Cymoxanil was purchased from Heowns, Tianjin, China. As a commercial product, CYM/chlorothalonil dispersible concentrate (DC) was obtained from Jiadeli Science and Technology Company, Sichuan, China. Cholesterol (Chol), ethylenediaminetetraacetic acid (EDTA), tris(hydroxymethyl)aminomethane (TRIS), 2-morpholinoethanesulfonic acid monohydrate (MES) and dialysis membrane (3.5 kDa) were purchased from Solarbio, Beijing, China. Stearylamine (SA) and benzene (analytical grade) were supplied by Macklin, Shanghai, China. Methanol (analytical grade) and sodium chloride (NaCl) were purchased from Kermel, Tianjin, China. Yeast extract peptone dextrose (YPD) medium was used to culture yeast cells. Yeast extract was purchased from Oxoid, Hants, United Kingdom. Peptone was obtained from Aobox, Beijing, China. Dextrose was purchased from Kermel, Tianjin, China. Dimethyl sulfoxide (DMSO) and acetonitrile (HPLC grade) were purchased from Sigma-Aldrich, Saint Louis, MO, United States. Deionized water was obtained from a Milli-Q water system (Master Q15, Hitech Instruments CO., Shanghai, China) and was used for all reactions and treatment processes.

Sterosomes were prepared as previously described (Cui et al., 2017). Briefly, dissolve SA and Chol (1/1 molar ratio) in benzene/methanol 90/10 (v/v). Freeze the solution in liquid nitrogen and lyophilize for at least 16 h. Hydrate the freeze-dried powder mixtures with a MES/TRIS buffer (TRIS 50 mM, MES 50 mM, NaCl 130 mM, EDTA 0.5 mM, pH = 5.0). Cycle temperature five times from liquid nitrogen (LN) temperature to ∼70°C (about 1 min in LN and heat for 10 min), and vortex (∼30 s) between successive cycles. After hydration, measure and readjust the pH to 5.0. Sonicate the hydrated suspensions at 40% intensity and an 80% duty circle for 20 min. The final concentration of sterosomes suspensions was 20 mg/mL. CYM-loaded sterosomes were prepared by adding CYM (dissolved in DMSO) to sterosome suspensions, and stirring overnight at room temperature.

The concentration of CYM was detected by absorbance (240 nm) with HPLC (Agilent 1200, Beijing, China). The details of the method are listed in Table 1 (Morrica et al., 2004). An equal volume of acetonitrile was added to CYM-loaded sterosome suspensions and shaken for 10 min to lyse the sterosomes. The supernatant was passed through a 220-nm filter and the flow-through was injected into the HPLC. The amount of CYM in the drug-loaded sterosomes was detected and calculated by comparing to a standard curve. The loading efficiency (LE) was defined as the ratio of actual versus theoretical amount of CYM in the drug-loaded sterosomes, as described by Equation 1. The drug to lipid ratio (D/L) was defined as the ratio of actual amount of CYM versus total amount of lipids, as described by Equation 2.

Size and ζ-potential were measured with a ZS90 (Malvern Instruments, United Kingdom). The sample was prepared with 1 mL sterosomes and CYM-loaded sterosomes. The change of size and ζ-potential after CYM loading showed dispersion and stability properties.

Before morphology analysis, sterosomes and CYM-loaded sterosome suspensions were freeze-dried overnight to yield uniform powders. Field emission scanning electron microscope (FE-SEM) images were recorded on a NOVA NanoSEM 450 (FEI, United States) under operation at an acceleration voltage of 20 kV.

X-ray powder diffraction (XRD, SmartLab 9KW, Japan) confirmed the crystallinity of sterosomes and CYM-loaded sterosomes in a 2θ range of 10∼80° with Cu-Kα radiation of 1.54 Å, an operating voltage of 40 kV, and a current of 40 mA.

Fourier-transform infrared (FT-IR) spectroscopy was used to analyze the functional groups of sterosomes and CYM-loaded sterosomes with an FTIR Spectrometer (6700, Thermo Fisher Scientific, United States).

A concentration of 1 mg/mL CYM in the drug-loaded sterosomes was transferred into a dialysis membrane (3.5 kDa), which was placed in a beaker containing 200 mL MES/TRIS buffer (pH = 5.0), at room temperature. An aliquot of the sample (20 μL) was collected from the beaker at the pre-determined time points to measure the CYM concentration, and an equal volume of fresh MES/TRIS buffer was replenished. Free CYM solution served as a negative control group and CYM/chlorothalonil DC (D/L 0.0204) was used as a commercial positive control group, which is mainly applied for the control of cucumber downy mildew. Three independent experiments were carried out. The quantification was calculated with the following equation:

where E_ris the cumulative release of CYM, %; C_t is the concentration of CYM in the MES/TRIS buffer at time t, mg/mL; V₀ is the volume in the membrane, mL; and M_p is the mass of CYM in the carrier, mg.

Saccharomyces cerevisiae (S. cerevisiae) served as a model fungus system to evaluate the sustained release of CYM-loaded sterosomes resulting from the statistics of cytotoxicity assay (Ribeiro et al., 2000; Fai and Grant, 2009). Each sample (CYM solution, CYM-loaded sterosomes, and CYM/chlorothalonil DC) was prepared and dispersed in YPD medium via dilutions at the effective testing concentrations of CYM, 50 mg/L (Ribeiro et al., 2000). The same concentration of sterosomes was added to evaluate the toxicity to yeast cells. The blank group was only cells in YPD medium, displaying the normal growth cycle of cells. Then S. cerevisiaegrew at 30°C, 200 rpm, and the growth status of the cells was detected by OD₆₂₀.

The morphology of prepared CYM-loaded sterosomes was characterized using FE-SEM (Figure 1). The well-distributed nanoparticles were observed after lyophilization. A few crystals from dried MES/TRIS buffer were present among sterosome powders. The size of sterosome particles fell under the nanometer scale, resulting in a high specific surface area, which provides more space for interactions between drugs and nano-carriers. After loading with CYM, the appearance of sterosomes became less regular, with the reduction of sphericity, indicating the successful loading of CYM, modifying the surface of sterosomes.

CYM-loaded sterosomes were prepared by adding CYM to sterosome suspensions. Various amounts of CYM were added to evaluate the effects on particle size distribution and ζ-potential properties. With the increase of the CYM concentrations over the range of 0.25∼1 mg/mL, the particle size of sterosomes exhibited no obvious change, while the polydispersity index increased after CYM loading (Figure 2A). Moreover, the ζ-potential of sterosomes after CYM loading decreased, indicating that some of the CYM was complexed on the surface of sterosomes (Figure 2B). The electrostatic interactions between the SA/CholSterosomes and the cymoxanil were the main driving force for complexation.

Dynamic light scattering (DLS) methods revealed more details of the distribution of sterosomes in suspensions. The nanoparticle size of sterosomes ranged between 120 and 130 nm (Figure 2C). One of the most unique properties of Chol in relation to the SA/Chol structure is its ability to induce the formation of a liquid-ordered (lo) lamellar phase (Carbajal et al., 2013). In the equimolar mixture at pH < 9, protonated SA is well mixed with Chol molecules, leading to the formation of stable lo lamellar phases (Cui and Lafleur, 2014). Then homogeneous sterosomes were obtained after ultrasonic dispersion. The ζ-potential for empty sterosomes was +42.4 mV, arising from the protonated amine groups of SA. A small decrease of ζ-potential for CYM-loaded sterosomes (+40.4 mV) was associated with the loading of CYM, covering the exposed protonated of SA (Figure 2D). According to the data of PDI, the dispersity of the sterosomes in buffer was relatively uniform because of the electrostatic repulsion among particles. CYM-loaded sterosomes are uniformly dispersed in the MES/TRIS buffer (pH = 5.0) and present good stability.

The XRD patterns depict the crystalline structure of sterosomes to show self-assembly behaviors with SA and Chol. It is shown that SA and Chol presented different crystalline structures (Figure 3). The mixture showed overlapped diffraction patterns of SA and Chol, which proved that only physical mixing of SA and Chol took place without any intermolecular assembly arrangement. However, sterosomes presented a completely different crystalline structure, suggesting that successful self-assembly between SA and Chol occurred during hydration and a new sterosome structure was formed. After CYM loading, the crystalline structure of CYM-loaded sterosomes at 2θ values barely changed (Figure 3). The addition of CYM did not alter the structure and integrity of sterosomes.

Fourier-transform infrared (FT-IR) spectroscopic analysis was carried out for further characterization of CYM-loaded sterosomes. FT-IR spectra of SA/Chol mixtures before and after hydration showed peaks of SA around 1,420∼1,480 cm^–1 and 2,700∼3,000 cm^–1 (Figure 4), indicating the structure of the aliphatic chain. The peaks in the area of 3,333 cm^–1 showed the presence of an -NH₂ group in SA. The peak at 1,056 cm^–1is ascribed to the ring deformation of Chol. Compared with the mixture, sterosomes obtained by hydration showed the same peaks, which confirmed that the structural change caused by the self-assembly of SA and Chol belonged to the change of physical phase transition without the change of chemical functional groups. Furthermore, CYM showed a peak at 1,706 cm^–1, associated with the ketone (C = O) groups, and CYM-loaded sterosomes also presented ketone (C = O) groups (Figure 4), indicating prominent cover of CYM on the sterosomes.

The CYM loading capacity of sterosomes is listed in Table 2. With the increased concentrations of CYM, the LE and D/L rose to 92.6% and 0.0761, respectively. The predominant interaction between drugs and sterosomes was surface electrostatic attraction. Typically, higher positive surface charge density leads to higher drug uptake efficiency and a more stable system (Cui et al., 2012). After CYM was added to sterosome dispersion, it was stirred overnight to facilitate its complete distribution in the buffer. Then, we measured the actual CYM content in the system. The results confirmed that CYM-loaded sterosomes with 1 mg/mL CYM achieved the highest LE and D/L among all of the experimental groups over the investigated concentration range, and a value much higher than that of the commercial product CYM/chlorothalonil DC (D/L 0.0204). Due to the limited solubility of CYM, ∼1 mg/mL, no higher concentration was accessible to reach a loading plateau.

Comparison of the sustained release capacities of CYM-loaded sterosomes, free CYM solution and CYM/chlorothalonil DC at room temperature was performed (Figure 5). At the same concentration of CYM, the release rate of free CYM solution group was the fastest, on account of rapid partition under high osmolar pressure. In the first 6 h, both CYM-loaded sterosomes and CYM/chlorothalonil DC showed significant sustained release capacity. After 1 day, 80% of CYM was released from CYM/chlorothalonil DC and the loaded drug could be released completely within 3 days. However, the equilibrium was essentially reached in 24 h for CYM-loaded sterosomes and nearly 80% of the drug after 3 days was released. The surface interaction between CYM and sterosomes effectively reduced the drug release rate. The remainder of CYM remained on the surface of sterosomes, supposedly to improve its pharmacological action with carrier shift. Our newly developed CYM exhibited enhanced drug stability and better sustained release behavior compared to the commercially available product.

CYM exhibited high efficacy against the yeast strain S. cerevisiae with an IC₅₀ of 50 mg/L. Hence. in the pharmacological activity of CYM-loaded sterosome experiments, S. cerevisiaewas used as the model fungus by detecting OD₆₂₀ of culture medium to reflect the growth status of the cells (Figure 6). As the blank curve, after 48 h of normal yeast growth, the logarithmic growth stage ended, and it reached a plateau growth stage. However, affected by free CYM solution, the inhibition of yeast growth lasted for only 48 h, which confirmed that CYM was only effective for a relatively short duration. Then, after 72 h, the yeast growth reached a stable stage and remained constant. CYM/chlorothalonil DC showed the same inhibition effect as the CYM group. Surprisingly, the effective inhibition period of CYM-loaded sterosomes was extended to 120 h, thus significantly increasing the efficacious duration. Although the positive charge of sterosomes can facilitate intracellular uptake, it also associates with cytotoxicity resulting from the generation of reactive oxygen intermediates, toxic oxidative bursts, and a disruption of cellular and sub-cellular membrane functions (Campbell, 1983; Dokka et al., 2000; Romoren et al., 2004). It is supposed to have no significant effect on cell viability with sterosomes added (Cui et al., 2015). Interestingly, a certain inhibitory effect was observed with the pure sterosomes added during the early stage of yeast growth, resulting in the right shift of the yeast growth curve, and it was then consistent with the blank group after 48 h. After sterosomes were added, yeast cells aggregated in the culture medium, which could be caused by the electrostatic interactions between yeast cells and sterosomes. This is because of the negative charges on the surface of the cell membrane, which facilitates the complexation of cationic sterosomes to accumulate on the cell surface. This behavior is supposed to affect cell mass transfer and metabolism, thereby delaying yeast growth.