Fabrication a controlled-release pesticide for improving UV-shielding properties and reducing toxicity via coating polydopamine

Abstract Controlled-release formulations (CRFs) have potential applications in modern agriculture, for it can not only prolong the duration of agrochemicals but also alleviate the adverse effect on non-target organism. In this study, we constructed pyraclostrobin@SiO2@polydopamine microcapsule (Pyr@SiO2@PDA MC). The resulting microcapsule is a near-rod shape (about 1.15 μm), which has a drug-loading efficiency of 55%. Fourier transform infrared (FTIR) and thermogravimetric analysis (TG) revealed the successful entrapment of the pesticide. The coating of polydopamine (PDA) endowing the microcapsule with superior UV-shielding properties than pyraclostrobin@SiO2 microcapsule (Pyr@SiO2 MC). Compared with pyraclostrobin emulsifiable concentrate (EC), the Pyr@SiO2@PDA MC exhibited 9.07-, 5.50-, 4.93- and 4.16-fold higher fungicidal activity against Rice blast fungus (Pyricularia oryzae) at concentrations of 0.5, 1, 2 and 4 mg/L. Moreover, acute toxicity tests demonstrated that the sample on zebrafish with lower toxicity on the first day. These results showed that the obtained microcapsule may process broader application potential in agriculture.


Introduction
Pyraclostrobin ( Figure 1) is a methoxy acrylate fungicide developed by BASF in 1993. [1] It acts on the cytochrome bcl complex in the fungal mitochondrial respiratory chain, preventing electron transfer and inhibiting mitochondrial respiration, making mitochondria unable to produce the energy (ATP) required for normal metabolism of cells, leading to cell death. [2,3] Pyraclostrobin (Pyr) has a very broad bactericidal spectrum with protective and therapeutic effects on crops. However, Pyr has a high ecological safety risk when used in rice fields, which limits its application to a certain extent. [4,5] A viable strategy to reducing the accumulation of pesticides in a water environment is to develop controlled release formulations (CRFs). Li et al. fabricated Pyr microcapsules (MCs) with Fe 3þ and tannic acid, which showed excellent efficacy on rice blast and significantly lower toxicity to four model organisms. [6] Controlled release technology (CRT) has been extensively employed in industries and fields such as pharmaceuticals, coatings, cosmetics, etc. [7][8][9] In the field of pesticide formulation processing, CRT is also an ideal choice to advance the efficiency of pesticides and reduce environmental pollution. Therefore, it has become an important direction for the development of new pesticide formulation. [10,11] To date, a host of inorganic materials like SiO 2 , and various polymers are invoked as a carrier to deliver the drug in order to achieve controlled-release. [12] Study on silica has attracted a wide range of attention due to its prominent properties, such as facile preparation, low cost, polymer coatings, etc. [13][14][15] More importantly, it has a significant impact on enhancing plant tolerance against biotic and abiotic stresses. [16] These advantages render it an ideal and suitable choice for drug delivery in agriculture.
Dopamine (DA) can form an adherent polydopamine (PDA) coating in a weakly alkaline solution through oxidative self-polymerization. [17] In addition, PDA coating can also be used for other reactions because of its abundant functional groups (amino, imino, and catecholyl). [18,19] Xin et al. prepared a PDA coated avermectin microcapsules (Av@PDA MC) with prominent sustained-release performance and good adhesion properties. [20] Gao et al. fabricated a novel imidacloprid microcapsule with PDA followed by polyurea (PU) exhibited excellent dispersion stability. [21] Undeniably, PDA is considered to be a promising pesticide encapsulant for obtaining novel controlled release formulation. [22,23] Microcapsules are one of the microparticle systems with great potential. As a representative of CRFs, it can improve the environment security and persistence of agrochemicals. [24,25] Pesticide microcapsules made of reliable material as a carrier could increase the length of activity as well as reduce side effects on the environment. In the present work, a simple method has been tried to encapsulate Pyr for acquiring high drug-loading efficiency. The preparation of microcapsules and the drug loading process are performed simultaneously to obtain Pyr@SiO 2 MC. This can effectively enhance the preparation efficiency. The deposition of PDA on the surface can give microcapsules excellent UV shielding properties. Prepared Pyr@SiO 2 @PDA MC was characterized on physicochemical properties including size, morphology, drug loading, release behavior and UV-shielding. The fungicidal activities against Pyricularia oryzae as well as the acute toxicity to zebrafish were also investigated.

Materials
Pyraclostrobin technical (97% TC) was provided by Jiangsu Subin Agrochemical Co., Ltd (Jiangsu China). 250 g/L Pyraclostrobin emulsifiable concentrate (EC) was purchased from BASF (Germany). Dopamine hydrochloride (DA) and Tris-HCl (1.5 M, pH ¼ 8.8) were supplied by Beijing Solarbio Science & Technology Co., Ltd. Cetyl trimethyl ammonium bromide (CTAB) and tetraethyl orthosilicate (TEOS) were purchased from Aladdin Reagent (Shanghai) Co., Ltd. Ethyl acetate, tween-80 emulsifier, methanol and the other reagents were came from Sinopharm group chemical reagent co. Ltd and used as received without further purification. The chemicals that we used for the experiments were of analytical grade.

Preparation of Pyr@SiO 2 @PDA microcapsules
Microcapsules were prepared as reporting of literature with some modification. [26] Firstly, 0.5% w/v water phase was obtained by dissolving 0.5 g cetyl trimethyl ammonium bromide in 100.0 mL of deionized water. Next, 0.5 g Pyr was dissolved in a mixture ethyl acetate and tetraethyl orthosilicate to prepare the oil phase. Then, the oil phase was mixed with the aforementioned water solution, ammonia water (25%-28%, v/v) was added dropwise to adjust its pH to 8. The mixture was agitated by a magnetic stirrer (C-MAG HS 7, IKA Company, Germany) with the rotation speed 600 rpm for 2 h, placed at room temperature overnight. Subsequently, DA and 200 ll Tris-HCl was added and stirred at room temperature for 24 h (Figure 2). After centrifugation, the solution was washed at least three times with deionized water and dried under vacuum at 45 C. The dried sample was named as Pyr@SiO 2 @PDA MC. In order to obtain the highest loading content of Pyr, different amount of DA were used to prepare the microcapsules ( Table 1). The preparation process of Pyr@SiO2 MC was the same as above, but without adding DA and Tris-HCl.
Characterization of the Pyr@SiO 2 @PDA microcapsule Particle size of Pyr@SiO 2 @PDA MC was measured by Mastersizer 2000 (Malvern instruments Ltd., Britain). The particles were suspended in deionized water for measurement, and each sample was measured three times. The appearance of the sample was observed by a JMS À 6360 scanning electron microscope (SEM; Tokyo, Japan) at 25 KV. Nicolet-IS 5 (United States) was used to record the characteristic absorption peak of the products in the range of 4000-500 cm À1 at room temperature. For purpose of confirming the existence of the Pyr and determine drug-loading efficiency, thermogravimetric analysis (TG) was conducted over the temperature range from 25 to 600 C at 10 C/min rate by mean of Mettler Toledo thermal analyzer (TGA 2). Zeta potential was tested by micro-electrophoresis apparatus (JS94H2 Shanghai Zhongchen Digital Technology Apparatus Co., Ltd., China). Equation (1) was utilized to calculate the effective components in the microcapsule. [27] Samples containing the same quality of Pyr, including Pyr TC, Pyr@SiO 2 MC and Pyr@SiO 2 @PDA MC were enclosed in quartz tube. Ultraviolet (UV) light was selected as light source, with the irradiance of 3.00 mW/cm 2 , rated power of 1.5 W Â 2, at a distance of 15 cm, and set the dark control. Samples were collected at regular intervals and content was determined. The released amounts of Pyr were monitored by HPLC (Agilent Technologies Inc., America) and the photodegradation rate of samples was calculated according to Eq. (2).
In vitro release studies The drug-loaded microcapsule was placed in a dialysis bag (MWCO 3500 Da) and immersed in 200.0 mL of release medium (phosphate buffer saline, ethanol and tween-80 emulsifier, 140:59:1 v/v/v), maintaining the temperature at 25 ± 1 C. Some of the solutions were withdrawn at appropriate intervals for the HPLC assay, and the same amount of fresh release medium was added after each sampling at predetermined time intervals to keep a constant volume. The chromatographic separation was carried out using an XBridge C18 column (250 mm Â 4.6 mm, 5 lm). The flow rate was set to 1 mL/min with a mobile-phase composition of chromatographic methanol and water (75:25, v/v). The injection volume was 10 lL, and the column temperature was 35 C. Meanwhile, for the control study, 97% Pyr TC and Pyr@SiO 2 MC were used as a reference under the same condition.

Indoor virulence assay
Fungicidal activity of Pyr@SiO 2 MC and Pyr@SiO 2 @PDA MC was assessed by the growth rate method using Pyricularia oryzae as the tested strain, and the 250 g/L Pyr EC was used as a control dosage form. [28] The tested strain was isolated and purified by the Laboratory of Pesticide Science, College of Plant Protection, Hunan Agricultural University (Changsha, China). Briefly, mycelial disks (7 mm in diameter) of Pyricularia oryzae were grown on potato dextrose agar plates, which were treated with different concentrations of 250 g/L Pyr EC, Pyr@SiO 2 MC and Pyr@SiO 2 @PDA MC. Finally, the cross method was applied to determine the colony diameter, and the concentration for 50% of maximal effect (EC 50 ) was calculated according to the Equation 3. The control group was treated with sterile water of the same volume. Each treatment was performed at least in triplicate.

Acute toxicity tests on zebrafish
In order to evaluate the acute toxicity of the sample to aquatic organisms, zebrafish, which respond quickly and inexpensively, was used as a model organism. The zebrafish was the same as the zebrafish used in the previous experiments, [29] purchased from a commercial supplier in Changsha China, weighing 0.2 to 0.4 g and having an average length of 2.0 to 3.0 cm. Acute toxicity tests were carried out according to guideline for "environmental safety evaluation tests of chemical pesticides standards." [30] The zebrafish were carried out in accordance with the basic principles of animal experiments at Hunan Agricultural University, and were approved by the Pesticide Chemistry Laboratory of the College of Plant Protection. Acute toxicity studied were performed in a 96 h semi-static test. [31] Briefly, placed 10 tails of adult fish in 3.00 L aerated water with diverse concentrations of agents(Pyr@SiO 2 @PDA MC and Pyr EC) ranging from 0.02 to 0.12 mg/L. Control experiments with only aerated water were consistent with the test group. Three repetitions were set for each experiment. Keep room temperature at 25 ± 1 C and light/dark cycle (16 hour light/8 hour dark) constant. Dead fish is being removed in order to avoid affecting the test. With SPSS, software available in the computer, LC 50 values and their 95% confidence limits could be obtained at 24, 48, 72, and 96 h, respectively.

Results and discussion
Effect of dopamine addition on microcapsules Particle size of microcapsules is increased with the addition of DA, but decreased when DA increased to 80 mg (Table 1).This may be due to the limited specific surface area that can be contacted by the microcapsule. As the covered PDA reaches an extreme value, increasing the dosage Mycelium groth inhibitin rate % ð Þ ¼ Control group colony growth diameter À Treatment group colony growth diamter Control group colony growth diameter Â 100 (3) of DA, particle size will not continue to increase. Besides, the drug-loading efficiency will first increase and then decline with the increase of DA. When the addition amount of DA was 40 mg, the drug-loading efficiency could reach 55%, which was higher than the single-and double-shelled cyhalothrin/SiO 2 microcapsules (about 50.0% and 25.0%, respectively). [32] As depicted in Figure 3(a), the color of the solution gradually deepens with the increase of DA. After tableting, samples S1, S2 and S3 were grayish white, grayish black and dark gray, respectively (Figure 3b). The color of microcapsule samples became darker with the increase of DA content.
Characterization of samples Figure 4 shows the morphology of samples under different magnifications (1000x/2000x/5000x). When magnified a thousand times (Figure 4a, 4d, and 4f), the difference between different samples is not obvious, and the overall microcapsules demonstrate a nearly rod-like structure. Compared with Figure 4(e), Figure 4(b) and 4(h) showed a more serious reunion phenomenon. After further magnification of the sample, we can observe that when the amount of DA added is increased to 80 mg, the excess DA causes more obvious agglomeration and the rod-like structure of the microcapsule is destroyed. This may be due to the limited surface area that the microcapsules can contact. When the covered PDA reaches saturation, the DA can no longer be deposited, resulting in obvious agglomeration. Finally, combining the results of drug loading and SEM, sample S2 (Table 1) was selected for further study. TG analysis of SiO 2 @PDA MC and Pyr@SiO 2 @PDA MC in nitrogen atmosphere have been carried out to further verify the presence of the Pyr. As showed in Figure 5(a), TG analysis was also carried out for quantitative analysis. The curves of SiO 2 @PDA MC and Pyr@SiO 2 @PDA MC showed weight loss in the range of 300-600 C due to the decomposition of PDA. [33] A new weight loss started at about 200 C, which assigns to the decomposition of Pyr ( Figure A1). The weight loss of bare SiO 2 @PDA MC was about 15%, but that of Pyr@SiO 2 @PDA MC ascended to 73%. Therefore the weight of Pyr loaded in the samples was about 58%. It also illustrated the successful encapsulation of Pyr in the Pyr@SiO 2 @PDA MC. The calculation found that the drug loading efficiency was only 55%, which was significantly lower than that obtained by TG analysis (58%). This may be because despite grinding and ultrasound, the samples were not completely destroyed and some drugs were not completely released, resulting in a low calculated drug loading efficiency.
Zeta potential was used to monitor the change of particle potential before and after PDA coating. Potential beginning with the silica was À16.05 mV and turned to À20.11 mV after the deposit of PDA layer (shown in Figure 5b). Since the Pyr@SiO 2 MC have a large amount of Si-OH groups, the zeta potential values appeared to be negative at first, and when the PDA is coated, the PDA also has a large amount of -OH and -NH showing negative zeta potential so that the negative potential of the particle band increases. [34] Zeta potential values are shifted with the cladding of the PDA layers, which indicated the successfully deposition of the PDA.

FTIR spectrum analysis
The infrared spectroscopy of DA, PDA, Pyr, SiO 2 , Pyr@SiO 2 MC and Pyr@SiO 2 @PDA MC was performed by FTIR spectra, which were utilized to verify successful encapsulation of pesticide. DA had many narrow peaks, which were characteristic of small molecule ( Figure 6a); while PDA showed only a few main peaks: the main peak of the aromatic ring was about 1623 cm À1 , and the main peak of the catechol-OH group was about 3446 cm À1 . [35] In the FTIR spectra of the microcapsule, the main peaks of SiO 2 and PDA overlapped with that of Pyr (Figure 6b). The infrared spectrum of the SiO 2 exhibited a typical peak at 1100 cm À1 (Si-O-Si) corresponding to the primary silica bonds [36] Compared with SiO 2 , the characteristic absorption peak of drug-loaded microcapsules appeared at 1717 cm À1 , which was attributed to the stretching vibration of C ¼ O in Pyr. Besides, a peak  corresponding to the stretching vibration of Si-O-Si appeared at 1100 cm À1 . In the Pyr@SiO 2 @PDA MC curve, the width of the -OH peak at 3446 cm À1 became larger, which was due to the increase in the number of oxygen-containing functional groups by the PDA coating. The results stated that Pyr was encapsulated into the microcapsule. Also, no new IR peaks indicated no chemical reactions occurred. [37] Studies of the UV-shielding properties of the PDA layer Statistically, plenty of applied pesticides are lost due to hydrolysis and photolysis. [38] Effectively encapsulation can protect the active ingredients and prolonging the duration. The UV-shielding properties of the PDA carrier was studied in ethanol/water solution with different samples. Figure 7 shows the photolysis curves of Pyr TC, Pyr@SiO 2 MC and Pyr@SiO 2 @PDA MC. In the first 2 h, the photodegradation rate of Pyr TC was 2.18 times and 2.84 times than that of Pyr@SiO2 MC and Pyr@SiO2@PDA MC. The photodegradation rate of the unencapsulated Pyr was significantly faster than that of the encapsulated Pyr. The photodegradation rate increased significantly within 24 h, and the photodegradation rate of Pyr TC, Pyr@SiO 2 MC and Pyr@SiO 2 @PDA MC were 85.23%, 69.26% and 39.98%, respectively. However, the content of Pyr in Pyr TC cannot be detected after 24 h. Therefore, microencapsulation significantly reduces the photolysis rate of the active ingredients. After 120 irradiation, the photolysis rate of Pyr@SiO 2 MC exceeded 80%, which was 1.25 times than that of Pyr@SiO 2 @PDA MC. These results clearly showed that the second coating with PDA can block the transmission of ultraviolet light, giving the microcapsules more excellent anti-photolysis performance. In addition, Tong found that graphene oxide (GO) with PDA layer had obvious adhesion properties. [39] When PDA coating was added, the persistence of pesticides on cucumber leaves was improved after simulated rain-wash test. Therefore, PDA carrier was an ideal carrier to extend the service life of pesticides.

Drug release property
The cumulative release curves of Pyr from TC, Pyr@SiO 2 MC and Pyr@SiO 2 @PDA MC are shown in Figure 8. In the first 12 h, the release rate of microcapsule without PDA coating was close to that of Pyr TC, but much higher than that of Pyr@SiO 2 @PDA MC, and their cumulative release rates were 33%, 38.68% and 10.34% respectively (Figure 8a). This may be due to the large difference between the internal and external concentrations of the microcapsules in the initial release of the drug. In addition, without the secondary coating of PDA, the drugs on the surface and shallow layer of the microcapsules are easily released quickly under the thrust of different concentrations. After 120 h, the Pyr released from Pyr@SiO 2 MC was over 72% w/w; however, the Pyr@SiO2@PDA MC released about 15% w/w of Pyr. It seems that Pyr@SiO2@PDA MC liberated much lesser Pyr molecules than Pyr@SiO2 MC at the same period. Result revealed that both Pyr@SiO 2 MC and Pyr@SiO2@PDA MC had sustained release properties, but the former had sudden release at the early stage of release, while the PDA coated microcapsules released more slowly and had better release control effect. Due to the presence of the PDA coating, the microcapsule was a good option for controlling the longterm release of the Pyr, which also helped to extend the duration.
Fungicidal activity of Pyr@SiO 2 @PDA microcapsule The fungicidal activity of Pyr@SiO 2 @PDA MC against Pyricularia oryzae was studied using mycelium growth rate method. The concentrations of Pyr EC, Pyr@SiO 2 MC and Pyr@SiO 2 @PDA MC were set at 0.5, 1, 2 and 4 mg/L for   different agents. On the 9th day after the administration, the fungicidal activity of EC was significantly inferior to that of the Pyr@SiO 2 @PDA MC ( Figure 9). As a result of microencapsulation, both microcapsules had noticeable bioactivities against Pyricularia oryzae. Because the PDA coating had good adhesion and photothermal effect, [40] the microcapsules coated with PDA had better fungicidal activity than unencapsulated microcapsules. At a concentration of 4 mg/L, the inhibitory rate of the microcapsules was more than four times that of the EC ( Figure A2). Thus, the findings clearly indicate that the virulence of the microcapsule surpassed that of the EC, showing a very obvious sustained release effect.

Toxicity evaluation
During the experiment, no death or abnormal behavior was detected in the blank control group of zebrafish. In contrast, the death started in the treatment group on the first day. At the beginning, after contact with the high-concentration EC, the zebrafish immediately turned upside down, swam rapidly, and jumped out of the water. Subsequently, poisoned zebrafish reacted dull and the swimming speed was slow.
When fish died, their abdomen and cheeks turned reddish ( Figure 10) The LC 50 values for 24-96 h and their 95% confidence limits were expressed in Table 2. LC 50 values of Pyr EC and Pyr@SiO 2 @PDA MC in 24 h were 0.065 and 0.118 mg/L. The value of LC 50 was between 0.1 mg a.i./L and 1 mg a.i./L. Thus, according to guideline for "environmental safety evaluation tests of chemical pesticides standard" (GB/T 31270.12-2014), virulence of MC on zebrafish was high toxicity. While the Pyr EC was extreme toxicity (LC 50 Ϲ 0.1 mg a.i./L). The toxicity of MC to zebrafish was lower in the first 48 h compared with Pyr EC (Table 2). This indicated that effective encapsulation can reduce the toxicity of pesticides to non-target organisms to some extent. This conclusion was similar to the result reported by Xu et al. He used poly(2-dimethylaminoethylmethacrylate) (PDMAEMA) and chitosan (CS) fabricated CS-g-PDMAEMA microcapsules. LC 50 (24 h) values of Pyr@CS-g-PDMAEMA were 0.1020 mg/L, which was less toxic than the control. [41] While, in the next 48 h, the acute toxicity of MC against zebra fish became comparable with that of Pyr EC. LC 50 (96 h) values of Pyr EC and Pyr@SiO 2 @PDA MC were 0.049 and 0.062 mg/L, respectively.

Conclusion
In this work, we have fabricated the Pyr@SiO 2 @PDA microcapsule in a straightforward way. The pyr-loaded microcapsules with maximum drug loading of 55% demonstrated sustained release for up to 200 hours. Compared with Pyr@SiO 2 MC, it had better release control effect. Contrasting with free Pyr, PDA could effectively improve the photostability of pesticides under ultraviolet light. It comes up with an effective strategy for protecting such photounstable pesticide. The obtained microcapsule proceeded with longer duration against Pyricularia oryzae compared to traditional formulation (250 g/L Pyr EC). On the other hand, a reduction in the amount of pesticide used helps to alleviate environmental pollution. Moreover, microcapsulation could provide a degree of protection against zebrafish on the first 24 h.
Consequently, this formulation could enhance utilization efficiency while exert less unintended negative effects on environment. Therefore, successful development of Pyr@SiO 2 @PDA microcapsule has made it possible to improve its application properties.

Disclosure statement
The authors declare that they have no competing interests.

Funding
This work received financial support from the National Key Research and Development Program of China: Special project on comprehensive technology development for chemical fertilizers and pesticides to reduce application and increase efficiency (2016YFD0201200).