YIN Ming-ming, ZHENG Yu, CHEN Fu-liang

Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, P.R.China

Abstract We used poly (lactic-co-glycolic acid) (PLGA) as a carrier polymer for pyraclostrobin-loaded nanospheres. Using the ultrasound emulsification-solvent evaporation method, the physicochemical characteristics and release properties of the pyraclostrobin-loaded nanospheres were studied by dialysis. The optimal nanospheres prepared had a diameter of 0.6 μm,an active ingredient loading of 17.2%, and a loading rate of 89.7%. Infrared spectroscopy data and differential scanning calorimetry revealed that pyraclostrobin was successfully embedded in the carrier PLGA, and photostability tests indicated enhanced ultraviolet resistance of pyraclostrobin-loaded PLGA nanospheres nanospheres. Release property testing indicated that smaller particles had a faster release rate. Nanospheres also had a faster release rate in slightly acidic and slightly basic environments than in a neutral condition. Agitated nanospheres had a faster release rate than immobile nanospheres. The cumulative release kinetics of pyraclostrobin-loaded nanospheres was consistent with the first order kinetic equation and the Weibull equation.

Keywords: nanospheres, pyraclostrobin, PLGA, physical and chemical properties, slow release

1. Introduction

Pesticides are a key component of modern agricultural production. However, many pesticides contain active ingredients that cause environmental pollution in the atmosphere, soil, water and other environments (Wanget al.2015). Controlled release formulations offer a relatively safer type of pesticide delivery system (Zheng 2013; Kharbet al.2014; Meyeret al.2014; Royet al.2014). One form of pesticide controlled release delivery isviamicrospheres.Microspheres are uniform, solid spheres bearing active ingredients which were embedded in chitosan, polylactic acid, gelatin or other carrier polymers. The microsphere diameter generally ranges between 1 and 300 μm, while nanosphere diameters cover less than 1 μm. Importantly,the microsphere micrometer particle size offers a long release time, but they are slow acting. On the other hand,smaller nanosphere sizes allow for faster release rates compared to the conventional microspheres. As such,they possess both relatively fast-acting and extendedrelease properties (Eacuteet al.2009; Alishahiet al.2011;Linet al.2013; Zhanget al.2013). These biodegradable polymers generally have good environmental compatibility and work similarly to their microsphere analogs. As the carrier materials begin to degrade, the active ingredient is released, providing highly efficient pesticide utilization (Vertet al.2012; Xuet al.2017).

Pesticide-based microsphere test began in USA agricultural crops for use as a novel insecticide approach. In 1985, naled-loaded polycarbonate microspheres and gypsy moth sex attractant-loaded polysulfone microspheres were developed. Subsequent bioassay results showed that both microsphere formulations had relatively long environmental persistence (Baker 1987). Later, aldicarb-loaded carboxymethyl cellulose microspheres were developed(Darvari and Hasirci 1996). Anothersuccessfully developed controlled release formulation was the norflurazon-loaded ethylcellulose microspheres (Pérez-Martínezet al.2001).Many insect-directed microsphere formulations have been prepared in recent years including avermectin-loaded gelatin microspheres, acetamiprid-loaded carboxymethyl chitosan microspheres, spinosad-loaded polylactic acid microspheres and emamectin benzoate loaded polylactic acid microspheres (Tanget al.2002; Shanget al.2007;Wuand Liu 2008; Huaiet al.2009; Guoet al.2011; Huanget al.2011; Liet al.2011; Zhaoet al.2011; Zhenget al.2012; Xiaet al.2014; Yinet al. 2018). These formulations were created using emulsification-condensation, solvent evaporation, and orifice-based methods and subsequent bioassays showed promising insecticidal performance.However, all of the aforementioned formulations were conventional microspheres and nanoparticle-based spheres have not been reported for pesticidal efficacy.

Pyraclostrobin (CAS no.: 175013-18-0) was developed by BASF (Germany) in 1993. It is a strobilurin fungicide with a pyrazole structure that is photo-unstable. The International Union of Pure and Applied Chemistry (IUPAC)data show that the photolysis half-life of pyraclostrobin in water is 1.7 days. The photo-susceptibility of pyraclostrobin restricts its application in many conventional formulations and because of this, its fungicidal efficacy cannot be fully realized. Developing a pyraclostrobin-based extended release formulation could be an effective way to improve its stability and test its fungicidal limits. Pyraclostrobin-loaded nanospheres could be used during foliar spray applications for the prevention or treatment of fungal pathogens in crop diseases. In this study, we used poly (lactic-co-glycolic acid) (PLGA) as a carrier polymer and an ultrasound emulsification-solvent evaporation method to prepare pyraclostrobin-loaded nanospheres.

2. Materials and methods

2.1. Agents and equipments

The chemical agents used in this experiments were:97.2% (w/w) pyraclostrobin standard (Taizhou Dapeng Pharmaceutical Industry Co., Ltd., China), 97.5% (w/w)pyraclostrobin technical material (Wuhan Yihuaheng Technology Development Co., Ltd., China), poly(lacticco-glycolic acid); mass ratio of polylactic acid to 2-hydroxyethanoic acid: 75/25, (Shandong Medical Instruments Institute, China), polyvinyl alcohol (PVA)(analytical grade, Tianjin Guangfu Fine Chemical Research Institute, China), dichloromethane (analytical grade, Beijing Chemical Reagent Factory, China), acetonitrile (high performance liquid chromatography grade; Fisher, USA),and potassium bromide (Thermo Scientific, USA).

The instruments used in this experiment were: magnetic stirrer 79-1 with heating (Changzhou Guohua Electric Appliance Co., Ltd., China), Ultrasonic Cell Disruptor JYD-650 (Shanghai Zhisun Instrument Co., Ltd., China), N-1001 Rotary Evaporator (Eyela, Japan), Sigma 3-15 High-speed Centrifugal Machine (Sigma, Germany), Dura-Dry TM MP Freeze Dryer (FTS, England), Agilent 1220 Infinity HPLC System (Agilent Technologies, USA), BT-9300H Laser Particle Size Distribution Analyzer (Dandong Bettersize Instrument Co., Ltd., China), HCT-1/2 Integrated Thermal Analyzer (Beijing Heijiu Experimental Equipment Co., Ltd.,China), Fourier transform infrared (FTIR) spectrometer(Thermo Nicolet 6700, USA), and Quanta 200 FEG field emission environmental scanning electron microscope(SEM) (FEI, USA), and ultraviolet (UV) lamp LUYOR-3115(Wavelength 365 nm, Wuxi Junda Instrument Co., Ltd.,China).

2.2. Preparation of pyraclostrobin-loaded PLGA nanospheres

Pyraclostrobin dichloromethane solution was mixed with PLGA dichloromethane solution. While stirring, the polymer solution was dripping into the PVA aqueous solution, and the resultant mixture was rapidly stirred. The mixture was ultrasonicated to give it an oil-in-water (O/W) emulsion properties. This emulsion was diluted with PVA aqueous solution, followed by stable dispersion. The organic solvent was removed using a rotary evaporator and the sample was subsequently centrifuged. The precipitate at the bottom of the tube was collected, washed three times with deionized water, and freeze-dried to yield the nanospheres. Three different pyraclostrobin-loaded PLGA nanosphere particle sizes (1#, 0.6 μm; 2#, 0.9 μm; 3#, 1.4 μm) and a blank PLGA nanosphere without pyraclostrobin (CK, 0.6 μm) were prepared using the above steps. The preparation conditions are shown in Table 1.

2.3. Measurement of the physicochemical properties and structural characterization of pyraclostrobin-loaded PLGA nanospheres

Morphology An appropriate sample of pyraclostrobin-loaded PLGA nanosphere suspension was collected and one drop of solution was placed on a clean coverslip. The sample was dried at room temperature and then fixed on a tape. After gold spraying using an ion plating apparatus under a vacuum, the surface morphology of the nanospheres was examined by SEM.

Table 1 Preparation conditions of pyraclostrobin-loaded poly (lactic-co-glycolic acid) (PLGA) nanospheres

Particle size and distributionA laser particle size distribution analyzer was used to measure the nanosphere particle size and distribution. Particle size and particle size distribution were characterized by median volume diameter(D50) and span, respectively. The smaller the span, the narrower the particle size distribution. Span=(D90?D10)/D50,whereD10,D50, andD90are the particle diameters when the nanosphere volumes were 10, 50, and 90%, respectively.

Loading rate and pesticide loadingHPLC was used to measure the pesticide loading and loading rate of the nanospheres. The pesticide loading (mass fraction) and loading rate of pyraclostrobin-loaded PLGA nanospheres were calculated according to the following equations:

Pesticide loading (%)=Mass of pyraclostrobin in the test sample/Mass of the test sample×100

Loading rate (%)=Actual pesticide loading/Theoretical pesticide loading×100

AnalysisAppropriate amounts of KBr were mixed with dried pyraclostrobin, blank nanospheres, and pyraclostrobinloaded PLGA nanospheres, respectively. Tablets were prepared to conduct FTIR analysis.

Differential scanning calorimetry analysisAn appropriate sample amount from each of the nanosphere treatments was taken for measurement. All scanning calorimetry analyses were carried out under nitrogen. A programmed temperature increase was performed from 25 to 300°C, with a heating rate of 10°C min–1and a gas flow rate of 30 mL min–1. Weight change and the heat flux curve were recorded.

2.4. Measurement of the photolysis properties of pyraclostrobin-loaded nanospheres

Appropriate amounts of pyraclostrobin and pyraclostrobinloaded PLGA nanospheres were placed in a volumetric flask to prepare a 0.1 mg mL–1suspension in distilled water.A 1-mL sample of the solution was transferred to a 5-mL centrifuge tube. The tube was placed under UV lamp, at a vertical distance of 13 cm, and samples were collected after 10, 20, 30, 40, 50, and 60 min of UV radiation. The centrifuge tube was washed with acetonitrile (3 mL, 3 times)and nanospheres were dissolved by ultrasonication for 20 min. The solution volume was fixed using acetonitrile.Filtration was performed using a 0.45-μm membrane filter,and samples were measured using HPLC. Calculations for the photolysis rate and related equations were:

Pesticide photolysis was fitted to the first order kinetics equation:

Where,Ctis the residual concentration or mass of the pesticide after photolysis at timet,C0is the initial concentration or mass of pesticide in photolysis,kis the rate constant of pesticide photolysis andt1/2is the photolysis half-life.

2.5. Measurement of the extended release of pyraclostrobin-loaded PLGA nanoparticles

A sample of pyraclostrobin-loaded PLGA nanospheres was weighed and placed in a dialysis bag with 100 mL of release medium solution. The release container was covered with foil wrap to prevent light exposure and then placed into a bath oscillator at 25°C, 100 r min–1. Sampling was made at a certain time interval. A 2-mL sample of medium solution outside the dialysis bag was collected and an equal amount of fresh medium solution was added back. The medium solution sample was diluted with acetonitrile and the active ingredient concentration was quantitatively determined using HPLC. Each experiment was repeated 3 times. The amount of pyraclostrobin released from the pyraclostrobin-loaded PLGA nanospheres was calibrated using the degradation rate of the generic pesticide in the medium solution. This was calculated using the following formula:

Where,Qis the cumulative release percentage (%),CTis the measured active ingredient concentration in the released medium solution at the release time point (mg mL–1),Wis the total mass of active ingredient loaded (mg),V0is the total volume of the release medium solution (mL), andVis the volume of sample taken each time (mL).

3. Results

3.1. Physicochemical properties and structural characterization of pyraclostrobin-loaded PLGA nanospheres

MorphologyThe scanning electron microscope images illustrated that created pyraclostrobin-loaded PLGA nanospheres have uniform distribution, no adhesion, and a rounded shape with a smooth surface (Fig. 1).

Particle size and distributionD50of the pyraclostrobinloaded PLGA nanospheres was 0.6 μm and the span range was 1.58 μm. This indicated that the nanosphere particle size was uniformly and normally distributed (Fig. 2).

Fig. 1 Scanning electron microscope images of emamectin benzoate-loaded polylactic acid microspheres. A, multiple microspheres shown at 10 000× magnification. B, individual microsphere shown at 20 000× magnification.

Loading rate and pesticide loadingThe particle size,pesticide loading, and loading rate of the nanospheres were 0.6 μm, 17.2%, and 89.7%, respectively.

Spectral analysisSpectra of the characteristic functional groups in pyraclostrobin were measured to confirm the fungicides configuration (Fig. 3-A). A strong absorption at 1 716.47 cm–1was associated with the stretching vibration of the C=O bond attached to the benzene ring. Five strong peaks were measured at 1 598.82, 1 548.46, 1 505.38, and 1 479.63 cm?1and reflected the stretching vibration of the benzene ring skeleton. The peaks at 1 439.12, 1 255.66,and 1 107.18 cm?1corresponded to the absorption of N-C=O bond, N-C stretching vibration, and C-O bond stretching vibration, respectively. The peaks at 825.81 and 818.82 cm?1originated from the vibration of the Cl-Ar bond.

Fig. 2 The particle size distribution of pyraclostrobin poly (lactic-co-glycolic acid) nanosphere 1#.

Fig. 3 Infrared spectrogram. A, infrared spectrogram of pyraclostrobin technical material. B, infrared spectrogram of blank poly(lactic-co-glycolic acid) (PLGA) nanosphere. C, infrared spectrogram of pyraclostrobin PLGA nanosphere 1#.

Spectra of the characteristic functional groups in the blank PLGA nanospheres were measured to confirm the PLGA molecular structure (Fig. 3-B). Two small peaks were observed at 2 997.55 cm?1and arose from the stretching vibration of the methyl group and methylene group. The peaks found at 1 758.62, 1 456.26, and 1 385 cm?1were assigned to the C=O stretching vibration, the O-H bending vibration (in-plane), and CH bending vibration, respectively.The broad absorption bands that were seen at 1 185.83 and 1 091.45 cm?1were designated to the ester bond absorption.Finally, the peak observed at 865.8 cm?1was due to the OH bond bending vibration (out-of-plane).

Spectra for the pyraclostrobin-loaded PLGA nanosphere 1# was measured to confirm molecular structure (Fig. 3-C).The peaks associated with the characteristic functional group C-H stretching and bending vibrations were seen at 2 946.94 and 1 456.27 cm?1in PLGA remained. The peak at 1 758.84 cm?1was due to the C=O stretching vibration and the peaks at 1 185.74 and 1 091.61 cm?1were due to the ester bond absorption also remained, but the ester bond absorption ratio was reduced. The absorption peaks of the functional groups of pyraclostrobin were seen at 1 547.99, 1 425.57, and 1 262.87 cm?1as well as in the fingerprint region. Red and blue shifts were observed for the N-C=O bond absorption and the N-C bond absorption,respectively. The characteristic absorption peaks from the blank nanospheres and active ingredients simultaneously appeared in the spectrum of PLGA nanospheres. This indicated that the active ingredient was embedded in the carrier and that the functional groups did not change nor was there a chemical reaction. A wavenumber shift occurred for individual absorption peaks, indicating that the carriers and the active ingredient were mixed with each other, affecting absorptions.

Differential scanning calorimetry pattern analysisThe differential scanning calorimetry patterns of pyraclostrobinloaded PLGA nanospheres and a mixture of blank PLGA nanospheres with pyraclostrobin powder were measured(Fig. 4). Pyraclostrobin showed an endothermic peak at 65.3°C in the differential thermal analysis (DTA) curve which measured the melting point. This indicated a crystal structure in the pyraclostrobin powder. There was also a strong exothermic peak at about 216°C. This was expected to be the exothermic peak for pyraclostrobin degradation. The blank PLGA pattern did not exhibit obvious melting peaks (Fig. 4-B). However, the glass-phase transition temperature occurred at 54°C, indicating that the PLGA was a non-crystalline material. Comparisons between the scanning calorimetry patterns of pyraclostrobin-loaded PLGA nanosphere #1 (Fig. 4-A) and a mixture of blank PLGA nanospheres with pyraclostrobin powder (Fig. 4-B)suggested that the melting peak of the pyraclostrobin in the nanospheres at 65°C disappeared and only the glass-phase transition peak remained. This indicated that pyraclostrobin was uniformly dispersed inside the PLGA nanospheres in an amorphous form rather than as crystals. This finding also verified the difference between microspheres and microcapsules, the two extended release dosage forms.

3.2. Photolysis properties of pyraclostrobin-loaded nanospheres

The photolytic kinetic parameters of pesticide-loaded nanospheres with different particle sizes or the pyraclostrobin powder was calculated (Table 2). The half-life of the UV-degraded aqueous pesticide solution was about 18.7 min whereas the half-life values of nanosphere #1, #2, and #3 were 38.5, 49.5, and 53.3 min, respectively. The half-life values were significantly extended after pyraclostrobin was processed into nanospheres. Additionally, the larger the nanosphere diameter, the longer the half-life. The initial degradation rate of the PLGA nanosphere formulations was significantly slower than that of the generic pesticide(Fig. 5). After 60 min of UV treatment, the photolysis rate of the pyraclostrobin powder was 88.4%, while the photolysis rates of PLGA nanospheres were 71.1% (#1),58.1 % (#2), and 54.7% (#3), respectively. The photolysis rate was substantially lower, indicating that pyraclostrobin had increased ultraviolet resistance after nanosphere processing.

3.3. Extended release properties of pyraclostrobin-loaded PLGA nanospheres

The release profile of pesticide-loaded nanospheres in the medium solution was determined. The release was divided into three stages. The first stage was the initial burst release of the active ingredient. Since free active ingredients were attached to the nanosphere surface, they began the extended release into the medium solution at the start of the release process. Therefore, the cumulative release rate showed a high initial value. The second stage was another stage of rapid release, where the volume of the release medium solution dissolved five times the amount of active ingredient. At this point, the active ingredient entered into the medium along the concentration gradient of the pesticide-loaded nanospheres. The third stage was the slow release. The slow release was mainly due to degradation of the carrier material degraded and the release of the active ingredient into the medium. Additionally, a small amount of active ingredient diffused into the carrier materials at this stage.

Fig. 4 Differential scanning calorimetry thermogram. A, differential scanning calorimetry thermogram of pyraclostrobin poly (lacticco-glycolic acid) (PLGA) nanosphere 1#. B, differential scanning calorimetry thermogram of mixture of pyraclostrobin technical material and blank PLGA nanosphere. DSC, differential scanning calorimetry; TG, thermogravimetric analysis; T, temperature.

Table 2 The photodecomposition rate of pyraclostrobin technical material (TC) and pyraclostrobin poly (lactic-co-glycolic acid)nanospheres of three particle sizes

Extended release properties of pyraclostrobin-loaded PLGA nanospheres with different particle sizesThe extended release behavior of pyraclostrobin-loaded PLGA nanospheres with different sizes were measured over time(Fig. 6). With increasing nanosphere particle size, the release rate of the nanospheres decreased. After 200 h,the cumulative pyraclostrobin release rates from the PLGA nanospheres were 89.0% (#1), 79.7% (#2), and 66.4% (#3).This indicated that the release rate can be controlled by preparing different sized pyraclostrobin-loaded PLGA.

Fig. 5 The photodecomposition rate curve of pyraclostrobin technical material (TC) and pyraclostrobin poly (lactic-coglycolic acid) nanospheres 1#, 2# and 3#.

Fig. 6 Releasing curve of pyraclostrobin poly (lactic-co-glycolic acid) nanospheres 1#, 2# and 3# in different particle sizes.

Effect of different pH on the pyraclostrobin-loadedPLGA nanosphere release rateThe pyraclostrobin-loaded PLGA nanospheres cumulative release behavior in different pH buffer solutions was measured (Fig. 7). During the initial release stage, the pyraclostrobin-loaded PLGA nanospheres had a relatively fast release in a slightly alkaline environment(pH 8). This was likely released quickly as PLGA is easily degraded in alkaline environments. At pH 5 and pH 8, the release rate was even faster than pH 6 and pH 7 during the initial stage. The extended release rate of pyraclostrobinloaded PLGA nanospheres at the neutral pH 7 was relatively slow, indicating that pH influences the release rate of the active ingredient.

Effect of temperature on the pyraclostrobin-loaded PLGA nanosphere release rateThe release curves of pyraclostrobin-loaded PLGA nanospheres at 25 and 35°C were measured (Fig. 8). After 96 h, the cumulative release rates of active ingredient from the pesticide-loaded PLGA nanospheres at 25 and 35°C were 70.5 and 87.5%,respectively. This indicated that temperature has a significant impact on the extended-release rate of the pyraclostrobinloaded PLGA nanospheres. Additionally, these results showed that high temperature promoted the release of the active ingredient. The influence of temperature should be considered in experiments to determine release rates.High temperatures could be useful for rapid nanospheres release rates.

Fig. 7 Releasing curve of pyraclostrobin poly (lactic-co-glycolic acid) nanosphere 1# in different pH solutions.

Effect of agitation on the pyraclostrobin-loaded PLGA nanosphere release rateThe cumulative release rate of pyraclostrobin from PLGA nanospheres under static or agitated conditions was measured (Fig. 9). Under static conditions, the active ingredient diffused along the concentration gradient and the actual release rate was slower than when agitated. Under agitation conditions (25°C,100 r min–1), the active ingredient was uniformly released by PLGA nanoparticles within a short time. Meanwhile,agitation also facilitated diffusion of the active ingredient into a low-concentration release medium, and accelerated the degradation of PLGA, resulting in a faster release rate than under static conditions. After 192 h, the cumulative release rate of pyraclostrobin from the PLGA nanospheres was 71.4% under static conditions. With agitation, the cumulative release rate of the active ingredient was 89.0%at 192 h. The release rate in static conditions was clearly slower than the release rate in agitation conditions.

Fig. 8 Releasing curve of pyraclostrobin poly (lactic-co-glycolic acid) nanosphere 1# in different temperatures (T).

Fig. 9 Releasing curve of pyraclostrobin poly (lactic-co-glycolic acid) nanosphere 1# in different processing modes.

Equation-based curve fitting of the pyraclostrobin release rate kineticsThe cumulative release curve of pyraclostrobin from the PLGA nanosphere was fitted with multiple kinetics equations to model the release behavior(Fig. 10). The pyraclostrobin-loaded PLGA nanospheres were fitted with the four models: the zero order kinetic equation (Q=a+bt), the first order kinetic equation (Q=a(1?ebt)), the Higuchi equation (Q=a+bt1/2), and the Weibull function (Q=1?e?(t?a)m/b). The first order kinetics and Weibull function exhibited a good fit for the cumulative release curve while the Higuchi equation and the zero-order kinetics fit poorly.

The fitting parameters for the different kinetic equations on the cumulative release curve of pyraclostrobin from PLGA nanospheres can be seen in Table 3. TheR2values of the first-order kinetic equation and Weibull function were over 0.99, showing an excellent fit. Both of these equations could serve as an effective prediction model for active ingredient release from PLGA nanospheres. TheR2values of Higuchi equation and the zero order kinetic equation were below 0.9, and both exhibited a significant difference from the cumulative release trend of pyraclostrobin. They showed a poor fit, failed to reflect the active ingredient release trend,and were not consistent with the curve obtained from actual measurements.

4. Discussion

Particle size of pyraclostrobin-loaded poly (lactic-co-glycolic acid) nanospheres had a significant influence on the cumulative release rate of the active ingredient. Here, the cumulative release rate of pyraclostrobin decreased with increasing particle size. This finding was consistent with previous findings, where microspheres that have smaller particle size provided a larger relative contact surface area between the microspheres and the release medium, offering a greater chance for the active ingredient to diffuse outward,thereby accelerating the release rate (Berklandet al.2001).

Fig. 10 Curve fitting of release equation on pyraclostrobin poly(lactic-co-glycolic acid) nanosphere 1#.

Table 3 The fitting parameters of release equation on pyraclostrobin poly (lactic-co-glycolic acid) nanosphere 1#

We also found that pH affects the release of pyraclostrobin from PLGA nanospheres. A study by Zolnikand Burgess(2007) found that degradation of PLGA could be accelerated in acidic and alkaline conditions. The alkaline release medium was able to undergo a neutralization reaction with the carboxyl group produced during PLGA degradation,promoting PLGA degradation. In an acidic environment,PLGA could undergo an autocatalytic reaction that accelerated degradation (Zolnik and Burgess 2007). In this experiment, the release behaviors of the pyraclostrobinloaded PLGA nanospheres under acidic and alkaline conditions were investigated and the initial release rates of pyraclostrobin-loaded PLGA nanospheres in both slightly acidic and slightly alkaline conditions were faster than under a neutral pH. The effects of different pH on the release rate of pesticide-loaded nanospheres may result from varying degradation rates of the carrier materials.

High temperatures were found to accelerate the release rate of pyraclostrobin-loaded PLGA nanospheres. This result relates to PLGA use as a polymeric material. During the nanosphere preparation process, the organic solvent evaporates and the hydrophobic macromolecular chain shrink and bind the active ingredient molecules tightly to form spherical entities. Water molecules can enter into the interior of the nanospheres through the gaps between the macromolecules or the cavities formed during evaporation.Chain scission and depolymerization occur at non-fixed points inside the macromolecules and PLGA is degraded.High temperature causes swelling of the PLGA carrier so that water can easily enter into the carrier promoting PLGA degradation and accelerating active ingredient release. In addition, the diffusion rate of pyraclostrobin from the PLGA carrier was found to increase at higher temperatures.

Pyraclostrobin-loaded PLGA nanospheres showed a significantly faster release rate under agitation conditions compared to static conditions. Nanospheres release the active ingredient through the dissolution and degradation of the carrier and the active ingredient diffuses into the outside medium along the concentration gradient or external force.In addition, the pyraclostrobin diffusion rate from the PLGA carrier was found to increase under agitation conditions.Under the static mode, the outward diffusion of the active ingredient relies solely on the concentration gradient. Under the agitation mode, the active ingredient can be uniformly distributed in the medium within a short time by external force action. In the static condition, the active ingredient distribution is uneven, with the highest concentration of active ingredient being near the nanosphere surface while the concentration in the medium away from the nanospheres is relatively low. A high concentration of active ingredient directly outside the nanospheres slows further diffusion of the active ingredient and the final result is a slower release rate. In the agitation mode, the concentration of active ingredient is relatively uniform throughout the entire medium, and the concentration of the active ingredient at the nanosphere surface is reduced. In this case, the outward diffusion of active ingredient from the nanosphere was more rapid. The cumulative release rate of the active ingredient simulated in the agitation mode was more similar to the pattern of nanosphere-based pesticide release. Lastly, the release rate shown in the static mode was closer to the efficacy trials in practical applications.

Generally, the release mechanism of conventional (nonmicroencapsulated) pesticide formulations follows zero order kinetics and the first order equation while that extendedrelease formulations tend to follow first order kinetics and the Weibull equation (Vertet al.2012). The cumulative release kinetics of emamectin benzoate polylactic acid microspheres prepared by Zhuet al.(2013) followed the first order kinetic release equation. Similarly, the cumulative release profile of pyraclostrobin-loaded PLGA nanospheres prepared in the current research also followed the first order kinetic equation as well as the Weibull equation.

5. Conclusion

Photostability tests showed that UV resistance of the active ingredient pyraclostrobin was enhanced in the pyraclostrobin-loaded PLGA nanospheres. The release properties of the pyraclostrobin-loaded PLGA nanospheres were studied using dialysis. After 200 h, the cumulative release rate of the active ingredient in buffer solution was 89.7%, showing a reasonable level of extended-release performance. The effects of particle size, medium pH,temperature, and agitation mode on the extended release properties of the nanoparticles were investigated and showed that particle size and release rate were negatively correlated. Nanoparticles had a faster release rate in acidic and basic pH than at neutral pH 7. High temperatures promoted nanosphere degradation and resulted in a more rapid release rate. Agitation increased the release rate compared to static conditions. The cumulative release kinetics of pyraclostrobin-loaded PLGA nanospheres was consistent with the first order kinetic equation and the Weibull equation.

Acknowledgements

This study was supported by the National Key R&D Program of China (2017YFD0200301, 2016YFD0200500).