Preparation, characterization, and evaluation of azoxystrobin nanosuspension produced by wet media milling
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To improve the bioavailability of the poorly water-soluble fungicide, an azoxystrobin nanosuspension was prepared by the wet media milling method. Due to their reduced mean particle size and polydispersity index, 1-Dodecanesulfonic acid sodium salt and polyvinylpyrrolidone K30 were selected from six conventional surfactants, the content only accounting for 15% of the active compound. The mean particle size, polydispersity index, and \(\zeta\) potential of the nanosuspension were determined to be 238.1 ± 1.5 nm, 0.17 ± 0.02 and − 31.8 ± 0.3 mV, respectively. The lyophilized nanosuspension mainly retained crystalline state, with only a little amorphous content as determined by powder X-ray diffraction. Compared to conventional fungicide formulations, the nanosuspension presented an increased retention volume and a reduced contact angle, indicating enhanced wettability and adhesion. In addition, the azoxystrobin nanosuspension showed the highest antifungal activity, with a medial lethal concentration of 1.4243 μg/mL against Fusarium oxysporum. In optical micrographs, hyphal deformations of thinner and intertwined hyphae were detected in the exposed group. Compared to the control group, the total soluble protein content, superoxide dismutase, and catalase activities were initially increased and then decreased with prolonged exposure time. The azoxystrobin nanosuspension reduced the defensive antioxidant capability of Fusarium oxysporum and resulted in the generation of excessive reactive oxygen species. This study provides a novel method for preparing nanosuspension formulation of poorly soluble antifungal agents to enhance the biological activity and decrease the negative environmental impact.
KeywordsAzoxystrobin Nanosuspension Antifungal activity Fusarium oxysporum Wet media milling
Basically, two approaches can be used to increase the solubility of a chemical component: physical techniques and chemical modifications (Mirza 2017). Physical techniques primarily include high-pressure homogenization, wet media milling, and carrier co-precipitation. In chemical modification, insoluble components are grafted with hydrophilic groups or transformed into salt forms. According to the Nernst–Brunner equation, a substance’s solubility in water is negatively correlated with its particle size (Brough and Williams 2013). Hence, reducing the particle size is an effective approach to improve the solubility of hydrophobic compounds. With the development of nanotechnology, the application of nanomaterials has attracted wide attention in the field of agriculture, such as pesticide and fertilizer (Kah and Hofmann 2014). Recently, nanosuspensions with increased particle surface area have become one of the most promising formulations to enhance solubility (Yadollahi et al. 2015; Kumar Singh et al. 2016). Wet media milling has been regarded as a top-down approach for the industrial production of nanosuspensions and benefits from high efficiency, low cost, and free of organic residue (Ghosh et al. 2012; Li et al. 2016). Based on these superiorities, wet media milling provides a novel and easy method to produce poorly soluble fungicides.
Azoxystrobin inhibits mitochondrial electron transport in the respiratory chain as most strobilurin fungicides. The inhibitors accelerate electrons escaping from mitochondria, which is hastened by the generation of reactive oxygen species (ROS) (Turrens and Boveris 1980; Olsvik et al. 2010). The fungi form a set of antioxidant defense system inclusive of superoxide dismutase (SOD) and catalase (CAT) (Azevedo et al. 2007). However, excess ROS at the early stages of mitochondrial disruption can lead to fungi death (Inoue et al. 2011).
In this study, an azoxystrobin nanosuspension was prepared by wet media milling. The particle size and \(\zeta\) potential of the nanosuspension were measured by dynamic light scattering (DLS). The morphology and structure of the nanoparticles were characterized by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The contact angle and retention volume on the leaves of cucumbers and cabbages were measured. Compared with commercially available formulations, antifungal activity of the nanosuspension was tested by potato dextrose agar (PDA) assay. The morphologies of Fusarium oxysporum were observed by optic microscopy and SEM. This study also evaluated the effects of the azoxystrobin nanosuspension on fungal protein content, SOD, and CAT.
Azoxystrobin (97%) was purchased from Hubei Sheng Tianheng record Biological Technology Co., Ltd. (Hubei, China). 1-Dodecanesulfonic acid sodium salt (SDS), polyvinylpyrrolidone K30 (PVP K30), hexadecyl trimethyl ammonium chloride (CTAC), and polyoxyethylene sorbitan monooleate (Tween 80) were provided by J&K Scientific Ltd. (Beijing, China). Poloxamer 188 (F68) was obtained from Sigma-Aldrich (Shanghai, China). Polycarboxylate was provided as a gift by Sinvochem S&D Co., Ltd. (Jiangsu, China). Commercially available water dispersible granules (WDG) were purchased from Jiangsu KWIN Group (WDG-A) and Jiangsu Huifeng Agrochemical Co., Ltd. (WDG-B). HPLC-grade methanol was obtained from Thermal Fisher Scientific (Tustin, CA, USA). Deionized Milli-Q water was used in all experiments (18.2 MΩ·cm, TOC ≤ 4 ppb).
Preparation of azoxystrobin nanosuspension
Azoxystrobin nanosuspension was manufactured by wet media milling. In brief, azoxystrobin powder was dispersed in an aqueous solution containing one of the six surfactants (SDS, PVP K30, F68, Tween 80, CTAC, and polycarboxylate), and the solution was subjected to mechanical stirring (Ika, mod. RW 20 digital, Germany) at 1000 rpm for 30 min. The suspension (6% azoxystrobin) was then milled with the milling media apparatus (WG-0.3 L, Vgreen nano-tech, China). The grinding chamber (0.3 L) was made of silicon carbide, and 80% of the chamber was filled with 0.3-mm zirconium oxide beads as the milling medium. The azoxystrobin crystals were fragmented into nanoparticles by the physical impact of the zirconium oxide beads at a speed of 2200 rpm.
Particle size and \(\zeta\) potential determination of the nanosuspension
The mean particle size, polydispersity index (PDI), and \(\zeta\) potential of the nanosuspension were analyzed by DLS with a Zetasizer Nano ZS90 (Malvern Instruments, UK) at room temperature. Each sample was measured three times for reliability.
Morphological characterization of the nanoparticles by SEM and TEM
The morphology of the azoxystrobin nanosuspension was evaluated using SEM (SU8010, Hitachi, Tokyo, Japan) at an acceleration voltage of 5 kV. The sample was placed on a clean silicon slice, dried at room temperature, and then sputtered with platinum under vacuum (EM ACE600, Leica, Germany). The size and morphology of the nanosuspension were characterized by TEM (HT7700, Hitachi, Tokyo, Japan) at an operational voltage of 80 kV. The sample was dropped on carbon-coated 300-mesh copper grids.
Powder X-ray diffraction analysis of the nanoparticles
For X-ray diffraction analysis, the water of azoxystrobin nanosuspension was removed by lyophilization (FD-81, EYELA, Tokyo, Japan). The patterns of samples were analyzed by a diffractometer (D8 ADVANCE, Bruker AXS Inc., Karlsruhe, Germany) with a Cu Kα radiation source, operated at 40-kV voltage and 40-mA current. Scans were recorded with a detector rate of 0.2°/min and 2θ range from 5 to 50°.
Wetting and spreading characteristics
Hydrophobic leaf of cabbage and hydrophilic leaf of cucumber were cultivated by light growth incubator. The contact angles of the droplets were measured with a contact angle apparatus (JC2000D2 M, Zhongchen Digital Technology Apparatus, Shanghai, China). The 7-μL diluted suspension was dropped onto the leaf using a 50-μL syringe.
The retention of the sample on the leaf was determined by the dipping method. A 15-mm-diameter leaf was perforated by a hole punch and weighed as M1. The leaf was immersed in the diluted suspension for 10 s. Until no more droplets falling, the leaf was removed and weighed as M2. The measurements were conducted at room temperature. Five tests were performed for each sample.
Azoxystrobin concentration analysis
The azoxystrobin concentration was estimated by high-performance liquid chromatography (HPLC) (Agilent 1260 series HPLC, Agilent Technologies) using a Zorbax Carbohydrate Analysis column (150 mm × 4.6 mm × 5 μm) and a 254-nm UV detector. The mobile phase consisted of methanol and water (75:25, v/v) at a flow rate of 0.8 mL/min.
In vitro dissolution
The suspensions commensurate with effective azoxystrobin were put into dialysis bags (2000 MWCO). The bags were then suspended in 100 mL of an ethanol/deionized water solution (50:50, v/v) as the release medium. The solution was shaken at 100 rpm at 28 °C using a constant temperature table (THZ-98C, Shanghai, China). A 2 mL of the outside release medium was removed at different timed intervals. Meanwhile, 2 mL of fresh mix solution was added back into the sustained release system. For quantitative analysis, the azoxystrobin concentration in the 2 mL of release medium was determined by HPLC.
Fungal antagonism assays
To examine fungal antagonism, Fusarium oxysporum was used for the antifungal activity test with PDA assay. Various azoxystrobin formulations containing the nanosuspension, WDG-A or WDG-B were precisely prepared with the following concentrations: 0.1, 0.25, 0.5, 1.0 and 5.0 μg/mL. A 5-mm-diameter mycelial disc was incubated on the test medium at 28 ± 1 °C for 2 days. The diameter of mycelium growth was determined by the criss-cross method. The toxicity regression equations and the medial lethal concentration (LC50) were calculated by probit analysis using SPSS 20 statistical software (IBM Corp., Armonk, NY, USA). Each experiment was implemented in triplicate.
Fungal hyphae microscopy analysis
Fusarium oxysporum was cultured with PDA, and then a 10-mm-diameter mycelial disc was incubated in the potato dextrose broth (PDB) medium at 28 ± 1 °C for 2 days. To evaluate the effect of the azoxystrobin nanosuspension on Fusarium oxysporum, the azoxystrobin nanosuspension with a final concentration of 5.0 μg/mL was added to the PDB medium. The hyphae from control and azoxystrobin-treated groups were collected after 24 h. Bright field microscopy images were observed using an inverted fluorescence microscope (Olympus IX71, Tokyo, Japan) with a 40-time objective lens. Furthermore, the hyphae were fixed with a 2.5% glutaraldehyde solution at 4 °C overnight. The fixed hyphae were washed with phosphate-buffered saline (PBS) solution (pH 7.4) three times for 10 min. Afterward, the washed hyphae were dehydrated with 50, 70, 90, and 100% ethanol for 10 min and then were further dehydrated with absolute ethanol for 20 min. The dehydrated hyphae were dropped onto clean silicon slices and sputtered with platinum under vacuum (EM ACE600, Leica, Germany). The morphology of the hyphae was recorded with SEM (SU8010, Hitachi, Tokyo, Japan) at an acceleration voltage of 5 kV.
SOD and CAT assays
The Fusarium oxysporum exposed to azoxystrobin nanosuspension (5 μg/mL) was collected to extract protein at different times after exposure for 1, 6, 18, 24, and 48 h, respectively. Protein was extracted by milling hyphae with a glass homogenizer in an ice bath. The homogenate was centrifuged (SORVALL ST16R, Thermo Scientific, MA, USA) at 10,000 rpm for 20 min at 4 °C. The supernatant was collected and used for the analysis of antioxidant enzyme activities. The concentration of soluble protein was measured by the Bradford method (1976) using bovine serum albumin (BSA) as a standard protein. SOD and CAT activities were determined by UV spectrophotometry (UV-2600, Shimadzu, Japan) using commercial assay kits (Jiancheng Institute, Nanjing, China).
Data analysis was performed using SPSS 20 statistical software (IBM Corp., Armonk, NY, USA). Data were presented as the mean ± standard deviation (SD). Quantitative data of contact angle and retention volume were evaluated with a one-way analysis of variance (ANOVA) followed by the least significant difference (LSD) method. A probability less than 0.05 was considered statistically significant. The other quantitative data were examined using a one-way ANOVA followed by the Student-Newman-Keuls (SNK) method.
Results and discussion
Preparation of the azoxystrobin nanosuspension
The effect of surfactant on the mean particle size and PDI of the nanosuspension
Mean size (d.nm)
2054.0 ± 216.20
0.52 ± 0.07
1233.3 ± 39.11
0.59 ± 0.07
307.5 ± 5.09
0.18 ± 0.01
854.7 ± 60.25
0.45 ± 0.07
344.9 ± 5.39
0.07 ± 0.02
661.0 ± 8.63
0.16 ± 0.03
The particle size and \(\zeta\) potential
In vitro dissolution of various azoxystrobin formulations
Stability of the nanosuspension
Wetting and spreading characteristics
Antifungal activity evaluation
Indoor toxicity of three azoxystrobin formulations against Fusarium oxysporum
y = 5.4764 + 0.1929 x
y = 5.4025 + 0.2440 x
y = 5.4433 + 0.1465 x
Effect on mycelial morphology
Effect on protein content, SOD, and CAT
For scale-up manufacture without organic residues, wet media milling technique has become more prevalent to enhance bioavailability of poorly soluble substance. In this study, the anionic surfactant SDS and polymeric PVP K30 were optimized from six conventional surfactants for the preparation of azoxystrobin nanosuspensions by wet media milling. The average diameter of the nanosuspension was approximately 200 nm in the dried state and the \(\zeta\) potential was –31.8 mV. Compared to WDG-A and WDG-B, the nanosuspension possessed the greater retention and the smaller contact angle on hydrophobic cabbage and hydrophilic cucumber leaves. Furthermore, the toxicity index of the azoxystrobin nanosuspension was approximately 1.7-fold that of the other formulations against Fusarium oxysporum. Morphological alterations of Fusarium oxysporum were observed by optical microscopy and SEM. The hyphal deformations disclosed that the azoxystrobin nanosuspension can disturb cell walls and enhance cell wall permeability. Antioxidant enzyme activities were affected by the azoxystrobin nanosuspension and Fusarium oxysporum was more susceptible to oxidative damage. In conclusion, these findings reveal that the nanosuspension produced by wet media milling is a desirable nanoformulation for azoxystrobin to improve the antifungal activity.
This study was financially supported by the Major National Scientific Research Program of China (No. 2014CB932200), the National Key Research and Development Program of China (2017YFD0201207, 2016YFD0200502), and the Agricultural Science and Technology Innovation Program (CAAS-XTCX2016004).
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