Preparation of MSNs-Chitosan@Prochloraz Nanoparticles for

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Preparation of MSNs-chitosan@prochloraz nanoparticles for reducing toxicity and improving release properties of prochloraz You Liang, Chen Fan, Hongqiang Dong, Wenbing Zhang, Gang Tang, Jiale Yang, Na Jiang, and Yongsong Cao ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/acssuschemeng.8b01511 • Publication Date (Web): 13 Jul 2018 Downloaded from http://pubs.acs.org on July 16, 2018

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ACS Sustainable Chemistry & Engineering

Preparation of MSNs-chitosan@prochloraz nanoparticles for reducing toxicity and improving release properties of prochloraz

You Liang, Chen Fan, Hongqiang Dong, Wenbing Zhang, Gang Tang, Jiale Yang, Na Jiang, Yongsong Cao* College of Plant Protection, China Agricultural University, Beijing, China *Corresponding author: NO.2 Yuanmingyuan West Road, China Agricultural University, Beijing, China, 100193 Telephone number: 86-10-62734302 (O), 86-10-62734302 (FAX) Email: [email protected], [email protected]

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ABSTRACT: Nanotechnology-based pesticide formulations can improve the utilization rate of pesticides and reduce their negative effects on the environment. In this work, prochloraz was encapsulated within the pores of mesoporous silica nanoparticles (MSNs) attached covalently chitosan on the surface as

gatekeepers

via a silane

coupling agent to

prepare

MSNs-chitosan@prochloraz nanoparticles. The results demonstrated that the obtained nanoparticles had a relatively high loading efficiency of prochloraz (25.4% w/w) and enhanced the light stability of prochloraz effectively. The nanoparticles showed excellent esterase and pH dual-responsive properties with controlled release behavior. And the biological activity survey conformed that the acid and enzyme produced by infected fruit can easily open the “gate” guarded by chitosan to achieve esterase and pH triggered on-demand pesticide release. Compared with prochloraz emulsifiable concentrate, preharvest application of MSNs-chitosan@prochloraz nanoparticles possessed a longer duration and a better antifungal activity against citrus diseases. The toxicity of the nanoparticles to zebrafish was reduced more than 6-fold compared with that of prochloraz technical. These results demonstrated that the MSNs-chitosan@prochloraz nanoparticles had potential as an environmentally-friendly preharvest treatment agent in agricultural application. KEYWORDS: prochloraz; mesoporous silica nanoparticles; dual-stimuli responsive; controlled release; citrus diseases; acute toxicity

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INTRODUCTION In modern agriculture, pesticides are widely used for improving the yield and quality of crop production. Approximately 4.6 million tons of pesticides are applied to prevent and control pests every year all over the world.1 However, less than 0.1% of the sprayed pesticides finally act on the harmfully biological targets owing to degradation, evaporation, leaching, and run off. Extensive and continuous spraying of pesticides over years has caused serious environmental pollution and health problems.2 Currently, controlled release formulations based on polymeric carriers have been developed extensively, which could not only impair pesticides toxic effects on the environment but also enhance pesticides utilization efficiency by prolonging their effective duration and reducing their evaporation and degradation.3 Unfortunately, polymer delivery system is not satisfactory, because some polymers can easily form hydrophobic structure and semicrystalline morphology, which is difficult to be degraded in the environment and causes secondary pollution.4 In recent years, controlled release formulations based on mesoporous silica delivery systems have attracted much attention due to their high loading capability, low production cost, excellent biocompatibility, and facile multi-functionalization.5 However, pure mesoporous silica carriers have been observed to have premature or burst pesticide release within several hours, which limits their practical application.6 To solve these problems, a series of double-shelled silica pesticide microcapsules were developed using natural polymers as the outer shell materials.7,8 However, the preparation of double-shelled silica pesticide microcapsules requires a precisely controlled synthetic process.9 In addition, due to lack valid trigger to control pesticide release, the initial concentration of active ingredient released from microcapsules is too low to control pests effectively.10,11 The employment of functional gatekeepers to block the pores of mesoporous silica nanoparticles (MSNs) are probably more convenient for the intelligent release of pesticide in the target site. For instance, Yi et al. established a novel redox-responsive functionalized MSNs using short-chain molecules as gatekeepers to control plant hormone (salicylic acid) release.12 Niedermayer et al. recently developed an enzymatic responsive controlled release insecticide using hydrolyzed α-CD derivative as gatekeepers on the surface of MSNs.13 These responsive release pesticide systems can improve the utilization and realize the precise positioning of 3



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pesticide, which has a great potential to be applied in the field of agriculture. Citrus, one of the largest production fruit in the world, is grown in over 100 countries and the production reached 131 million tons in 2012.14 However, citrus fruit is easily infected by microbial pathogens due to its high amount of water content, nutrients, and low pH value.15 Green mold rot (Penicillium digitatum (Pers.: Fr.) Sacc.), blue mold rot (Penicillium italicum Wehmer), and sour rot (Geotrichum candidum (syn. Geotrichum citri-aurantii)) are the three most devastating pathogenic fungi affecting citrus fruit, which can decrease local pH value of fruit and produce a large amount of hydrolase (such as esterase and pectinase) at the site of infection during decay development.16 In addition, the diseases may reduce the production of citrus fruit by as much as 30% during storage. These pathogenic fungi usually have been managed by pretharvest or postharvest treatment using fungicides such as prochloraz, imazalil and thiabendazole in citrus fruit.17,18 Although postharvest treatment by pesticides may be an effective way to control postharvest pathogens, a preharvest treatment is a more practical strategy because it can decrease the potential for damage and injuries which may occur during the postharvest treatments.19 Therefore, it is necessary to develop a preharvest treatment agent for control postharvest fruit diseases. Prochloraz

(N-propyl-N-[2-(2,4,6-trichlorophenoxy)ethyl]imidazole-1-carboxamide),

a

broad-spectrum imidazole fungicide, has superior antifungal activity against fruit postharvest pathogens. However, prochloraz is sensitive to sunlight, which leads to its degradation and failure to control the occurrence of plant diseases with enough time in the field.7 Meanwhile, the wide application of prochloraz may cause some adverse effects in the aquatic environment.20 Therefore, it is an urgent task to construct a novel stimuli-responsive pesticide delivery system, which can reduce the photodegradation, prolong the effective duration, the abate toxicity and realize controlled release of prochloraz. Herein,

a

silane

coupling

agent

3,3-dimethoxy-9-methyl-8-oxo-2,7-dioxa-11-thia-3-

silatetradecan-14-oic acid (DMTSO) was synthesized by thiol-ene click chemistry. The as-prepared silane coupling agent was immobilized on MSNs and cross-linked with chitosan to construct esterase and pH dual-stimuli responsive pesticide system. And the pesticide loading of MSNs-chitosan@prochloraz nanoparticles; the effects of temperature, pH values, and enzymes on 4


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the release properties; the bioactivity, and acute toxicity were also investigated.

EXPERIMENTAL Materials. Prochloraz technical (97% purity) and emulsifiable concentrate (EC, 25%) were supplied from Nanjing Red Sun Co., Ltd. (Nanjing, China). Cetyltrimethylammonium bromide (CTAB, 99%), 3-(trimethoxysilyl)propyl methacrylate (TMSPMA, 98%), 3-mercaptopropionic acid (MPA), ammonia aqueous solution, tetraethyl orthosilicate (TEOS), acetone, hydrochloric acid, acetic acid, N-hexane, acetone, and ethanol were analytical chemicals provided by Sinopharm Chemical Reagent Beijing Co., Ltd (Beijing, China). Esterase (≥20 U/g; from Rhizopus oryzae), benzoin dimethyl ether (DMAP), chitosan (Mw=160 kDa, deacetylation degree≥75%), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC●HCl), N-hydroxysuccinimide (NHS) were purchased from Sigma Aldrich. Acetonitrile (HPLC grade) was supplied from J. T. Baker. Deionized water used to prepare all solutions was collected from a Millipore water purification system (Milli-Q water).

Preparation of MSNs-chitosan@prochloraz nanoparticles. Synthesis of DMTSO. In a typical experiment, 0.1 mmol of photoinitiator DMAP was dissolved in 5 mmol of TMSPA and then reacted with 5 mmol of MPA. The mixture was irradiated at room temperature using a UV

light (365 nm) for 1 h under nitrogen atmosphere. After the reaction, the product was washed three times with 30 mL of N-hexane. The obtained colorless oil was DMTSO. Synthesis of mesoporous silica nanoparticles (MSNs). The MSNs were prepared by using the modified reference methods.21 First, 0.35 g of CTAB, 60 mL of ethanol, 130 mL of deionized water, and 1 mL of ammonia aqueous solution (25 wt%) were mixed and stirred for more than 30 min. After the reaction mixture turned into a clear surfactant solution, 2.2 mL of TEOS was added with rapid stirring and the resulting solution was heated to 45°C. After being reacted for 24 h, the white precipitates were centrifuged at 9,300 g for 10 min and purified by ethanol several times. To remove the surfactant (CTAB), 0.5 g of the product was transferred to 100 mL of ethanol solution containing 6 mL of concentrated hydrochloric acid (37 wt%). After stirring at 70°C for 24 h, the surfactant-free MSNs were washed with ethanol three times and dried overnight under high vacuum.

5



Synthesis of MSNs-COOH. Approximately 100 mg of MSNs was re-dispersed in 20 mL of anhydrous ethanol, then 600 µL of DMTSO (50% in anhydrous ethanol) was added into the suspension. The mixture was stirred for 16 h at room temperature and carboxy-functionalized MSNs (MSNs-COOH) were collected by centrifugation. After that, the samples were washed with ethanol several times and dried under vacuum.

Synthesis of MSNs-chitosan. To obtain chitosan solution, 1.0 g of chitosan was added to 50 mL of 2% of acetic acid solution (v/v) with continuous stirring for 6 h. Approximately 100 mg of MSNs-COOH in 10 mL of phosphate buffer solution (pH 6.0) was sonicated for 30 min. Then, 100 mg of EDC and 60 mg of NHS was added into the previous suspension to activate the carboxylic group of MSNs. After stirring the mixture for 20 min, 2.5 mL of chitosan solution was added dropwise into the reaction system with vigorous stirring. After further stirring at room temperature for 12 h, the chitosan end-capped MSNs (MSNs-chitosan) were isolated by centrifugation, washed three times with phosphate buffer solution (pH 7.0). Then, the dry MSNs-chitosan was collected after drying for 24 h in a vacuum oven at 45°C.

Characterization. The morphology and mesoporous structure of the samples were measured using a JEM-2100 transmission electron microscope (TEM) (JEOL, Japan) at an acceleration voltage of 200 kV. The variation of the functional groups present on the samples were recorded on a Nexus 670 Fourier transform spectrophotometer (FTIR) (Thermo-Fisher, USA) using the KBr pellet technique. The amount of prochloraz loaded in MSNs-chitosan was carried out by a SDT-Q600 thermogravimetric analyzer (TA Instruments-Waters LLC, USA) over a range of temperature from 25 to 800 °C at a rate of 10°C/min under nitrogen atmosphere. The zeta potential of the samples were performed by a Nano-zs90 Nanosizer (Malvern Instruments, UK). The nitrogen adsorption/desorption experiment was tested using a TriStar II 3020 analyzer (Micromeritics Instrument Corporation, USA). The surface areas and pore size distributions of synthesized nanoparticles were calculated by using Brunauer−Emmett−Teller (BET) approach and Barrett−Jyner−Halenda (BJH) method, respectively. The concentration of prochloraz was monitored by high performance liquid chromatography (HPLC) with an ultraviolet detector (Shimadzu, Japan) at 220 nm. The chromatographic separation was conducted by a Kromasil ODS C18 column (250 mm × 4.6 mm, 5 mm; Dikma, 6


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USA) equipped with a guard column (4 mm × 3 mm). A flow rate of 1 mL/min was carried out with a mobile phase composition of acetonitrile and 1 g/L acetic acid (70:30, v/v). The injection volume was 20 µL and the column temperature was at room temperature. All the solvents used for HPLC measurements were filtered with a 0.45 µm membrane filter.

Prochloraz

loading

and

chitosan

functionalization

of

MSNs

(MSNs-chitosan@prochloraz nanoparticles). Approximately 100 mg of MSNs-COOH was ultrasonically dispersed in 5 mL of acetone, and then a certain amount of prochloraz was introduced into the solution. The mixture was sealed and stirred at 25°C for 24 h. After that, the suspension was dried in a vacuum oven overnight to remove the acetone. The resulting solids (MSNs-COOH@prochloraz nanoparticles) were washed with deionized water and dried under vacuum. Then, the obtained MSNs-COOH@prochloraz nanoparticles were added to 10 mL of phosphate buffer solution (pH 6.0) and activated with 100 mg EDC and 60 mg NHS. The mixture was stirred at room temperature for 20 min, 2.5 mL of chitosan solution was introduced into the suspension and stirred at room temperature for 12 h. Finally, MSNs-chitosan@prochloraz nanoparticles were collected by centrifugation at 9,300 g for 10 min and further washed with phosphate buffer solution (pH 7.0) five times. The amount of the free prochloraz in the supernatant was measured by HPLC. The pesticide loading efficiency (PLC) was calculated as follow: PLC (%) =

initial amount of prochloraz -amount of free prochloraz × 100 amount of the nanoparticles

(1)

Controlled release kinetics. Approximately 10 mg of MSNs-chitosan@prochloraz nanoparticles was suspended with 100 mL of phosphate buffer solution (pH 7.0) and agitated at 25, 37, and 50°C for 30 days. At predetermined time intervals, 1.0 mL of the suspension was collected and centrifuged for 10 min at 11,000 g to obtain supernatant without MSNs-chitosan@prochloraz nanoparticles. Then the amount of prochloraz released from the nanoparticles was determined by HPLC. In

the

pH

triggered

prochloraz

release

experiment,

approximately

10

mg

of

MSNs-chitosan@prochloraz nanoparticles was dispersed in different pH phosphate buffer solutions (7.0, 6.0, 5.0, and 4.0). Subsequently, 1 mL of the solution was taken out at different 7



intervals. After centrifugation at 11,000 g for 10 min, the amount of released prochloraz in the supernatant was measured by HPLC. In the dual-stimuli responsive prochloraz release experiment, 10 mg of MSNs-chitosan@prochloraz nanoparticles was dispersed in 100 mL of four different release media (i: pH 5.0 in the presence of 1 mg/L esterase, ii: pH 5.0 in the absence of 1 mg/L esterase, iii: pH 7.0 in the presence of 1 mg/L esterase and iv: pH 7.0 in the absence of 1 mg/L esterase) and placed in shaker at 25°C for 30 days. The amount of prochloraz released into the solution was measured at each time interval. The release tests were repeated three times. The data of pesticide release were analyzed by the Ritger and Peppas equation: Mt = kt n Mz

(2)

where Mt/Mz represents the amount of prochloraz released at time t; k is the kinetic constant which depends on incorporate characteristics of prochloraz and the nanoparticicles system; and n is the diffusional exponent that characterizes the mechanism of pesticide release. For the spherical samples, n≤0.5 corresponds to a Fickian diffusion, 0.5 < n < 0.89 to a non-Fickian or anomalous diffusion, n≥0.89 to a zero-order transport.22

Light stability of MSNs-chitosan@prochloraz nanoparticles. To evaluate the light stability of MSNs-chitosan@prochloraz nanoparticles under UV irradiation, approximately 30 mg of MSNs-chitosan@prochloraz nanoparticles was dispersed in 30 mL of deionized water, and the suspension was packed in quartz tube. The tube was disposed at a fixed distance and exposed to a 300 W (Emax=365 nm) UV lamp. At various time intervals, an aliquot of 500 µL was removed from each tube, and the concentration of prochloraz in each sample was determined by HPLC. The stability of prochloraz technical was analyzed by the same procedure. To eliminate the influence of other factors, quartz tubes containing the samples, wrapped in aluminum foil, were also measured (dark controls). The experiment was performed in triplicate.

Bioactivity. Preharvest treatments. The prochloraz EC (100, 200, 400 mg/L) and MSNs-chitosan@prochloraz nanoparticles (100, 200, 400 mg/L) were sprayed on citrus (Citrus reticulata cv. Shatangju) trees by using a hand-sprayer until the trees were wet to runoff (about 2 L/tree), where all the concentrations were determined by the mass of prochloraz. Additional trees 8


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were sprayed with water as the control. The fruit was harvested at 10 days after treatment and each treatment was composed three replications of fifty fruit. The fruit was packed in cartons, stored at 20°C and 95% RH. The numbers of decayed fruit were counted after 30 days. Weight loss of citrus fruit in each treatment was measured by monitoring weight changes after 30 days storage at 20 °C. The soluble solids content in the fruit was determined by using a hand refractometer (AO Scientific Instrument, USA). Postharvest treatments. To further determine the inhibitory effects of prochloraz EC and MSNs-chitosan@prochloraz nanoparticles on pathogens, the decay severity of citrus fruit was investigated by postharvest treatment. In this experiment, citrus fruit was wounded with a steel rod to make a 1 mm wide and 2 mm deep wound, and 10 µL aliquot of mixed spore suspension containing 106 conidia/mL of P. digitatum, P. italicum and G. candidum was injected into each wound with a pipette. The fruit was dipped in different concentrations of prochloraz EC (100, 200, 400 mg/L) and MSNs-chitosan@prochloraz nanoparticles (100, 200, 400 mg/L) for 60 s, where all the concentrations were determined by the mass of prochloraz. Control fruit was treated with sterile distilled water. Each treatment included three replicates, with fifty fruit per replicate. Severity of decay for each fruit was evaluated after 10 days at 20°C (0, no sign of disease; 1, browning area lager than＜10%; 2, browning area 10-25%; 3, browning area 25-50%; 4, browning area 50-75%; 5, browning area＞75%).23

Acute toxicity to adult zebrafish. Zebrafish (Danio rerio) is well known as an ideal vertebrate model to assess the toxicity of pesticides in the environment due to its inexpensiveness, short lifecycle, and easy maintenance.24 In this experiment, the zebrafish was used as an efficient environment monitoring model to evaluate the potential toxicity of MSNs-chitosan@prochloraz nanoparticles. Adult wild type zebrafish (length 3 ± 0.5 cm; wet weight 0.25 ± 0.05 g) were purchased from a tropical fish wholesale (Beijing, China) and kept in flow through system containing charcoal-filtered water at 26°C with a photoperiod of 14:10 h (light/dark). After acclimating for at least two weeks in chlorine-free tap water (pH 6.8), ten fish were exposed individually in 2 L beakers containing 1.0, 2.0, 3.0, 4.0, 8.0, 12.0, 16.0, 32.0, and 64.0 mg/L of prochloraz technical and MSNs-chitosan@prochloraz nanoparticles, where all the concentrations were determined by the mass of prochloraz. Triplicate samples were applied to each control and 9



exposure treatment. All the tests were monitored daily to access the acute toxicity of the pesticides to zebrafish.

Data analysis. All statistical analyses were conducted using SPSS 23.0 statistical analysis software (SPSS, Chicago, IL, USA). The median lethal concentrations (LC50) values were calculated by probit regression model. The data were analyzed by Duncan multiple range test (p

Preparation of MSNs-Chitosan@Prochloraz Nanoparticles for

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