 4 h before blood collection. Hematological examinations were performed using an automatic hematology analyzer, Animal Blood Counter Vet (Horiba ABX Diagnostics) for the following parameters: white blood cell count (WBC), red blood cell count (RBC), hemoglobin concentration (HGB), hematocrit value (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC). Differential leukocytes count was obtained with an optical microscope for the following parameters: lymphocytes, neutrophils, monocytes, eosinophils and basophils. Serum biochemistry was performed using a spectrophotometer Cobas Mira Plus® (Roche Diagnostic System, Livingston, UK) for the following parameters: alanine aminotransferase (ALT), aspartate aminotransferase (AST), urea and creatinine.

All animals were subjected to necropsy, and heart, liver, kidney, spleen and thymus were excised, weighed to determination of indexes of organs [organ weight (mg)/body weight(g)] and then preserved in neutral buffered 10% formalin. Liver and kidney samples were submitted to microscopic examination. Portions of these organs were cut into small pieces (5 µm), followed by staining of the histological sections with hematoxylin-eosin and masson for liver. Histological analysis was performed by light microscopy to determine the presence and extent of liver or kidney lesions attributed to the CeNP. Systems of scores to grade liver and kidneys lesions were employed as previously described (Medeiros et al., 2010; Brunt et al., 1999).

2.9 Experimental design-acute toxicity on Artemia salina

A 25 cm long, 25 cm wide, and 25 cm high, glass aquarium, illuminated with LED lamp was used for hatching the cysts of A. salina (Linnaeus, 1758; Anostraca, Brachiopoda, Crustacea) in artificial seawater (3%, m/v) (Ozkan et al., 2016). The temperature was kept constant at 20 °C, and the pH between 7.0 and 8.0.

The toxicity test was carried out at different concentrations of CeNP 0.001, 0.01, 0.1, 1.0, 5.0, 10.0, 15.0, 20.0 and 25 mg/ mL. After 48 h, the number of dead nauplii were counted as previously described (Ozkan et al., 2016; Gambardella et al., 2014). All of the tests were performed in quadruplicate on different days.

2.10 Statistical analysis

The data collected were submitted to descriptive and inferential statistical analysis using the SPSS (Statistical Package for the Social Sciences) version 20.0 (SPSS Inc., Chicago, IL, USA). In general, data are presented as mean and standard deviation. For mice toxicity data mean ± standard error was used. Student t test was used to compare the means of the inhibition zones, and the ANOVA with Tukey post-test to distinguish between average absorbance differences and toxicity profile. Results were considered significant when p < 0.05.

3.1 Synthesis and characterization of cerium oxide nanoparticles

Through the BET analysis, it was determined that the CeNP sample synthesized by hydrothermal microwave method had a specific surface area of about 295 m²/g.

The SEM (Fig. 1A), reveals various particle sizes, presenting both linear structures, and thin (nm) overlapping layers.

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Fig. 1

Characterization of CeNP. (A) Scanning Electron Microscopy image: the general status of CeNP nanoparticles confirming the presence of nanocrystallites in agglomerates. (B) Transmission electron microscopy (TEM) panoramic image of CeNP with uniform size and rounded shapes of about 4 nm. (C) TEM reveals interplanar spacings in agreement with the CeNP structure; (D) X-ray diffraction pattern of CeNP exhibited six typical peaks corresponding to (1 1 1), (2 0 0), (2 2 0), (3 1 1), (2 2 2), (4 0 0) planes, which are typical of cubic fluorite structure of CeNP; (E) Typical X-ray energy dispersive spectroscopy (EDS) spectra of CeNP taken during TEM. (F) UV − VIS absorption spectra of CeNPs with maximum absorption at 300 nm, indicative of their higher Ce⁴⁺ content.

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The average crystallite size values were also determined by TEM investigation (Additional file 1). The size is determined by counting 100 particles. The mean ± SD size distribution of CeNP was 4.10 ± 0.99 nm diameter. The panoramic TEM image (Fig. 1B) shows nanoparticles.

The average calculated particle size by P_BET was found to be 3–5 nm. The average crystallite size calculated by the Debye-Scherrer formula was about 3.17 nm. These average particle sizes calculated reported are consistent with the TEM study. The TEM picture shown in Fig. 1B the strong agglomeration of small particles with a diameter on the order of about 4 nm, and interplanar distances of about 0.31 nm (Fig. 1C).

The cubic fluorite structure of cerium oxide and crystallite size was confirmed from XRD data and Fig. 1D shows the X-Ray Diffraction pattern. XRD pattern shows three main reflections (1 1 1), (2 0 0), (2 2 0), (3 1 1), (2 2 2), and (4 4 0) as characteristic of cerium oxide cubic phase with fluorite structure. The calculated lattice parameters was 5.404 Å.

Broad peaks of reflection were clearly visible, indicating small crystal size, and nanoscale dimension, thus proving the success of the proposed method. The phases found were indexed, after analysis and correlation, using the JCPDS 34-0394 datasheet (Joint Committee on Powder Diffraction Standards) for the structure, fluorite, CeO₂.

Fig. 1E was obtained by means of energy dispersive spectroscopy, and it confirm the presence of cerium in the CeNP.

The wavelength at which the maximum absorbance occurs around 300 nm (Fig. 1F).

The average zeta potential for CeNP was from − 12.6 ± 0.3 mV.

3.2 Minimum inhibitory concentration and minimum fungicidal concentration

CeNP was able to inhibit the growth of all Candida strains tested. C. albicans LM 65 and ATCC 76485 strains showed the lowest concentration of MIC (1.50 mg/mL). Miconazole Nitrate (concentration ≤ 62 µg/mL) was able to inhibit all strains (Table 1).

3.3 Growth kinetic study

The growth kinetics study was conducted on three strains of C. albicans (ATCC 1106, ATCC 76 485, and LM 38) randomly selected in the presence of CeNP with three concentrations (MIC ÷ 2, MIC, and MICx2) at 1, 4, 8, and 10 h. In the figure, it is clear that MICx2 had significant effect on strains growth (Fig. 2). At 10 h, CeNP reduced growth of the three strains. For ATCC 1106 (MIC), the survival yeast was 46.35% (Additional file – Fig. S2A). For ATCC 76,485 (MIC), the survival yeast was 49.8% (Additional file – Fig. S2B). LM 38 exposed by CeNP MIC has 59.33% of survival cells (Additional file – Fig. S2C). At 10 h, the highest inhibition of yeast growth was observed at a concentration of 15.0 mg/mL (MICx2) in the ATCC 1106 strain.

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Fig. 2

Growth kinetics of the strain Candida albicans in the presence of MIC ÷ 2, MIC, and MICx2 of CeNP. Ten hour growth curve showing the sensitivity of (A) ATCC 1106 to CeNP at 3.75 mg/mL (MIC ÷ 2), 7.5 mg/mL (MIC), and at 15.0 mg/mL (MICx2); (B) ATCC 76485 to CeNP 0.75 mg/mL (MIC ÷ 2), 1.50 mg/mL (MIC) and 3.0 mg/ml (MICx2); and (C) LM 38 to CeNP at 1.25 (MIC ÷ 2), 2.50 mg/mL (MIC), and 5.0 mg/ml (MICx2). In each case, the growth of the strain is control. Note different scales. (D) DPPH radical scavenging potential of CeNP remained low and constant regardless of the concentration of nanoparticles in the acellular system for 90 min of contact between CeNP e DPPH.

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3.4 Analysis of the substrate pH

The results showed higher pH with increasing CeNP in the suspension containing the fungus and the culture medium. The pH of the SDB + fungi + CeNP suspension was 10.0, higher than the pH of the SDB + fungi solution (pH 5.0). The SDB + CeNP suspension had a pH of 7.0.

3.5 DPPH radical scavenging activity

The results showed that CeNP showed an average antioxidant activity of 15.06 ± 0.78% (Fig. 2D). Ascorbic acid at a concentration of 9.23 ± 0.29 μg/mL exhibited a percentage inhibition of 50% and for 18 μg/mL 100%.

3.6 Acute oral toxicity study on mice

No behavioral changes were observed during the study. One death was recorded in the first group treated with 2000 mg/kg of CeNP.

3.6.4

3.6.4 Histopathology findings

Histopathological analyses of liver of control and treated groups showed normal histological architecture without periportal and periseptal activity, and fibrosis (Fig. 3). At 300 mg/kg of CeNP, it was observed discreet portoparenchymatous inflammatory processes, and rare foci of hepatocellular necrosis. At 2000 mg/kg, the livers of the animals treated with CeNP also presented portoparenchymatous inflammatory processes with multifocal lobular necrosis, probably of toxic nature, and multifocal sub-capsular granulomatous inflammatory processes with the absence of cellular atypia (Fig. 3). The changes observed are described in Table 6.

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Fig. 3

Histopathology analysis - the liver was examined: Control - Normal hepatocytes, isomorphic; CeNP (300 mg/kg) group - presence of inflammatory infiltrate around the lobular vein and rare foci of hepatocellular necrosis (arrows point); CeNP (2000 mg/kg) group - multifocal sub-capsular granulomatous inflammatory processes. Histopathology analysis - the kidneys were examined: Control - Glomerulus preserved with thin Bowman's capsule, capillary tuft supported by delicate mesangium; CeNP (300 mg/kg) group - Distal convoluted tubule normal, and discreet increase in the number of portal lymphocytes, along with some polymorphonuclear neutrophils in rare portal spaces (arrows point); CeNP (2000 mg/kg) group - focal interstitial nephritis (arrows point). Hematoxylin-Eosin. All the images were 400× the original magnification.

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Table 6 Effect of CeNP (300 or 2000 mg/kg) on histopathology changes in liver tissue of mice after a single dose of oral treatment.

Parameters	Groups	Histophatology changes	Score
Portal inflammatory infiltrate	Control	Normal histophatology	0
	300 mg/kg CeNP*	Discreet increase in the number of portal lymphocytes, along with some polymorphonuclear neutrophils in rare portal spaces	1
	2000 mg/kg CeNP	Moderate increase in the number of portal lymphocytes, along with polymorphonuclear neutrophils in some portal spaces	2
Parenchymal activity	Control	Normal histophatology	0
	300 mg/kg CeNP	Discreet hepatocytic alterations with lymphohistiocytic infiltrate and rare necrotic areas. The parenchymal necrosis foci are seen randomly in zones I, II and III	1
	2000 mg/kg CeNP	Focal hepatocyte necrosis surrounded by lymphohistiocytic aggregate. The parenchymal necrosis foci are seen randomly in zones I, II and III	2

* Cerium oxide nanoparticle.

Histopathological analyses of kidneys of control and 300 mg/kg CeNP groups showed normal histology. At 2000 mg/kg of CeNP, it was observed focal interstitial nephritis and necrosis of proximal tubular epithelium (Fig. 3).

3.7 Acute toxicity against Artemia salina

The cytotoxicity assay in the Artemia salina animal model did not reveal a LD50 or LC50 (lethal dose or minimum lethal concentration of 50%). Exposure to CeNP did not cause death to the naupli in concentrations of 0.001 up to 15.0 mg/mL. The mortality in the group exposed to concentrations of 20.0 and 25.0 mg/mL of CeNP was 4 and 10%, respectively, demonstrating the non-toxicity of CeNP against A. salina.