Polymeric Nanoparticles as a Metolachlor Carrier: Water-Based

Aug 7, 2017 - Pesticide formulation is highly desirable for effective utilization of pesticide and environmental pollution reduction. Studies of pesti...
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Polymeric Nanoparticles as a Metolachlor Carrier: Water-Based Formulation for Hydrophobic Pesticides and Absorption by Plants Yujia Tong,† Yan Wu,‡ Caiyan Zhao,‡ Yong Xu,† Jianqing Lu,‡ Sheng Xiang,† Fulin Zong,§ and Xuemin Wu*,† †

College of Science, China Agricultural University, 2 Yuanmingyuan West Road, Beijing 100083, China CAS Key Laboratory for Biomedical Effects of Nanomaterials & Nanosafety, CAS Center for Excellence in Nanoscience, National Center for Nanoscience and Technology (NCNST), 11 Beiyitiao, Zhongguancun, Beijing 100190, China § Institute for the Control of Agrochemicals, Ministry of Agriculture, Beijing 100125, China Downloaded via UNIV OF LOUISIANA AT LAFAYETTE on January 24, 2019 at 08:01:48 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

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ABSTRACT: Pesticide formulation is highly desirable for effective utilization of pesticide and environmental pollution reduction. Studies of pesticide delivery system such as microcapsules are developing prosperously. In this work, we chose polymeric nanoparticles as a pesticide delivery system and metolachlor was used as a hydrophobic pesticide model to study water-based mPEG−PLGA nanoparticle formulation. Preparation, characterization results showed that the resulting nanoparticles enhanced “water solubility” of hydrophobic metolachlor and contained no organic solvent or surfactant, which represent one of the most important sources of pesticide pollution. After the release study, absorption of Cy5-labeled nanoparticles into rice roots suggested a possible transmitting pathway of this metolachlor formulation and increased utilization of metolachlor. Furthermore, the bioassay test demonstrated that this nanoparticle showed higher effect than non-nano forms under relatively low concentrations on Oryza sativa, Digitaria sanguinalis. In addition, a simple cytotoxicity test involving metolachlor and metolachlor-loaded nanoparticles was performed, indicating toxicity reduction of the latter to the preosteoblast cell line. All of these results showed that those polymeric nanoparticles could serve as a pesticide carrier with lower environmental impact, comparable effect, and effective delivery. KEYWORDS: nanopesticides, formulation, mPEG−PLGA, absorption, metolachlor pharmaceutical field, such as drug-loaded nanoparticles for tumor therapy.11,12 Use of nanoparticles provides a wide range of solubility, biodegradability, large surface area and permeability.13,14 Tiny-sized nanoparticles such as dendrimers, carbon nanotubes, quantum dots, and fullerenes were investigated for phytotoxicity or genetic research, but they were still in their infancy in the loading of agrochemicals.15−17 In fact, the field of nanotechnology has great potential within agrochemicals application and plant systems and corresponding risk researches were carried out in recent years.18−20 During the past few years, several groups published studies on pesticide-loaded particles that facilitate sustained release of active ingredients, and some of those groups studied the pesticide effect as well.21−27 However, the application improvement in pesticide formulation and the influence of nanoparticles on pesticide delivery were rarely discussed. In nature, nanoparticles with proper size, surface properties, and particle composition influence formulation stability and pesticide delivery and have indirect influence on utilization.28−31 Moreover, solubility improvement and particle size stability contribute to a more concise composition of pesticide formulation, especially for a poorly water-soluble pesticide because they usually need solvent and cosolvent to form formulations.32 Metolachlor is widely used to

1. INTRODUCTION Pesticides, an indispensable tool to protect plants from pests and guarantee the quality of harvests, were widely used in crop protection. Unfortunately, the massive consumption of pesticides has led to environmental pollution and threats to human health.1 These detrimental effects arose from not only the low efficiency utilization of pesticide but also the organic solvent and surfactants in pesticide formulations, part of which contained volatile organic compounds (VOCs).2,3 These have led to a demand for progress in pesticide formulations with maximal efficacy and minimal detrimental effects of adjuvants.4,5 Thus, research in pesticide formulation has been directed toward the development and deployment of waterbased, granule-based, release-controlled, and multifunctional formulations.6,7 The development of formulations such as microemulsion (ME), suspension concentrate (SC), granule (GR), water dispersible granule (WDG), and microcapsule (MC) was a trend, enabling the formulation of pesticides with high utilization and less organic solvent.8,9 However, formulation diversity is not suitable for all types of pesticides and extra surfactants and other problems frequently appear in these formulations.10 Though things cannot be satisfactory to all sides, all these ameliorations are aimed at reducing pollution and maintaining the pesticide effect at least. Among methods of such endeavor, water-based copolymer nanoparticles hold a superior advantage in terms of good dispersibility and internalization. Nanoparticles have been widely used in the © 2017 American Chemical Society

Received: Revised: Accepted: Published: 7371

May 15, 2017 August 3, 2017 August 7, 2017 August 7, 2017 DOI: 10.1021/acs.jafc.7b02197 J. Agric. Food Chem. 2017, 65, 7371−7378

Article

Journal of Agricultural and Food Chemistry

Morphological inspection of the MNPs was performed by transmission electron microscopy (TEM), (HT7700, Hitachi Ltd., Tokyo, Japan). The samples were dried on copper grids overnight for observation. 2.4. Stability of MNPs in the Experiment Medium. To confirm the stability of metolachlor-loaded nanoparticles during the experiment period, the suspension of MNPs in Murashige and Skoog (MS) medium and DMEM were incubated at 25 and 37 °C, respectively, for 72 h. The concentration was the same as the maximum one in the cytotoxicity test (active ingredient 200 mg/L) and herbicide activity experiments (active ingredient 64 mg/L). The average size of MNPs was analyzed by DLS every 24 h. 2.5. Determination of Metolachlor Loading and Encapsulation Efficiency. To evaluate the encapsulation efficiency of MNPs, the resulting nanoparticles were dispersed in a certain amount of methanol followed by sonication for 30 min and measured by highperformance liquid chromatography (HPLC, Agilent 1100) with an ultraviolet detector. The determination of metolachlor was performed on a Spuril-C18 column (4.6 mm × 250 mm, 5 mm, Dikma Technologies Inc., China) with methanol−water (90/10, v/v) as the mobile phase. The sample (5 μL) was injected into the HPLC system with a constant flow rate of 1.0 mL min−1 under 230 nm wavelength. The encapsulation efficiency and pesticide loading was calculated as follows: encapsulation efficiency (EE) (%) = 100% × (quantity of metolachlor in nanoparticles)/(initial quantity of metolachlor); pesticide loading (PL) = 100% × (quantity of metolachlor in nanoparticles)/(quantity of metolachlor in nanoparticles + quantity of mPEG−PLGA) 2.6. In Vitro Release Experiment in Different Mediums. To evaluate the release behavior of metolachlor from MNPs under sinking conditions, a modified method from a previous paper was used.39 In order to investigate the way by which the release medium affects release behavior, a portion of 2 mL of nanoparticles suspension or metolachlor aqueous dispersion was added to a dialysis bag (Mw 3500 Da) which was immersed into 20 mL of three kinds of mediums, respectively: (i) MS medium, (ii) 10% acetonitrile MS medium, (iii) 1‰ sodium dodecyl sulfonate (SDS) MS medium. The centrifuge tubes were under moderate shaking at 25 °C using an orbital shaker incubator (Yate Co., Jiangsu Province, China) at 150 rpm. A volume of 1 mL of solution was taken out to be a sample and 1 mL of a fresh medium was added, respectively, at indicated time intervals in order to maintain the settled volume and unsaturated condition in the centrifuge tubes. The sampled solution was filtered through a cellulose membrane filter (diameter, 13 mm; pore size, 0.22 μm; Dikma Technologies Inc.) and then injected into the HPLC system under the same conditions mentioned in section 2.5. 2.7. Plant Model System. In our work, Oryza sativa, Digitaria sanguinalis, and Arabidopsis thaliana was chosen to be the test plants. All the seeds were sterilized and washed by sterile water as described by Mohamed H. Lahiani et al. with some modifications.40 Then Oryza sativa seeds were grown on MS medium in conical flasks. All the plants were grown in light incubator under conditions: 14 h light at approximately 250 μmol m−2 s−1 intensity, 10 h dark; 28 °C day, 25 °C night. Then germinant Oryza sativa seedlings were used for subsequent experiments. 2.8. Plant Root Cross Section Imaging Using Confocal Laser Scanning Microscopy. A portion of 15 rice seedlings were placed per conical flask on blank medium (MS medium) and the same medium supplemented with free Cy5, blank particles (contrast), Cy5 labeled nanoparticles, nanoparticles with large size and microparticles. All treatment groups had the same concentration of Cy5 (0.5 μg mL−1 medium). Then the seedlings were incubated for 24 h in dark at 25 °C and then washed by deionized water. From these growing seedlings, the cross sections of roots were cut by freezing microtome. Images of cross sections were taken with confocal laser scanning microscopy (CLSM, Carl Zeiss, Boston, MA). 2.9. Bioassay of MNPs. The germinant Oryza sativa seedlings, Digitaria sanguinalis seeds, and Arabidopsis seeds were placed on the MS medium (as blank group), MS medium with blank nanoparticles (nanoparticles without metolachlor, as control group), and the same

control Gramineae and some broadleaf weeds in domestic soybean and corn crops.33,34 However, owing to its strong hydrophobicity, most of its formulations are emulsifiable concentrates where hazardous organic solvents and surfactants are required to form the formulation and the utilization was low. In order to improve the utilization of metolachlor and minimize the detrimental effects of adjuvants, studies of metolachlor microcapsules and granules were reported. The granular and microcapsule formulation exhibited pesticide effect greater than or equal to that of emulsifiable concentrate formulation; unfortunately, it suppressed crop growth and showed greater soil persistence.35−37 Thus, there remains a need to develop better formulations of metolachlor and more generally, water-based formulations and high utilization formulations for hydrophobic pesticides. In this report, a water-based metolachlor nanoparticle formulation based on mPEG−PLGA was prepared. Nanoparticle morphology was investigated by transmission electron microscopy (TEM). Size distribution and stability was measured by dynamic light scattering (DLS). Absorption of nanoparticles into plants was observed by confocal laser scanning microscope (CLSM). Bioassays was performed to evaluate herbicidal activity of the formulation. Cytotoxicity of nanoparticles formulation was evaluated via cellular viability assays with the preosteoblast cell line. The goal was to develop a water-based nanoparticle formulation with high utilization and low adverse effect on the environment and human health.

2. MATERIALS AND METHODS 2.1. Materials. mPEG (Mw, 5 kDa)−PLGA (Mw 8 kDa, LA/GA = 75:25); mPEG (Mw, 5 kDa)−PLGA (Mw 45 kDa, LA/GA = 75:25); and mPEG (Mw 5 kDa)−PLGA (Mw 95 kDa, LA/GA = 75:25) were obtained from Daigang Biochemical Co. (Jinan, China). Metolachlor at a purity of 96% (original pesticide) was obtained from Zhejiang Heben Technology Co., Ltd. (China). DMEM culture medium and Cell Counting Kit-8 was provided by Dojindo Molecular Technologies (Tokyo, Japan). Acetonitrile and methanol at HPLC grade were supplied by Thermo Fisher Scientific Inc. Rice seeds (Oryza sativa japonica cv. Nipponbare or cv. Zhonghua11), Digitaria sanguinalis seeds, Arabidopsis seeds (Arabidopsis thaliana), and Cyanine 5 (Cy5) were provided by China Agricultural University (Beijing, China). 2.2. Preparation of Water-Based Metolachlor-Loaded Nanoparticles (MNPs). The metolachlor-loaded nanoparticles were fabricated by the dialysis method with some modified procedures.38 Briefly, 1 mL of the mPEG (Mw, 5 kDa)−PLGA (Mw 8 kDa, LA/DA = 75:25) solution (10, 20, 30 mg mL−1) in dimethyl sulfoxide with different concentrations of metolachlor (20, 10, 5, 2.5 mg mL−1) (at weight ratios of 1/1, 2/1, 4/1, 8/1, polymer/metolachlor) was added dropwise to 8 mL of deionized water, and the mixture was agitated at room temperature for 30 min. Then dialysis bags (Mw 3500 Da) were used to eliminate dimethyl sulfoxide and free metolachlor for 4 h, after which a suspension of metolachlor-loaded nanoparticles (MNPs) was obtained. The particles used as a contrast with different sizes were prepared by mPEG (Mw 5 kDa)−PLGA (Mw 45 kDa, LA/GA = 75:25) and mPEG (Mw, 5 kDa)−PLGA (Mw 95 kDa, LA/GA = 75:25) using the same method with the optimized conditions. Cy5 was added in the organic phase at a concentration of 50 μg per milliliter of dimethyl sulfoxide. All the preparation process of Cy5-labeled particles was carried out in darkness. 2.3. Characterization of the MNPs. The average size and size distribution of the MNPs were measured by dynamic light scattering (DLS), using a Zeta Sizer Nano series Nano-ZS (Malvern Instruments Ltd., Malvern, U.K.). The measurements were conducted at 633 nm with a constant angle of 90°. The nanoparticle suspension was diluted in deionized water at proper concentrations. Each sample was measured in triplicate. 7372

DOI: 10.1021/acs.jafc.7b02197 J. Agric. Food Chem. 2017, 65, 7371−7378

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Journal of Agricultural and Food Chemistry medium supplemented with MNPs, metolachlor-loaded nanoparticles with a large size (L-MNPs) and metolachlor-loaded microparticles (MMPs), respectively (as treatment groups). All the treatment groups have the same concentration of active ingredients. Then the plants were incubated for 7 days under conditions in 2.8. Then the seedling height of Oryza sativa and Digitaria sanguinalis and root length of Arabidopsis thaliana were measured. 2.10. Evaluation of Cytotoxicity Using CCK Test. To evaluate the cytotoxicity difference between metolachlor technical and MNPs, cytotoxicity assays were carried out using the cell MC3T3 preosteoblast, which was maintained in continuous DMEM culture medium (with 10% fetal bovine serum) under a humid atmosphere containing 5% CO2 at a temperature of 37 °C.41 Viable cell suspension was inoculated in 96-well plates and incubated for 24 h. Each plate contained 100 μL of cell suspension (about 5000 cells). Then the cells were exposed for 24 h to metolachlor technical and MNPs at the same concentrations of active ingredient ranging from 50 to 200 mg/L. 2.11. Statistical Analysis. Data were expressed as mean standard deviation (S.D.). Statistical analysis was determined using Student’s t test and *p < 0.05, **p < 0.01, and ***p < 0.001 were used to show statistical significance.

tration on the encapsulation efficiency and particle size distribution (Table 1). Our results showed that, with decreasing pesticide amount, particle size became smaller while the degree of encapsulation and pesticide loading decreased. With a comprehensive consideration of a certain encapsulation efficiency and particle size tiny enough, the optimal copolymer/pesticide ratio was 4/1. The optimized concentration of mPEG−PLGA was 20 mg mL−1. The size of contrast particles made by mPEG (Mw, 5 kDa)−PLGA (Mw, 45 kDa, LA/GA = 75:25) and mPEG (Mw, 5 kDa)−PLGA (Mw, 95 kDa, LA/GA = 75:25) with the optimized conditions was 478.41 and 2322.15 nm, respectively. They were metolachlorloaded nanoparticles with a large size (L-MNPs) and metolachlor-loaded microparticles (MMPs). TEM images and DLS size distribution of optimum metolachlor-loaded nanoparticles were presented in Figure 2.

3. RESULTS AND DISCUSSION 3.1. Preparation and Characterization of MetolachlorLoaded Nanoparticles. 3.1.1. Preparation Process and Size Distribution of MNPs. The preparation process of metolachlorloaded nanoparticles was illustrated in Figure 1. The organic Figure 2. TEM images (A) and DLS size distribution (B) of metolachlor-loaded nanoparticles.

The figure showed that the spherical shapes of MNPs were retained after drying and a good dispersion in aqueous solution was observed. DLS measured the hydrodynamic size of MNPs swelling in aqueous solution. As a consequence, the size determined by TEM was slightly smaller than that measured by DLS. The nanoparticles suspension formulation was translucent to transparent with no visible particles or precipitation, being a relatively stable system. Referring to Stokes’ law of resistance 1, sedimentation rate of particles in the liquid depends on the radius of the particles (r), viscosity of fluid (η), and differentials of density (ρ − ρ′), among which the radius make a big difference because of the quadratic relation. We can infer that particles with smaller size exhibit much lower sedimentation rate. These may rationalize the fact that nanoparticles could show relative stability without involvement of surfactants. In this way, the “solubility” of metolachlor in water was also improved.

Figure 1. Preparation procedure of metolachlor-loaded nanoparticles.

phase with mPEG−PLGA and metolachlor was injected into the aqueous phase under magnetic stirring. With the rapid diffusion of the organic solvent, polymer deposition occurred immediately on the interface between water and the organic solvent, leading to instantaneous formation of nanoparticles.42 The hydrophilic moieties of the copolymer are outside and hydrophobic ones with pesticide are inside. After nanoparticles formation, the organic solvent and free pesticide were removed by dialysis. Some preparation factors are crucial to formation of the nanoparticles suspension. In the work, we studied the effect of different polymer/pesticide ratio and copolymer concen-

Table 1. Effect of Copolymer/Pesticide Weight Ratio on the Encapsulation Efficiency, Pesticide Loading, and Particle Size Distribution

a

sample

copolymer [mg mL−1]

metolachlor [mg mL−1]

1 2 3 4 5 6 7 8

20 20 20 20 30 30 10 10

20 10 5 2.5 3.75 2.5 2.5 1.25

average size [nm] 125.6 121.3 97.87 90.90 116.3 128.7 90.49 91.03

± ± ± ± ± ± ± ±

3.3 0.8 5.5 2.1 3.7 5.0 4.8 5.3

PDIa 0.100 0.149 0.128 0.121 0.208 0.127 0.209 0.212

± ± ± ± ± ± ± ±

0.011 0.005 0.020 0.031 0.016 0.004 0.005 0.033

EEb [%]

PLc [%]

± ± ± ± ± ± ± ±

46.37 ± 3.39 28.04 ± 2.58 14.95 ± 1.20 7.80 ± 1.04 7.45 ± 2.11 2.96 ± 0.86 4.50 ± 1.19 2.94 ± 2.41

86.47 77.92 70.31 68.58 64.44 36.55 21.05 24.26

3.12 6.43 3.05 4.91 5.25 6.16 2.84 4.11

Polydispersity index. bEncapsulation efficiency. cPesticide loading. 7373

DOI: 10.1021/acs.jafc.7b02197 J. Agric. Food Chem. 2017, 65, 7371−7378

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Journal of Agricultural and Food Chemistry Stokes law:

v = 2gr 2(ρ − ρ′)/9η

(1)

3.1.2. Infrared Spectrum of MNPs. In order to confirm pesticide loading, FTIR spectra of metolachlor, mPEG−PLGA, and MNPs were measured and shown in Figure 3. The peaks at

Figure 4. In vitro release profile of metolachlor from MNPs in different mediums.

Table 2. Constants of Fitting Korsmeyer−Peppas Model to the Release of Metolachlor from MNPs under Different Conditions

Figure 3. FTIR spectra of metolachlor, mPEG−PLGA, and metolachlor-loaded mPEG−PLGA nanoparticles (MNPs).

2875 and 1758 cm−1 are attributed to C−H stretching of CH3 and ester CO stretching, respectively. The primary absorption bands for metolachlor were presented in the region of 1672 and 2926 cm−1. C−H stretching and the aromatic group were responsible for these peaks, respectively. The peaks at 2883 cm−1, 1760 and 1668 cm−1 in the spectra of MNPs represent the stretching of methyl, the stretching vibrations of the carbonyl group from mPEG−PLGA, and the aromatic group from metolachlor, respectively. As a whole, the spectrum of MNPs contained not only characteristic peaks of mPEG− PLGA but also metolachlor, and in consideration of the TEM image and measurement of encapsulation efficiency, we considered MNPs were successfully prepared. 3.2. In Vitro Release of MNPs in Different Mediums. The release profile of metolachlor from MNPs was investigated at 30 °C in MS culture medium, 10% acetonitrile MS culture medium, and 0.1% SDS MS culture medium, respectively. The addition of acetonitrile changed the polarity of the medium and the incorporation of SDS represented the situation with surfactants. These mediums provided the different environments to influence the release behaviors of pesticide. As shown in Figure 4, approximately 48%, 51%, and 60% of metolachlor can be released from MNPs within the first 24 h in MS culture medium, 10% acetonitrile MS culture medium, and 0.1% SDS MS culture medium, respectively, which were followed by continuous slow release behaviors. In comparison to metolachlor technical aqueous dispersion, MNPs showed sustained release behaviors. The rapid release in the first several hours was related to metolachlor absorbed/encapsulated on or near the nanoparticles’ surface.43 The sustained release indicated the release was dependent on pesticide diffusion and/or matrix erosion. 44 The release data were analyzed by fitting Korsmeyer−Peppas model “Mt/M∞ = ktn” and the results were presented in Table 2. All the R2 exceeded 0.9. The release

medium

n

lg k

R2

MS 10% acetonitrile MS 0.1% SDS MS

0.1947 0.2409 0.2054

7.2655 5.6484 7.3238

0.9314 0.9458 0.9251

mechanism could be inferred by the value of n. The n values in Table 2 were less than 0.45. That means diffusion was the main mechanism of MNPs during the 72 h of release experiment. It was revealed by calculated data that medium containing SDS promoted faster metolachlor release than pure MS medium and 10% acetonitrile MS medium. The water uptake of mPEG−PLGA, based on contact angle between water and copolymer, has an influence on metolachlor release.45,46 Whereas SDS, a surfactant, enhanced the contact between nanoparticle surface and water to contribute to the result. 3.3. Stability of MNPs in Plant Culture Solution. The stability of MNPs was first examined under conditions similar to those used in absorption experiment, CCK test, and bioassay test. We measured the size of MNPs at indicated time intervals in 72 h. The obtained data in Figure 5 showed that no significant change of MNPs size was observed for up to 3 days (p > 0.05), indicating the stability of MNPs during the experiment period. 3.4. Absorption Experiment of MNPs into Rice Seedlings. Figure 6 showed photographs of the cross section of roots of untreated rice seedlings, rice seedlings incubated with free Cy5, and Cy5-labeled nanoparticles/microsized particles for 1 day, respectively. As shown in the figure, no fluorescence was observed in the untreated root at 633 nm, and the root treated with free Cy5 had hardly any fluorescence. It revealed that when Cy5 was released from nanoparticles, it could not induce fluorescence in the root. It was only visible in the root when loaded in the nanoparticles indicating the whole carrier system was in the root. A distinct fluorescence was observed in the root treated with Cy5-labeled nanoparticles. In contrast, rice seedlings treated with nanoparticles with large size and microparticles had invisible fluorescence. It shows clearly that Cy5-labeled nanoparticles have permeated the rice roots. The reports about the internalization of different nanoparticles 7374

DOI: 10.1021/acs.jafc.7b02197 J. Agric. Food Chem. 2017, 65, 7371−7378

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Journal of Agricultural and Food Chemistry

Figure 5. Stability of metolachlor-loaded nanoparticles (MNPs) in deionized water, MS medium, and DMEM.

into plants, such as fullerene, carbon nanotube, nanosilicon, and Ag, have confirmed that nanomaterials could be internalized by plants.16,47,48 Furthermore, Liu et al. reported that the internalization of the pesticide delivery system into the target organism can keep the bioactivity of the pesticide.49 In this work, the hydrophobic droplets of metolachlor was “surface modified” when loaded into the polymeric nanoparticles. The tiny size and hydrophilic segment of mPEG−PLGA nanoparticle made metolachlor “soluble” in water. Then the metolachlor-loaded nanoparticles penetrated into the plant after root exposure.50 In consideration of the thick cell wall, the tiny wall pore on it, and the photographs of the cross section of roots, the possible way by which nanoparticles internalize into plant was the apoplast way.51 This provided a pathway by which pesticide formulations could enter the plant after root exposure. In this process, metolachlor was internalized along with the uptake of water by plant, increasing the absorption of metolachlor even it was not released from the carriers. The actual absorption mechanism still needs to be studied in future work. 3.5. Bioassay Test of MNPs. The inhibition of seedlings by the prepared nanoparticles was shown in Figure 7. As shown in the control group, mPEG−PLGA nanoparticles without metolachlor exhibited negligible inhibition to plants. Metolachlor is used in the soybean field to control Gramineae weeds and some broad-leaved weeds. The seedling height and root length data showed that the MNPs treatment groups exhibited higher inhibition on Oryza sativa and Digitaria sanguinalis seedlings than L-MNPs and MMPs under relatively low concentrations. However, it showed no significant inhibition or even less inhibition comparing to L-MNPs and MMPs treatment groups under relatively high concentrations. On Arabidopsis thaliana, herbicide activity improvement was not all significant compared with L-MNPs and MMPs. The above results showed that MNPs did have efficiency improvement, but it had its limited scope. The controlled release maintained the persistence effectiveness of MNPs during the experiment time. MNPs exhibited high-activity owing to its absorption into plants at low concentrations. This process along with water absorption guaranteed the utilization of metolachlor. More mechanism and applied range need to be studied in the future work.

Figure 6. Plant root cross section imaging using confocal laser scanning microscope. The left rank was recorded at 633 nm by a confocal microscope. The middle rank was in the bright field mode using white light, and the right rank was the superposition of the two: (A) untreated rice seedlings, (B) rice seedlings incubated with free Cy5, (C) rice seedlings incubated with Cy5-labeled nanoparticles prepared by mPEG (Mw, 5 kDa)−PLGA (Mw, 8 kDa), (D) rice seedlings incubated with nanoparticles prepared by mPEG (Mw, 5 kDa)−PLGA (Mw, 45 kDa), (E) rice seedlings incubated with microsized particles prepared by mPEG (Mw, 5 kDa)−PLGA (Mw, 95 kDa).

3.6. Cytotoxicity of MNPs to Mammal Cell Line. As a pharmaceutical adjuvant certified by FDA, mPEG−PLGA− based nanoparticles may exhibit excellent toxicity reduction in mammals.52 In this work, apart from the morphological characterizations and absorption into plants, the in vitro cellular viability test was performed to make a preliminary exploration about the cytotoxicity of metolachlor-loaded nanoparticles and metolachlor technical. The preosteoblast cell line (MC3T3) was employed to evaluate the percentage of cell viability at test concentrations. The results of cell viability showed that metolachlor-loaded nanoparticles are less toxic than the free metolachlor (Figure 8). Nanomaterials with good biocompatibility may have contributed to this result. 7375

DOI: 10.1021/acs.jafc.7b02197 J. Agric. Food Chem. 2017, 65, 7371−7378

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Journal of Agricultural and Food Chemistry

Figure 7. Herbicide activity of MNPs, L-MNPs, and MMPs. (A) Herbicide activity on Oryza sativa and (B) Oryza sativa under metolachlor concentration 0.1 mg/L. (C) Herbicide activity on Digitaria sanguinalis. (D) Digitaria sanguinalis under metolachlor concentration 16 mg/L. (E) Herbicide activity on Arabidopsis thaliana. (F) Arabidopsis thaliana under metolachlor concentration 0.02 mol/L.

common formulations. In this way, “water-solubility” enhancement of strongly hydrophobic pesticide, such as metolachlor, is realized and it contributes to safer and cleaner pesticide formulation. The results of TEM and DLS revealed the

In summary, mPEG−PLGA nanoparticles were used as a carrier for metolachlor. The water-based mPEG−PLGA nanoparticles with proper size and surface property eliminated the requirement of hazardous organic solvent and surfactants in 7376

DOI: 10.1021/acs.jafc.7b02197 J. Agric. Food Chem. 2017, 65, 7371−7378

Article

Journal of Agricultural and Food Chemistry

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Figure 8. Cytotoxicity of MNPs and metolachlor-tech at different concentrations: (a) MNPs and (b) metolachlor technical.

morphology and size distribution. Sustainable release of metolachlor was observed from the polymeric nanoparticles. Furthermore, absorption of the resulting nanoparticles into rice suggested a possible way by which pesticide formulation could transmit into the plant and guarantee utilization. Resulting nanoparticles exhibited higher herbicide activity than those in non-nano forms at relatively low concentrations on Oryza sativa and Digitaria sanguinalis. Finally, lower toxicity to MC3T3 cell line was observed when metolachlor was loaded in the nanoparticles, indicating a possible way of toxicity reduction. As a whole, this work provided a potential solution to mitigate the pollution and risk of common pesticide formulations. It also demonstrated the promising applications of polymeric nanoparticles in pesticide delivery and increase the utilization of pesticide. Further work will be needed to study the mechanism and applications of pesticide-loaded nanoparticles.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Yan Wu: 0000-0001-8508-7077 Xuemin Wu: 0000-0002-6606-2074 Funding

This work was supported by The National Key Research and Development Program of China (Grant 2017YFD 0200301). We thank Prof. Zhenhua Zhang (China Agricultural University) for providing some fluorescent reagents (National Key Technologies R&D Program of China, Grant 2015BAK45B01, CAU). Notes

The authors declare no competing financial interest.

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DOI: 10.1021/acs.jafc.7b02197 J. Agric. Food Chem. 2017, 65, 7371−7378

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