Development of a new generation of miniemulsion based on cottonseed oil with α-tocopherol and ZnO and evaluation of its adjuvant activity

Minioemulsion development and characterization

Zinc oxide nanoparticles synthesis

The zinc oxide (ZnO) nanoparticles were obtained by the coprecipitation method. Zinc sulfate monohydrate (ZnSO₄ • H₂O) was dissolved in deionized water at 0.5 M that remained under stirring at 300 rpm while sodium hydroxide solution (2 M NaOH) was added in drops. After, the supernatant was discarded, and the precipitate formed was washed with deionized water and centrifuged at 8,000 rpm for 10 min. Precipitate was dried in a hot air oven at 60 °C for 24 h. Then, the obtained powder was calcinated at 300 °C for 2 h, in a muffle.

Miniemulsion formulation

Two miniemulsions were produced as follow: (1) cottonseed oil miniemulsion (CN) (to form the oil phase of 275 mg), in which α-tocopherol (25 mg), Tween 20 (35 mg) and DMSO (50 ml) were dissolved, and (2) cottonseed oil miniemulsion + ZnO nanoparticles (CNZ) was formulated in the same way as described above plus the addition of ZnO nanoparticles (2.5 µg) in the oil phase. The oil phase was calibrated with saline solution (1 mL) and mixed at 12,000 rpm in homogenizer for 10 min (ULTRA-TURRAX^®; IKA Works Inc., Wilmington, NC, USA).

Miniemulsion characterization

Droplet size and polydispersity index (PDI) were determined by dynamic light scattering (DLS) and zeta potential (PZ) was determined by electrophoretic mobility measurements in a ZS90-Nano Zetasizer (Malvern Instruments, Malvern, UK). All results were measured in triplicate at room temperature (25 °C). The ZnO nanoparticles were characterized by X-ray diffraction (XRD) and the Fourier Transform Infrared Spectroscopy (FT-IR), ZnO nanoparticles morphology and size were confirmed by scanning electron microscopy (SEM), and the droplet size was confirmed by atomic force microscopy (AFM), images were analyzed with Gwyddion software. The characterization of miniemulsions functional groups and their components (cottonseed oil, α-tocopherol, and ZnO nanoparticles) were determinate by attenuated total reflection (ATR) in conjunction with FT-IR technique. The samples were placed onto the diamond ATR crystal, and spectra was recorded by a FT-IR (Perkin Elmer, Waltam, MA, USA), with an accumulation of 16 scans at a resolution of 4 cm⁻¹ between 4,500–500 cm⁻¹. Data were analyzed with the software Perkin Elmer Spectrum 10.

Animals

Male BALB/c mice (6–8 weeks old, weighing 23 ± 2 g) were used for all in vivo and ex vivo experiments. The animals were obtained from the Bioterium of Facultad de Ciencias Biológicas of the Universidad Autónoma de Nuevo León, (UANL, FCB) under the approval of Bioethics committee protocol No. CEIBA-2019-015. Animals were housed eight per cage (polypropylene) under constant specific conditions with temperature at 24 °C, 50% relative humidity and a controlled light and dark cycle (12 h:12 h) with ventilated caging systems, wood sawdust bedding and standardized environmental enrichment. Feed (pellet total mix ration) and clean water were supplied ad libitum. For sample (serum, macrophage, thymocyte and splenocyte) collection, animals were euthanized by overdoses of anesthesia (ketamine-xylazine protocol).

Cell obtention and culture conditions

Thymus, spleen, and peritoneal macrophages primary cultures were obtained from healthy mice. Cell cultures were maintained in a medium containing 5% fetal bovine serum (FBS), antibiotic and antimycotic 1%, under an atmosphere of 5% CO₂ and 95% air at a constant temperature of 37 °C.

Ethical statement

All the experiments pertaining to animal use and their care strictly followed the guidelines of the Official Mexican Standard (NOM-062-ZOO-1999) on technical specifications for production, care and use of laboratory animals. All the protocols were approved by UANL FCB Animals Ethics Committee (protocol CEIBA-2019-015).

Cell toxicity

The miniemulsions toxicity was evaluated in vitro on thymus, spleen, and peritoneal macrophages primary cultures. Cells were seeded in 96-well plates at a density of 2 × 10⁵ cells/well with 100 µL of DMEM culture medium. After 24 h of incubation culture medium was replaced by new one containing CN or CNZ (0, 1.25, 2.5, 5 and 10 µL/ml). Cell viability was evaluated 24 h after treatment using the technique of MTT.

The spectrophotometry measurement was performed in triplicate at 570 nm in a plate reader (Synergy HT; Biotec, Emmerich, Germany), the percentage of viability was determined by the following equation:

$\mathrm{V}\mathrm{i}\mathrm{a}\mathrm{b}\mathrm{i}\mathrm{l}\mathrm{i}\mathrm{t}\mathrm{y}(\mathrm{\%})=(\mathrm{A}\mathrm{b}\mathrm{s}\mathrm{o}\mathrm{r}\mathrm{b}\mathrm{a}\mathrm{n}\mathrm{c}\mathrm{e}\mathrm{o}\mathrm{f}\mathrm{t}\mathrm{h}\mathrm{e}\mathrm{s}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e}/\mathrm{A}\mathrm{b}\mathrm{s}\mathrm{o}\mathrm{r}\mathrm{b}\mathrm{a}\mathrm{n}\mathrm{c}\mathrm{e}\mathrm{o}\mathrm{f}\mathrm{t}\mathrm{h}\mathrm{e}\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l})\ast 100$

Macrophage NO (nitric oxide) and antioxidants production

For the miniemulsions effect over nitric oxide production (as nitrite) and total antioxidant capacity (TAC), murine peritoneal macrophages (MPM) were used. As positive control lipopolysaccharide (LPS) was used in a concertation of 5 µL/mL (Sigma-Aldrich Inc., St Louis, MO, USA).

Nitrite concentration in treated cells supernatant was determined by Griess reaction (Griess Reagent, Catalog No. ab234044; Abcam, Cambridge, UK). MPM were cultured in the same way as mentioned in the cell toxicity assays section described above. Supernatants (50 μL) were collected, centrifuged at 600× g for 5 min, mixed with 100 μL of Griess reagent (0.1% N-(1-naphthyl)-ethylene diamine, 1% sulfanilamide in 5% phosphoric acid) and incubated at room temperature for 10 min. Absorbance was measured at 540 nm (Bie et al., 2019).

TAC was determinate in MPM culture supernatants. MPM were cultured and supernatants were collected under conditions described above. The Antioxidant assay kit (Catalog No. KA1622; Abnova, Taipei, Taiwan) was used according to manufacturer’s instructions. Assay measures TAC by the reduction of Cu⁺² to Cu⁺ observed by a colored complex formation. Trolox (6-hydroxyl-2,5,7,8-tetra methyl chroman-2-carboxylic) standard curve was measured at 540 nm.

Macrophage uptake and phagocytosis

Two assays were designed to determine the miniemulsions uptake and phagocytic activity effect in MPM. CN and CNZ (10 µL/mL) were used as cell stimulation treatment, for phagocytosis inhibition control disodium clodronate (0.6 mg/ mL) was used and DMEM medium as negative control. MPM quantitative uptake capacity was determinate by neutral red assay (Bie et al., 2019) using the Neutral Red Assay Kit-Cell Viability/Cytotoxicity (Catalog No. ab234039; Abcam, Cambridge, UK) according to manufacturer’s instructions. For the assay, 2 × 10⁵ murine peritoneal macrophages were seeded on 96-well plates, washed three times with PBS, and then incubated with 10% neutral red for 2 h. After three washes, neutral red incorporated into lysosomes was dissolved by the lysis solution provided by the kit, and the absorbance was read at 540 nm. Qualitative phagocytic activity was determined using Phagocytosis assay kit (IgG FITC) (Catalog No. 500290; Cayman chemical, Ann Arbor, MI, USA). MPM were seeded on coverslips in six-well plates and incubated for 24 h in DMEM medium. After 1 h of treatments cells were incubated with latex beads-rabbit IgG-FITC complex for 2 h. Photographies were observed in fluorescence microscope (Axioscope 5 Pol; Zeiss, Oberkochen, Germany).

Candida Albicans macrophages phagocytosis

For the evaluation of the MPM phagocytic capacity, C. albicans (ATCC 10261) was used. MPM, obtained from three healthy mice were seeded under the previously described conditions in the cell toxicity assay. Phagocytosis assay was performed based in the method proposed by Alieke et al. (2002). Co-culture supernatant (macrophages (1 × 10⁶ cells)/C. albicans (1 × 10⁶–1 × 10⁸ cells) proportion) was collected to assess the increase or decrease of colony forming units (CFU) by the Mc-Farland method (Vasconcelos et al., 2014). The percentage of phagocytized microorganisms was defined as following equation:

$$(1-(\mathrm{N}\mathrm{u}\mathrm{m}\mathrm{b}\mathrm{e}\mathrm{r}\mathrm{o}\mathrm{f}\mathrm{u}\mathrm{n}-\mathrm{p}\mathrm{h}\mathrm{a}\mathrm{g}\mathrm{o}\mathrm{c}\mathrm{y}\mathrm{t}\mathrm{e}\mathrm{d}\mathrm{C}\mathrm{F}\mathrm{U}/\mathrm{C}\mathrm{F}\mathrm{U}\mathrm{a}\mathrm{t}\mathrm{t}\mathrm{h}\mathrm{e}\mathrm{s}\mathrm{t}\mathrm{a}\mathrm{r}\mathrm{t}\mathrm{o}\mathrm{f}\mathrm{i}\mathrm{n}\mathrm{c}\mathrm{u}\mathrm{b}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}))\ast 100.$$

Death of C. albicans by phagocytes was assessed in the same monolayers. The nitic oxide (as nitrite) production was also determined as mentioned previously.

Immunization and sample collection

Three independent experiments were designed. Ovalbumin (OVA), (Sigma-Aldrich Inc., Saint Louis, MO, USA) was used as antigen (10 mg per animal in 50 µL of saline solution). All immunizations were subcutaneously applied (s.c.) in a total volume of 100 µL per animal (50 µL of miniemulsion and 50 µL of antigen). Freund’s Adjuvant Complete (FCA; Sigma-Aldrich Inc., Saint Louis, MO, USA) was used as positive control; a group of only antigens was used as control (ovalbumin 10 mg per dose in 100 µL of saline solution).

Experiment A. Mice inoculated with CN, CNZ, FCA and control were sacrificed by cervical dislocation at 24 h post-treatment. The administration site (subcutaneous tissue over the femoral area) was fixed in 10% formaldehyde solution and after that, covered with hematoxylin and eosin (H & E) to determine leukocyte infiltration. For the quantification and cell typing, mice were treated intraperitoneally with miniemulsions without antigen. The cells (cells/µL) were located by size and granularity to stablish the gate, and the CD14⁺ expression was determined by flow cytometry. These assays were carried out by flow cytometry (Accuri C6; DB Biosciences, San Jose, CA, USA).

Experiment B. Mice were immunized with OVA, and its combination with CN, CNZ, and FCA or control (saline solution) on days 0 and 14. Blood samples were taken on days 7, 14, 28, and 42. The blood samples were obtained from cardiac punction in anesthetized mice. Blood serum was separated and frozen at −80 °C until the time of isotype antibody determination (IgG1, IgG2a, IgG2b, IgG3, IgA and IgM) and determination of cytokine profile TH1 (IFN-γ, IL-2 and TNF-α)/TH2 (IL-4, IL-6 and IL-10)/TH17 (IL 71a). Cytokines were measured by flow cytometry (Accuri C6; DB Biosciences, San Jose, CA, USA) using a commercial Cytometric Bead Array (CBA) Mouse Th1/Th2/Th17 Cytokine kit (Catalog No. 560485; DB Biosciences, San Jose, CA, USA).

Experiment C. Mice were immunized at days 0 and 14 with the different miniemulsions (CN or CNZ) previously mentioned and challenged with OVA at day 42 and, blood samples were taken at 72 h after. The blood serum was separated to evaluate specific antibodies and frozen at −80 °C until analysis.

ELISA assay

Anti-ovalbumin antibodies were evaluated by ELISA, as previously described (Thommen Maciel Sartor, Moleta Colodel & Albuquerque, 2011). Briefly, polystyrene 96 well-plates (Falcon) were coated overnight at 4 °C with 0.5 mg of OVA per well in carbonate-bicarbonate buffer pH 9.6, after that washed with PBS 0.05%-Tween-20, and then blocked with 1%-BSA/PBS for 1 h at room temperature. Mouse antiserum from day 45 was added by triplicate (serial dilutions from 1:100) and incubated overnight at 4 °C. Plates were washed and incubated for 1 h at 37 °C with goat anti-mouse IgG-HRP (Catalog No. sc-2005; Santa Cruz Biotechnology Inc., Dallas, TX, USA). After that wells were washed, and tetramethylbenzidine (TMB) was added. The reaction was stopped with H₂SO₄. Plate was read at 450 nm in a plate reader (Synergy HT; Biotec, Emmerich, Germany), values indicating optical density (DO). The antibodies isotypes (IgG1, IgG2a, IgG2b, IgG3, IgA and IgM) were determinate by commercial kit Ig Isotyping Mouse Uncoated ELISA (Catalog No. 88-50630; Thermo Fisher Scientific Inc., Vienna, Austria).

Statistical analysis

All data analysis was performed with Minitab^® (2014) software. Data was expressed as the mean ± standard error (SE). For the variables of nitric oxide production and antioxidant capacity, a Pearson correlation test was performed. Statistical significance (α = 0.05) was determined by ANOVA. The comparison of means was performed by Tukey test.

Miniemulsion development and characterization

The miniemulsion CN and CNZ presented stability for 90 days, and this was confirmed by DLS analysis (Table 1 and Figs. 1A and 1B). Immediately after preparation, the CN miniemulsion presented a higher particle size (543.1 nm) with a zeta potential = −26.6 mV and PDI = 0.074, in contrast to CNZ that presented a size of 320 nm, a zeta potential = −28.1 mV and PDI = 0.449. However, after 90 days slightly differences were observed, the CN emulsion presented a decrease in zeta potential (−13 mV) and increase of PDI (0.225), and CNZ presented a decrease in size (259.4 nm) and PDI (0.312) but increase of zeta potential (−29.5 mV). The particle size difference of CN and CNZ was confirmed by atomic force microscopy (AFM) (Fig. 1C), where CN showed big semispherical structures and CNZ small spherical structures.

The FT-IR spectra of ZnSO₄ • H₂O and ZnO nanoparticles is shown in Figs. 2A and 2B, respectively. In Fig. 2A, can be observed the characteristic peaks of ZnSO₄ • H₂O, a hydroxyl group (OH) at 3,135 cm⁻¹, a carbonyl group (C=O) at 1,506 cm⁻¹, and sulfate groups (SO⁻²₄) at 1,090 y 855 cm⁻¹. The spectra of ZnO nanoparticles (Fig. 2B) showed a peak at 576 cm⁻¹, characteristic of the ZnO stretching mode. Figure 2C indicates the principal groups of ZnSO₄ • H₂O and ZnO nanoparticles observed in FT-IR spectra. The photography by scanning electron microscopy (SEM) (Fig. 2C) showed agglomerates of ZnO nanoparticles with spherical morphology.

There is a qualitative difference between two samples in the size of the emulsions, considering that in Fig. 1 the emulsions are larger compared to Fig. 2, in addition a homogeneous size is observed in the case of the emulsions with CNZ, evidence of a large amount of residues in the first sample probably due to the inefficiency of the synthesis system, which indicates that in CNZ there is an improvement in the synthesis by adding zinc nanoparticles, probably due to its hydrophilic characteristics.

The Fig. 3 showed the FT-IR spectra of miniemulsions and their precursors. In cottonseed oil (Fig. 3A) was observed the C-H group strongly stretched at 2,922 and 1,465 cm⁻¹; stretching vibrations of carboxyl group (COOH) at 2,856 cm⁻¹, ketone group (C=O) at 1,744 cm⁻¹, and C-O group at 1,156 cm⁻¹. The spectra of α-tocopherol (Fig. 3B) showed peaks at 2,927 and 2,867 cm⁻¹ that correspond to the asymmetric and symmetric stretching vibrations of CH₂ and CH₃, respectively. The peak was observed at 1,758 cm⁻¹ for an alkene group (C=C), at 1,461 cm⁻¹ for an asymmetric bending of a phenyl group (C₆H₅), and methyl and methylene symmetric bending peaks at 1,365 and 1,203 cm⁻¹, respectively.

The miniemulsion CN FT-IR spectra showed interaction of functional groups COOH, C=O and C-O of cottonseed oil with the C=C and C₆H₅ functional groups of α-tocopherol (Fig. 3C) in addition to the interaction of ZnO nanoparticles in CNZ miniemulsion (Fig. 3D).

Figure 3E represent the FT-IR spectra comparative of miniemulsions CN, CNZ, and cottonseed oil, and Table 2 showed the principal functional groups of precursors.

Effect of miniemulsions on immune cell function

CN and CNZ miniemulsions at concentrations of 1.25 to 10 µL/mL had no effect on the viability of macrophages (p = 0.108), splenocytes (p = 0.413), and thymocytes (p = 0.923) in vitro (Fig. 4). All doses tested of CN and CNZ induced nitric oxide production in dose dependent manner when compared with the control (0 µL). The CN induced a similar production of nitric oxide at doses of 1.25 (p = 0.998) and 2.5 (p = 0.112) µL/mL and highest at doses of 5 and 10 µL/mL (p = 0.0001) when compared with the positive control (LPS). No difference (p = 0.935) in nitric oxide production was found at all doses tested of CNZ when compared with LPS (Fig. 5).

The antioxidant capacity

All CN and CNZ doses tested had the capacity to induce the production of antioxidants by dose dependent manner when compared with the control (p = 0.0001). The CN 5–10 µL/mL doses induced a major antioxidant production (p = 0.0001) than LPS and 2.5 µL/mL (p = 0.979)/1.25 µL/mL (p = 1.0) doses induced similar productions levels. The CNZ treatment at 10 µL/mL (p = 0.002) dose induced a higher antioxidant total production than LPS, but at minor doses 5 µL/mL (p = 0.115), 2.5 µL/mL (p = 0.925) and 1.25 µL/mL (p = 1.0) no difference was found (Fig. 6). There is a strong correlation between the production of nitric oxide and antioxidants in macrophages treated with CN (r = 0.905, p = 0.0001) as in those treated with CNZ (r = 0.804, p = 0.002).

Histological evaluation

The histological evaluation of the tissue at the inoculation site (subcutaneous tissue on femoral area) with CN or CNZ with ovalbumin showed inflammation with higher leukocyte infiltration compared with CFA (Fig. 7). The intraperitoneal inoculation with CN, CNZ miniemulsion and CFA showed a high recruitment of total intraperitoneal cells with the CD14⁺ phenotype, after 24 h of inoculation compared with control (p = 0.0001), however no difference was found between CN, CNZ and FCA (Fig. 8).

Macrophage uptake and phagocytosis

The CN (p = 0.158) and CNZ (p = 0.495) miniemulsions evaluated by neutral red assay did not affect the macrophages uptake activity when compared with untreated macrophages (Fig. 9). However, in the macrophages Candida albicans-phagocytosis assay the CN and CNZ miniemulsions increased the phagocyte capacity with respect to the untreated macrophages (Fig. 10). Next, the evaluation of residual colony forming units indicated that only CN significantly decreased (p = 0.004) this value respect CNZ, but at 4 h no difference was found with respect to the untreated macrophages (Fig. 11A). By other side, only CN miniemulsion increased (p = 0.002) the nitric oxide production in all times evaluated (Fig. 11B).

Cytokine production

The CNZ miniemulsion stimulated a major IFNγ secretion compared with FCA at day 7 (p = 0.0001), this difference was not observed at day 14 (p = 1.0) (Fig. 12). A major secretion of IL-2 was observed at days 7 (p = 0.027) and 14 (p = 0.007), stimulated with the CN and CNZ miniemulsions (Fig. 12). However, the IL-10 was not affected (Fig. 12).

Switching isotype evaluation

The CN and CNZ miniemulsions did not affect the production of IgG1. However, the FCA stimulated a major production of IgG1 at day 42 compared with the untreated mice (p = 0.044) (Fig. 13). The CN and CNZ miniemulsions did not affect the production of IgG2b at all times evaluated, only significant difference (p = 0.044) was found with FCA at day 28 (Fig. 13). At the last, the miniemulsions did not affect the production of antibody isotypes (IgG1, IgG2a, IgG3, IgA and IgM) at days 7, 14, 28 and 42.

IgG specific OVA

The CN miniemulsion induced a significant production of IgG against OVA, but lesser than FCA. CNZ miniemulsion did not affect this, when compared with the OVA treatment (p = 0.0001) (Fig. 14).