Polymeric Nanoparticles as a Metolachlor Carrier: Water-Based

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Polymeric nanoparticles as 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 J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b02197 • Publication Date (Web): 07 Aug 2017 Downloaded from http://pubs.acs.org on August 10, 2017

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Journal of Agricultural and Food Chemistry

Polymeric nanoparticles as metolachlor carrier: water-based formulation for hydrophobic pesticides and absorption by plant

Yujia Tong 1, Yan Wu 2, Caiyan Zhao 2, Yong Xu 1, Jianqing Lu 2, Sheng Xiang 1, Fulin Zong 3, and Xuemin Wu 1, * 1

College of Science, China Agricultural University, 2 Yuanmingyuan West Road, Beijing

100083, China [email protected] 2

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 3

Institute for the Control of Agrochemicals, Ministry of Agriculture, Beijing 100125, China

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Abstract:

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Pesticide formulation is highly desirable for effective utilization of pesticide and

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environmental pollution reduction. Studies of pesticide delivery system such as microcapsules

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are developing prosperously. In this work, we chose polymeric nanoparticles as a pesticide

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delivery system and metolachlor was used as a hydrophobic pesticide model to study water-

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based mPEG-PLGA nanoparticle formulation. Preparation, characterization results showed

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that the resulting nanoparticles enhanced “water solubility” of hydrophobic metolachlor and

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contained no organic solvent or surfactant which represent one of the most important sources

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of pesticide pollution. After release study, absorption of Cy5-labelled nanoparticles into rice

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roots suggested a possible transmitting pathway of this metolachlor formulation and increased

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utilization of metolachlor. Furthermore, the bioassay test demonstrated that this nanoparticle

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showed higher effect than non-nano forms under relatively low concentrations on Oryza

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sativa, Digitaria sanguinalis. In addition, a simple cytotoxicity test involving metolachlor and

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metolachlor-loaded nanoparticles was performed, indicating toxicity reduction of the latter to

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the preosteoblast cell line. All of these results showed that those polymeric nanoparticles

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could serve as a pesticide carrier with lower environmental impact, comparable effect and

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effective delivery.

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Keywords: Nanopesticides; Formulation; mPEG-PLGA; Absorption; Metolachlor

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1. Introduction

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Pesticides, an indispensable tool to protect plants from pests and guarantee the quality of

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harvests, were widely used in crop protection. Unfortunately, the massive consumption of

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pesticides has led to environmental pollution and threats to human health.1 These detrimental

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effects arose from not only the low efficiency utilization of pesticide but also the organic

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solvent and surfactants in pesticide formulations, part of which contained volatile organic

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compounds (VOCs).2,3 These have led to a demand for progress in pesticide formulations with

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maximal efficacy and minimal detrimental effects of adjuvants.4,5 Thus, research in pesticide

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formulation has been directed toward the development and deployment of water-based,

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granule-based, release-controlled, and multi-functional formulations.6,7

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The development of formulations such as microemulsion (ME), suspension concentrate

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(SC), granule (GR), water dispersible granule (WDG) and microcapsule (MC) was a trend,

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enabling the formulation of pesticides with high utilization and less organic solvent.8,9 But

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formulation diversity is not suitable for all types of pesticides and extra surfactants and other

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problems frequently appear in these formulations.10 Though things cannot be satisfactory to

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all sides, all these ameliorations are aimed at reducing pollution and maintaining the pesticide

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effect at least. Among methods of such endeavor, water-based copolymer nanoparticles hold a

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superior advantage in terms of good dispersibility and internalization. Nanoparticles have

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been widely used in the pharmaceutical field, such as drug-loaded nanoparticles for tumor

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therapy.11-12 Use of nanoparticles provides a wide range of solubility, biodegradability, large

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surface area and permeability.13,14 Tiny-sized nanoparticles such as dendrimers, carbon

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nanotubes, quantum dots, and fullerenes were investigated for phytotoxicity or genetic

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research, but they were still in their infancy in the loading of agrochemicals.15-17 In fact, the

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field of nanotechnology has great potential within agrochemicals application and plant

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systems and corresponding risk researches were carried out recent years.18-20 During the past 3 ACS Paragon Plus Environment


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few years, several groups published studies on pesticide-loaded particles that facilitate

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sustained release of active ingredients, and some of those groups studied the pesticide effect

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as well.21-27 However, the application improvement in pesticide formulation and the influence

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of nanoparticles on pesticide delivery were rarely discussed. In nature, nanoparticles with

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proper size, surface properties and particle composition influence formulation stability,

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pesticide delivery and have indirect influence on utilization.28-31 Moreover, solubility

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improvement and particle size stability contribute to a more concise composition of pesticide

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formulation, especially for poorly water-soluble pesticide because they usually need solvent

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and cosolvent to form formulations.32 Metolachlor is widely used to control Gramineae and

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some broadleaf weeds in domestic soybean and corn crops.33,34 However, owing to its strong

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hydrophobicity, most of its formulations are emulsifiable concentrates where hazardous

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organic solvents and surfactants are required to form the formulation and the utilization was

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low. In order to improve the utilization of metolachlor and minimize the detrimental effects of

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adjuvants, studies of metolachlor microcapsules and granules were reported. The granular and

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microcapsule formulation exhibited pesticide effect greater than or equal to that of

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emulsifiable concentrate formulation; unfortunately, it suppressed crop growth and showed

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greater soil persistence.35-37 Thus, there remains a need to develop better formulations of

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metolachlor and more generally, water-based formulations and high utilization formulations

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for hydrophobic pesticides.

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In this report, a water-based metolachlor nanoparticle formulation based on mPEG-

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PLGA was prepared. Nanoparticle morphology was investigated by transmission electron

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microscopy (TEM). Size distribution and stability was measured by dynamic light scattering

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(DLS). Absorption of nanoparticles into plants was observed by confocal laser scanning

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microscope (CLSM). Bioassays was performed to evaluate herbicidal activity of the

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formulation. Cytotoxicity of nanoparticles formulation was evaluated via cellular viability


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assays with preosteoblast cell line. The goal was to develop a water-based nanoparticle

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formulation with high utilization and low adverse effect on environment and human health.

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2. Method

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2.1. Materials

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mPEG (Mw:5kDa) – PLGA (Mw:8kDa, LA:GA=75:25), mPEG (Mw:5kDa) – PLGA

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(Mw:45kDa, LA:GA=75:25) and mPEG (Mw:5kDa) – PLGA (Mw:95kDa, LA:GA=75:25)

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were obtained from Daigang Biochemical Co. (Jinan, China). Metolachlor at a purity of 96%

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(original pesticide) was obtained from Zhejiang Heben Technology Co., Ltd. (China). DMEM

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culture medium and Cell Counting Kit-8 was provided by Dojindo Molecular Technologies

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(Tokyo, Japan). Acetonitrile and methanol at HPLC grade were supplied by Thermo Fisher

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Scientific Inc. (USA). Rice seeds (Oryza sativa japonica cv. Nipponbare or cv. Zhonghua11),

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Digitaria sanguinalis seeds, Arabidopsis seeds (arabidopsis thaliana) and Cyanine 5 (Cy5)

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were provided by China Agricultural University (Beijing, China).

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2.2. Preparation of water-based metolachlor-loaded nanoparticles (MNPs)

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The metolachlor-loaded nanoparticles were fabricated by the dialysis method with some

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modified procedures.38 Briefly, 1 mL of the mPEG (Mw:5kDa) – PLGA (Mw:8kDa,

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LA:DA=75:25) solution (10,20,30 mg mL-1) in dimethyl sulfoxide with different

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concentration of metolachlor (20, 10, 5, 2.5 mg mL-1) (at weight ratios of 1/1, 2/1, 4/1, 8/1,

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polymer/metolachlor) was added dropwise to 8 mL of deionized water and the mixture was

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agitated at room temperature for 30 min. Then dialysis bags (Mw 3500D) were used to

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eliminate dimethyl sulfoxide and free metolachlor for 4 h, after which a suspension of

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metolachlor-loaded nanoparticles (MNPs) was obtained. The particles used as contrast with

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different sizes were prepared by mPEG (Mw:5kDa) – PLGA (Mw:45kDa, LA:GA=75:25)

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and mPEG (Mw:5kDa) – PLGA (Mw:95kDa, LA:GA=75:25) using the same method with the 5 ACS Paragon Plus Environment


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optimized conditions. Cy5 was added in the organic phase at a concentration of 50µg per

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milliliter of dimethyl sulfoxide. All the preparation process of Cy5-labelled particles was

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carried out in darkness.

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2.3. Characterization of the MNPs

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The average size and size distribution of the MNPs were measured by dynamic light

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scattering (DLS), using a Zeta Sizer Nano series Nano-ZS (Malvern Instruments Ltd, Malvern,

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UK). The measurements were conducted at 633 nm with a constant angle of 90°. The

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nanoparticle suspension was diluted in deionized water at proper concentrations. Each sample

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was measured in triplicate.

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Morphological inspection of the MNPs was performed by transmission electron

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microscopy (TEM), (HT7700, HITACHI Ltd., Tokyo, Japan). The samples were dried on

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copper grids overnight for observation.

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2.4. Stability of MNPs in the experiment medium

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To confirm the stability of metolachlor-loaded nanoparticles during the experiment

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period, the suspension of MNPs in Murashige and Skoog (MS) medium, and DMEM was

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incubated at 25 °C and 37°C respectively for 72 hours. The concentration was the same as the

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maximum one in the cytotoxicity test (active ingredient 200 mg/L) and herbicide activity

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experiments (active ingredient 64 mg/L). The average size of MNPs was analyzed by DLS

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every 24 hours.

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2.5. Determination of metolachlor loading and encapsulation efficiency

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To evaluate the encapsulation efficiency of MNPs, the resulting nanoparticles were

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dispersed in a certain amount of methanol followed by sonication for 30 min and measured by

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high-performance liquid chromatography (HPLC, Agilent 1100, USA) with an ultraviolet 6 ACS Paragon Plus Environment

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detector. The determination of metolachlor was performed on a Spuril-C18 column (4.6

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mm×250 mm, 5 mm, Dikma Technologies Inc., China) with methanol–water (90/10, v/v) as

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the mobile phase. The sample (5 µL) was injected into the HPLC system with a constant flow

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rate of 1.0 mL min-1 under 230 nm wavelength. The encapsulation efficiency and pesticide

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loading was calculated as follows: Encapsulation Efficiency (EE) (%) = 100% × (quantity of

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metolachlor in nanoparticles) / (initial quantity of metolachlor); Pesticide Loading (PL) =

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100% × (quantity of metolachlor in nanoparticles) / (quantity of metolachlor in nanoparticles

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+ quantity of mPEG-PLGA)

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2.6. In vitro release experiment in different mediums

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To evaluate the release behaviour of metolachlor from MNPs under sink condition, a

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modified method from a previous paper was used.39 In order to investigate the way by which

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the release medium affects release behaviour, a portion of 2 mL nanoparticles suspension or

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metolachlor aqueous dispersion was added to a dialysis bag (Mw: 3500D) which was

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immersed into 20 mL of three kinds of mediums, respectively: (i) MS medium, (ii) 10%

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acetonitrile MS medium, (iii) 1‰ Sodium Dodecyl Sulfonate(SDS) MS medium. The

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centrifuge tubes were under moderate shaking at 25 °C using an orbital shaker incubator

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(Yate Co., Jiangsu Province, China) at 150 rpm. 1 mL of solution was taken out to be a

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sample and 1 mL of a fresh medium was added respectively at indicated time intervals in

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order to maintain the settled volume and unsaturated condition in the centrifuge tubes. The

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sampled solution was filtered through a cellulose membrane filter (diameter: 13 mm; pore

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size: 0.22 µm; Dikma Technologies Inc.) and then injected into the HPLC system under the

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same conditions mentioned in section 2.5.

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2.7. Plant model system



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In our work, oryza sativa, digitaria sanguinalis and arabidopsis thaliana was chosen to be

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the test plants. All the seeds were sterilized and washed by sterile water as described by

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Mohamed H. Lahiani et al with some modifications.40 Then oryza sativa seeds were grew on

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MS medium in conical flasks. All the plants were grown in light incubator under conditions:

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14 h light at approximately 250 µmol m-2 s-1 intensity, 10 h dark; 28 °C day, 25 °C night.

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Then germinant oryza sativa seedlings were used for subsequent experiments.

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2.8 Plant root cross section imaging using confocal laser scanning microscope

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A portion of 15 rice seedlings were placed per conical flask on blank medium (MS

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medium) and the same medium supplemented with free Cy5, Cy5-labelled nanoparticles or

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blank particles as contrast. All treatment groups had the same concentration of Cy5 (0.5 µg

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mL-1 medium). Then the seedlings were incubated for 24 hours in dark at 25 °C and then

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washed by deionized water. From these growing seedlings, the cross sections of roots were

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cut by freezing microtome. Images of cross sections were taken with confocal laser scanning

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microscopy (CLSM, Carl Zeiss, Boston, MA, USA).

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2.9 Bioassay of MNPs

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The germinant Oryza sativa seedlings, Digitaria sanguinalis seeds and Arabidopsis seeds

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were placed on the MS medium (as blank group), MS medium with blank nanoparticles

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(nanoparticles without metolachlor, as control group) and the same medium supplemented

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with MNPs, L-MNPs and MMPs respectively (as treatment groups). All the treatment groups

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have the same concentration of active ingredients. Then the plants were incubated for 7 days

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under conditions in 2.8. Then the seedling height of Oryza sativa, Digitaria sanguinalis and

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root length of Arabidopsis thaliana were measured.

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2.10 Evaluation of cytotoxicity using CCK test 8 ACS Paragon Plus Environment

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To evaluate the cytotoxicity difference between metolachlor technical and MNPs,

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cytotoxicity assays were carried out using the cell MC3T3 preosteoblast, which was

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maintained in continuous DMEM culture medium (with 10% fetal bovine serum) under a

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humid atmosphere containing 5% CO2 at temperature of 37 °C.41 Viable cell suspension was

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inoculated in 96-well plates and incubated for 24 h. Each plate contained 100 µL cell

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suspension (about 5000 cells). Then the cells were exposed for 24 h to metolachlor technical

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and MNPs at the same concentrations of active ingredient ranging from 50-200 mg/L.

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2.11 Statistical Analysis.

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Data were expressed as mean standard deviation (S.D.). Statistical analysis was

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determined using Student’s t test and *p < 0.05, **p < 0.01, and ***p < 0.001 were used to

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show statistical significance.

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3. Results and discussion

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3.1 Preparation and Characterization of metolachlor-loaded nanoparticles

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3.1.1 Preparation process and size distribution of MNPs

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The preparation process of metolachlor-loaded nanoparticles was illustrated in Figure 1.

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The organic phase with mPEG-PLGA and metolachlor was injected into the aqueous phase

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under magnetic stirring. With the rapid diffusion of the organic solvent, polymer deposition

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occurred immediately on the interface between water and organic solvent, leading to

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instantaneous formation of nanoparticles.42 The hydrophilic moieties of copolymer are outside

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and hydrophobic ones with pesticide are inside. After nanoparticles formation, the organic

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solvent and free pesticide were removed by dialysis. Some preparation factors are crucial to

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formation of the nanoparticles suspension. In the work, we studied the effect of different

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polymer/pesticide ratio and copolymer concentration on the encapsulation efficiency and

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particle size distribution (Table 1). Our results showed that, with decreasing pesticide amount, 9 ACS Paragon Plus Environment


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particle size became smaller while the degree of encapsulation and pesticide loading

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decreased. With a comprehensive consideration of a certain encapsulation efficiency and

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particle size tiny enough, the optimal copolymer/pesticide ratio was 4/1. The optimized

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concentration of mPEG-PLGA was 20 mg mL-1. The size of contrast particles made by mPEG

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(Mw:5kDa) – PLGA (Mw: 45kDa, LA:GA = 75:25) and mPEG (Mw: 5kDa) – PLGA (Mw:

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95kDa, LA:GA = 75:25) with the optimized conditions was 478.41 nm and 2322.15 nm,

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respectively. They were metolachlor-loaded nanoparticles with large size (L-MNPs) and

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metolachlor-loaded micro-particles (MMPs).

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TEM images and DLS size distribution of optimum metolachlor-loaded nanoparticles

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were presented in Figure 2. The figure showed that the spherical shapes of MNPs were

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retained after drying and a good dispersion in aqueous solution was observed. DLS measured

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the hydrodynamic size of MNPs swelling in aqueous solution. As a consequence, the size

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determined by TEM was slightly smaller than that measured by DLS.

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The nanoparticles suspension formulation was translucent to transparent with no visible

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particles or precipitation, being a relatively stable system. Referring to Stokes' law of

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resistance (1), sedimentation rate of particles in the liquid depends on radius of the particles

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(r), viscosity of fluid (η) and differentials of density (ρ – ρ’), among which the radius make a

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big difference because of the quadratic relation. We can infer that particles with smaller size

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exhibit much lower sedimentation rate. These may rationalize the fact that nanoparticles could

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show relative stability without involvement of surfactants. In this way, the “solubility” of

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metolachlor in water was also improved.

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Stokes law: v = 2gr2 (ρ – ρ’) / 9η (1)

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3.1.2 Infrared spectrum of MNPs

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In order to confirm pesticide loading, FTIR spectra of metolachlor, mPEG-PLGA and

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MNPs were measured and shown in Figure 3. The peaks at 2875 cm-1 and 1758 cm-1 are

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attributed to C–H stretching of CH3 and ester C=O stretching, respectively. The primary 10 ACS Paragon Plus Environment

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absorption bands for metolachlor were presented in the region of 1672 cm-1 and 2926 cm-1.

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C–H stretching and aromatic group were responsible for these peaks, respectively. The peaks

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at 2883 cm-1, 1760 cm-1 and 1668 cm-1 in the spectra of MNPs represent the stretching of

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methyl, the stretching vibrations of carbonyl group from mPEG-PLGA and the aromatic

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group from metolachlor, respectively. As a whole, the spectrum of MNPs contained not only

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characteristic peaks of mPEG-PLGA, but also metolachlor and in consideration of TEM

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image and measurement of encapsulation efficiency, we considered MNPs was successfully

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prepared.

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3.2 In vitro release of MNPs in different mediums

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The release profile of metolachlor from MNPs was investigated at 30 °C in MS culture

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medium, 10% acetonitrile MS culture medium, and 0.1% SDS MS culture medium,

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respectively. The addition of acetonitrile changed the polarity of the medium and the

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incorporation of SDS represented the situation with surfactants. These mediums provided the

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different environments to influence the release behaviours of pesticide. As shown in Figure 4,

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approximately 48%, 51% and 60% of metolachlor can be released from MNPs within the first

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24 h in MS culture medium, 10% acetonitrile MS culture medium, and 0.1% SDS MS culture

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medium, respectively, which were followed by continuous slow release behaviours. In

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comparison to metolachlor technical aqueous dispersion, MNPs showed sustained release

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behaviours. The rapid release in the first several hours was related to metolachlor

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absorbed/encapsulated on or near the nanoparticles’ surface.43 The sustained release indicated

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the release was dependent on pesticide diffusion and/or matrix erosion.44The release data were

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analysed by fitting Korsmeyer–Peppas model “Mt / M∞ = ktn” and the results were presented

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in Table 2. All the R2 exceeded 0.9. The release mechanism could be inferred by the value of

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n. The n values in Table 2 were less than 0.45. That means diffusion was the main mechanism

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of MNPs during the 72 hours of release experiment. 11 ACS Paragon Plus Environment


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It was revealed by calculated data that medium containing SDS promoted faster

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metolachlor release than pure MS medium and 10% acetonitrile MS medium. The water

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uptake of mPEG-PLGA, based on contact angle between water and copolymer, has an

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influence on metolachlor release.45,46 Whereas SDS, a surfactant, enhanced the contact

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between nanoparticle surface and water to contributed to the result.

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3.3 Stability of MNPs in plant culture solution

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The stability of MNPs was first examined under conditions similar to those used in

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absorption experiment, CCK test and bioassay test. We measured the size of MNPs at

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indicated time intervals in 72 hours. The obtained data in Figure 5 showed that no significant

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change of MNPs size was observed for up to 3 days (p>0.05), indicating the stability of

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MNPs during the experiment period.

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3.4 Absorption experiment of MNPs into rice seedlings

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Figure 6 showed photographs of the cross section of roots of untreated rice seedlings,

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rice seedlings incubated with free Cy5, Cy5-labelled nanoparticles/micro-sized particles for 1

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day, respectively. As shown in the figure, no fluorescence was observed in the untreated root

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at 633 nm, and the root treated with free Cy5 had hardly any fluorescence. It revealed that

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when Cy5 was released from nanoparticles, it could not induce fluorescence in the root. It was

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only visible in the root when loaded in the nanoparticles indicating the whole carrier system

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was in the root. A distinct fluorescence was observed in the root treated with Cy5-labelled

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nanoparticles. In contrast, rice seedlings treated with L-MNPs and MMPs had invisible

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fluorescence. It shows clearly that Cy5-labelled nanoparticles have permeated the rice roots.

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The reports about the internalization of different nanoparticles into plants, such as fullerene,

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carbon nanotube, nano-silicon and Ag, have confirmed that nanomaterials could be 12 ACS Paragon Plus Environment

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internalized by plants.16,47,48 Furthermore, Liu, X. et al reported that the internalization of the

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pesticide delivery system into the target organism can keep the bioactivity of pesticide.49 In

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this work, the hydrophobic droplets of metolachlor was “surface modified” when loaded into

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the polymeric nanoparticles. The tiny size and hydrophilic segment of mPEG-PLGA

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nanoparticle made metolachlor “soluble” in water. Then the metolachlor-loaded nanoparticles

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penetrated into plant after roots exposure.50 In consideration of the thick cell wall, the tiny

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wall pore on it and the photographs of the cross section of roots, the possible way by which

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nanoparticles internalize into plant was the apoplast way.51 This provided a pathway by which

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pesticide formulations could enter the plant after root exposure. In this process, metolachlor

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was internalized along with the uptake of water by plant, increasing the absorption of

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metolachlor even it was not released from the carriers. The actual absorption mechanism still

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need to be studied in the future works.

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3.5 Bioassay test of MNPs

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The inhibition of seedlings by the prepared nanoparticles was shown in Figure 7. As

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shown in control group, mPEG-PLGA nanoparticles without metolachlor exhibited negligible

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inhibition to plants. Metolachlor is used in soybean field to control Gramineae weeds and

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some broad-leaved weeds. The seedling height and root length data showed that the MNPs

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treatment groups exhibited higher inhibition on Oryza sativa and Digitaria sanguinalis

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seedlings than L-MNPs and MMPs under relatively low concentrations. However, it showed

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no significant inhibition or even less inhibition comparing to L-MNPs and MMPs treatment

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groups under relatively high concentrations. On Arabidopsis thaliana, herbicide activity

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improvement was not all significant comparing with L-MNPs and MMPs. The above results

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showed that MNPs did have efficiency improvement but it had its limited scope. The

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controlled release maintained the persistence effectiveness of MNPs during the experiment

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time. MNPs exhibited high-activity owing to its absorption into plant at low concentrations. 13 ACS Paragon Plus Environment


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This process along with water absorption guaranteed the utilization of metolachlor. More

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mechanism and applied range need to be studied in the future work.

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3.4 Cytotoxicity of MNPs to mammal cell line

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As a pharmaceutical adjuvant certified by FDA, mPEG–PLGA–based nanoparticles may

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exhibit excellent toxicity reduction in mammals.52 In this work, apart from the morphological

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characterizations and absorption into plants, the in vitro cellular viability test was performed

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to make a preliminary exploration about the cytotoxicity of metolachlor-loaded nanoparticles

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and metolachlor technical. The preosteoblast cell line (MC3T3) was employed to evaluate the

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percentage of cell viability at test concentrations. The results of cell viability showed that

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metolachlor-loaded nanoparticles are less toxic than the free metolachlor (Fig 8).

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Nanomaterials with good biocompatibility may have contributed to this result.

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In summary, mPEG-PLGA nanoparticles was used as a carrier for metolachlor. The

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water-based mPEG-PLGA nanoparticles with proper size and surface property eliminated the

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requirement of hazardous organic solvent and surfactants in common formulations. In this

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way, “water-solubility” enhancement of strongly hydrophobic pesticide, such as metolachlor,

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is realized and it contributes to safer and cleaner pesticide formulation. The results of TEM

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and DLS revealed the morphology and size distribution. Sustainable release of metolachlor

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was observed from the polymeric nanoparticles. Furthermore, absorption of the resulting

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nanoparticles into rice suggested a possible way by which pesticide formulation could

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transmit into plant and guarantee utilization. Resulting nanoparticles exhibited higher

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herbicide activity than those in non-nano forms at relatively low concentrations on Oryza

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sativa and Digitaria sanguinalis. Finally, lower toxicity to MC3T3 cell line was observed

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when metolachlor was loaded in the nanoparticles, indicating a possible way of toxicity

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reduction. As a whole, this work provided a potential solution to mitigate the pollution and

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risk of common pesticide formulations. It also demonstrated the promising applications of 14 ACS Paragon Plus Environment

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polymeric nanoparticles in pesticide delivery and increase the utilization of pesticide. Further

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work will be needed to study the mechanism and applications of pesticide-loaded

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nanoparticles.

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Acknowledgements

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This work was supported by The National Key Research and Development Program of

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China (2017YFD 0200301). We thank Prof. Zhenhua Zhang (China Agricultural University)

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for providing Cy-5 reagents (National Key Technologies R&D Program of China,

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2015BAK45B01, CAU). The authors declare no competing financial interest.

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References

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1. Voinova,O.N.; Kalacheva, G.S.; Grodnitskaya, I.D.; Volova, T.G. Microbial polymers as a

341

degradable carrier for pesticide delivery. Appl. Biochem. Micro. 2009,45, 384-388.

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2. Engelskirchen, S.; Maurer, R.; Levy, T.; Berghaus, R.; Auweter, H.; Glatter, O. Highly

343

concentrated emulsified microemulsions as solvent-free plant protection formulations. J.

344

Colloid Interface Sci. 2012, 388, 151-161.

345

3. Li, J.; Fan, T.; Xu, Y.; Wu, X. Ionic liquids as modulators of physicochemical properties

346

and nanostructures of sodium dodecyl sulfate in aqueous solutions and potential application in

347

pesticide microemulsions. Phys. Chem. Chem. Phys. 2016, 18, 29797-29807.

348

4. Garrido-Herrera, F.J.; Daza-Fernandez, I.; Gonzalez-Pradas, E.; Fernandez-Perez, M.

349

Lignin-based formulations to prevent pesticides pollution. J. Hazard. Mater. 2009, 168, 220-

350

225.

351

5. Guo, M.; Zhang, W.; Ding, G.; Guo, D.; Zhu, J.; Wang, B.; Punyapitak, D.; Cao, Y.

352

Preparation and characterization of enzyme-responsive emamectin benzoate microcapsules

353

based on a copolymer matrix of silica–epichlorohydrin–carboxymethylcellulose. RSC Adv.

354

2015, 5, 93170-93179.

355

6. Liu, Y.; Sun, Y.; He, S.; Zhu, Y.; Ao, M.; Li, J.; Cao, Y. Synthesis and characterization of

356

gibberellin-chitosan conjugate for controlled-release applications. Int. J. Biol. Macromol.

357

2013, 57, 213-217.

358

7. Sopena, F.; Maqueda, C.; Morillo, E. Norflurazon Mobility, Dissipation, Activity, and

359

Persistence in a Sandy Soil As Influenced by Formulation. J. Agric. Food. Chem. 2007, 55,

360

3561-3567.

361

8. Elek, N.; Hoffman, R.; Raviv, U.; Resh, R.; Ishaaya, I.; Magdassi, S. Novaluron

362

nanoparticles: Formation and potential use in controlling agricultural insect pests. Colloids

363

and Surfaces A: Physicochem. Eng. Aspects 2010, 372, 66–72


Page 16 of 34

Page 17 of 34


364

9. Chevillard, A.; Angellier-Coussy, H.; Guillard, V.; Gontard, N.; Gastaldi, E. Controlling

365

pesticide release via structuring agropolymer and nanoclays based materials. J. Hazard. Mater.

366

2012, 205-206, 32-39.

367

10. Liu, Y.; Ni, Z.; Mo, R.; Shen, D.; Zhong, D.; Tang, F. Environmental behaviors of

368

phoxim with two formulations in bamboo forest under soil surface mulching. J. Environ. Sci.

369

2015, 35, 91-100.

370

11. Zhao, C.; Shao, L.; Lu, J.; Zhao, C.; Wei, Y.; Liu, J.; Li M.; Wu, Y. Triple Redox

371

Responsive Poly(Ethylene Glycol)-Polycaprolactone Polymeric Nanocarriers for Fine-

372

Controlled Drug Release. Macromol. Biosci. 2016, DOI: 10.1002/mabi.201600295.

373

12. Davis, M.E.; Chen, Z.G.; Shin, D.M. Nanoparticle therapeutics: an emerging treatment

374

modality for cancer, Nature reviews. Drug discovery. Nat. Rev. Drug Discov. 2008, 7, 771-

375

782.

376

13. Arasoglu, T.; Mansuroglu, B.; Derman, S.; Gumus, B.; Kocyigit, B.; Acar, T.;

377

Kocacaliskan, I. Enhancement of Antifungal Activity of Juglone (5-Hydroxy-1,4-

378

naphthoquinone) Using a Poly(D,L‑lactic-co-glycolic acid) (PLGA) Nanoparticle System. J.

379

Agric. Food Chem. 2016, 64, 7087−7094.

380

14. Bouwmeester, H.; Dekkers, S.; Noordam, M.Y.; Hagens, W.I.; Bulder, A.S.; Heer, C.;

381

Voorde, S.E.; Wijnhoven, S.W.; Marvin, H.J.; Sips, A.J. Review of health safety aspects of

382

nanotechnologies in food production. Regul. Toxicol. Pharmacol. 2009, 53, 52-62.

383

15. Shen, D.; Zhou, F.; Xu, Z.; He, B.; Li, M. Shen J.; Yin, M.; An, C. Systemically

384

interfering with immune response by a fluorescent cationic dendrimer delivered gene

385

suppression. J. Mater. Chem. B 2014, 2, 4653-4659.

386

16. Lin, S.; Reppert, J.; Hu, Q.; Hudson, J.S.; Reid, M.L.; Ratnikova, T.A.; Rao, A.M.; Luo,

387

H.; Ke, P.C. Uptake, translocation, and transmission of carbon nanomaterials in rice plants.

388

Small 2009, 5, 1128-1132.



389

17. Wild, E.; Jones, K.C. Novel Method for the Direct Visualization of in Vivo Nanomaterials

390

and Chemical Interactions in Plants. Environ. Sci. Technol. 2009, 43, 5290-5294.

391

18. Wang, P; Lombi, E; Zhao, F; Kopittke, P. Nanotechnology: A New Opportunity in Plant

392

Sciences. Trends in Plant Science 2016, 21, 699-712.

393

19. Popat, A; Liu, J; Hu, Q; Kennedy, M; Peters B; Lu, G; Qiao, S. Adsorption and release of

394

biocides with mesoporous silica nanoparticles. Nanoscale 2012, 4, 970-975.

395

20. Kookana, R; Boxall, A; Reeves, P; Ashauer, R; Beulke, S; Chaudhry, Q; Cornelis, G;

396

Fernandes, T; Gan, J; Kah, M; Lynch, I; Ranville, J; Sinclair, C; Spurgeon, D; Tiede, K; Brin,

397

P. Nanopesticides: Guiding Principles for Regulatory Evaluation of Environmental Risks. J.

398

Agric. Food Chem. 2014, 62, 4227-4240.

399

21. Yang, J.; Ren, H.; Xie, Y. Synthesis of amidic alginate derivatives and their application

400

in microencapsulation of lambda-cyhalothrin. Biomacromolecules 2011, 12, 2982-2987.

401

22. He, S.; Zhang, W.; Li, D.; Li, P.; Zhu, Y.; Ao, M.; Li, J.; Cao, Y. Preparation and

402

characterization of double-shelled avermectin microcapsules based on copolymer matrix of

403

silica–glutaraldehyde–chitosan. J. Mater. Chem. B 2013, 1, 1270-1278.

404

23. Zhang, W.; He, S.; Liu, Y.; Geng, Q.; Ding, G.; Guo, M.; Deng, Y.; Zhu, J.; Li, J.; Cao, Y.

405

Preparation and Characterization of Novel Functionalized Prochloraz Microcapsules Using

406

Silica−Alginate−Elements as Controlled Release Carrier Materials. ACS Appl. Mat. Interfaces.

407

2014, 6, 11783-11790.

408

24. Atta, S.; Bera, M.; Chattopadhyay, T.; Paul, A.; Ikbal, M.; Maiti, M.K.; Singh, N.D.P.

409

Nano-pesticide formulation based on fluorescent organic photoresponsive nanoparticles: for

410

controlled release of 2,4-D and real time monitoring of morphological changes induced by

411

2,4-D in plant systems. RSC Adv. 2015, 5, 86990-86996.

412

25. Cui, B.; Feng, L.; Pan, Z.; Yu, M.; Zeng, Z.; Sun, C.; Zhao, X.; Wang, Y.; Cui, H.

413

Evaluation of Stability and Biological Activity of Solid Nanodispersion of Lambda-

414

Cyhalothrin. PloS one 2015, 10, e0135953 18 ACS Paragon Plus Environment

Page 18 of 34

Page 19 of 34


415

26. Li, M.; Huang, Q.; Wu Y. A novel chitosan-poly(lactide) copolymer and its submicron

416

particles as imidacloprid carriers. Pest. Manag. Sci. 2011, 67, 831-836.

417

27. Ding, G.; Li, D.; Liu, Y.; Guo, M.; Duan, Y.; Li, J.; Cao, Y. Preparation and

418

characterization of kasuga-silica-conjugated nanospheres for sustained antimicrobial activity.

419

J. Nanopart. Res. 2014, 16, 1-10.

420

28. Wu, H.; Yang, W.; Zhang, Z.; Huang, T.; Yao, G.; Xu, H. Uptake and phloem transport of

421

glucose-fipronil conjugate in Ricinus communis involve a carrier-mediated mechanism. J.

422

Agric. Food. Chem. 2012, 60, 6088-6094.

423

29. Hu, L.; Yang, W.; Xu, H. Novel fluorescent conjugate containing glucose and NBD and

424

its carrier-mediated uptake by tobacco cells. J. Photochem. Photobiol. B 2010, 101, 215-223.

425

30. Torney, F.; Trewyn, B.G.; Lin, V.S.; Wang, K. Mesoporous silica nanoparticles deliver

426

DNA and chemicals into plants. Nat. Nanotech. 2007, 2, 295-300.

427

31. Deng, X.; Cao, M.; Zhang, J.; Hu, K.; Yin, Z.; Zhou, Z.; Xiao, X.; Yang, Y.; Sheng, W.;

428

Wu, Y.; Zeng, Y. Hyaluronic acid-chitosan nanoparticles for co-delivery of MiR-34a and

429

doxorubicin in therapy against triple negative breast cancer. Biomaterials 2014, 35, 4333-

430

4344.

431

32. Khandelwal, N.; Barbole, R.S.; Banerjee, S.S.; Chate, G.P.; Biradar, A.V.; Khandare, J.J.;

432

Giri, A.P. Budding trends in integrated pest management using advanced micro- and nano-

433

materials: Challenges and perspectives. J. Environ. Manage., 2016, 184, 157-169.

434

33. Nikoloff, N.; Escobar, L.; Soloneski, S.; Larramendy, M.L. Comparative study of

435

cytotoxic and genotoxic effects induced by herbicide S-metolachlor and its commercial

436

formulation Twin Pack Gold(R) in human hepatoma (HepG2) cells. Food Chem. Toxicol.

437

2013, 62, 777-781.

438

34. Burke, I.C.; Price, A.J.; Wilcut, J.W.; Jordan, D.L.; Culpepper, A.S.; Tredaway-Ducar, J.

439

Annual Grass Control in Peanut (Arachis hypogaea) with Clethodim and Imazapic. Weed

440

Technology 2004, 18, 88-92. 19 ACS Paragon Plus Environment


441

35. Potter, T.L.; Gerstl, Z.; White, P.W.; Cutts, G.S.; Webster, T.M.; Truman, C.C.;

442

Strickland, T.C.; Bosch, D.D.; Fate and efficacy of metolachlor granular and emulsifiable

443

concentrate formulations in a conservation tillage system. J. Agric. Food. Chem. 2010, 58,

444

10590-10596.

445

36. Sopena, F.; Maqueda, C.; Morillo, E. Formulation affecting alachlor efficacy and

446

persistence in sandy soils. Pest Manag. Sci. 2009, 65, 761-768.

447

37. Dowler, C.C. Polymeric Microcapsules of Alachlor and Metolachlor - Preparation and

448

Evaluation of Controlled-Release Properties .J. Agric. Food. Chem. 1999, 47, 2908-2913.

449

38. Zhang, Y.; Zhuo, R.X. Synthesis and drug release behavior of poly (trimethylene

450

carbonate)-poly (ethylene glycol)-poly (trimethylene carbonate) nanoparticles. Biomaterials

451

2005, 26, 2089-2094.

452

39. Xu, Y.; Chen, W.; Guo, X.; Tong, Y.; Fan, T.; Gao, H.; Wu, X. Preparation and

453

characterization of single- and double-shelled cyhalothrin microcapsules based on the

454

copolymer matrix of silica–N-isopropyl acrylamide–bis-acrylamide. RSC Adv. 2015, 5,

455

52866-52873.

456

40. Lahiani, M.H.; Chen, J.; Irin, F.; Puretzky, A.A.; Green, M.J.; Khodakovskaya, M.V.

457

Interaction of carbon nanohorns with plants: Uptake and biological effects. Carbon 2015, 81,

458

607-619.

459

41. Oliveira, J.L.; Campos, E.V.R.; Silva, C.M.G.; Pasquoto, T.; Lima, R.; Fraceto, L.F. Solid

460

Lipid Nanoparticles Co-loaded with Simazine and Atrazine:Preparation, Characterization, and

461

Evaluation of Herbicidal Activity. J. Agric. Food Chem. 2015, 63, 422−432.

462

42. Govender, T.; Stolnik, S.; Garnett, M.C.; Illum, L.; Davis, S.S. PLGA nanoparticles

463

prepared by nanoprecipitation drug loading and release studies of a water soluble drug. J.

464

Control. Release. 1999, 57, 171-185.


Page 20 of 34

Page 21 of 34


465

43. Alibolandi, M.; Sadeghi, F.; Abnous, K.; Atyabi, F.; Ramezani, M.; Hadizadeh, F. The

466

chemotherapeutic potential of doxorubicin-loaded PEG-b-PLGA nanopolymersomes in

467

mouse breast cancer model. Eur. J. Pharm. Biopharm. 2015, 94, 521-531.

468

44. Kao. W.J.; Fu, Y.; Drug release kinetics and transport mechanisms of non-degradable and

469

degradable polymeric delivery systems. Expert Opin. Drug Deliv. 2010, 7, 429-444.

470

45. Penco, M.; Marcioni, S.; Ferruti, P.; D'Antone, S.; Deghenghi, R. Degradation behaviour

471

of block copolymers containing poly(lactic-glycolic acid) and poly(ethylene glycol) segments.

472

Biomaterials. 1996, 17, 1583-1590.

473

46. Beletsi, A.; Leontiadis, L.; Klepetsanis, P.; Ithakissios, D.S.; Avgoustakis, K. Effect of

474

preparative

475

methoxypoly(ethyleneglycol) copolymers related to their application in controlled drug

476

delivery. Int. J. Pharm. 1999, 182, 187-197.

477

47. Nair, R.; Poulose, A.C.; Nagaoka, Y.; Yoshida Y.; Maekawa, T.; Kumar, D.S. Uptake of

478

FITC labeled silica nanoparticles and quantum dots by rice seedlings: effects on seed

479

germination and their potential as biolabels for plants. J. Fluoresc. 2011, 21, 2057-2068.

480

48. Larue, C.; Castillo-Michel, H.; Sobanska, S.; Cecillon, L.; Bureau, S.; Barthes, V.;

481

Ouerdane, L.; Carriere, M.; Sarret, G. Foliar exposure of the crop Lactuca sativa to silver

482

nanoparticles: evidence for internalization and changes in Ag speciation. J. Hazard. Mater.

483

2014, 264, 98-106.

484

49. Liu, X.; He, B.; Xu, Z.; Yin, M.; Yang, W.; Zhang, H.; Cao, J.; Shen, J. A functionalized

485

fluorescent dendrimer as a pesticide nanocarrier: application in pest control. Nanoscale 2015,

486

7, 445-449.

487

50. Nedosekin, D.A.; Khodakovskaya, M.V.; Biris, A.S.; Wang, D.; Xu, Y.; Villagarcia, H.;

488

Galanzha, E.I.; Zharov, V.P. Cytom. Part A 2011, 79, 855-865.

489

51. Steudle E, P.C. How does water get through roots. J. Exp. Bot. 1998, 49, 775-788.

variables

on

the

properties

of


poly(dl-lactide-co-glycolide)–


490

52. Song, Y.; Zhao, R.; Hu, Y.; Hao, F.; Li, N.; Nie, G.; Tang, H.; Wang, Y. Assessment of

491

the Biological Effects of a Multifunctional Nano-Drug-Carrier and Its Encapsulated Drugs.

492

J. Proteome. Res. 2015, 14, 5193-5201.

493


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494 495 496

Figure 1. The preparation procedure of metolachlor-loaded nanoparticles.

497 498

Figure 2. The TEM images (A) and DLS size distribution (B) of metolachlor-loaded nanoparticles.

499 500

Figure 3. The FTIR spectra of metolachlor, mPEG-PLGA and metolachlor-loaded mPEG-PLGA

501

nanoparticles (MNPs).

502 503

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

504 505

Figure 5. The stability of metolachlor-loaded nanoparticles (MNPs) in deionized water, MS medium

506

and DMEM.

507 508

Figure 6. Plant root cross section imaging using confocal laser scanning microscope. The left rank

509

was recorded at 633 nm by confocal microscope. The middle rank was in bright field mode using

510

white light and the right rank was the superposition of the two. A, untreated rice seedlings; B, rice

511

seedlings incubated with free Cy5; C, rice seedlings incubated with Cy5-labelled nanoparticles

512

prepared by mPEG (Mw: 5kDa)–PLGA (Mw: 8kDa); D, rice seedlings incubated with nanoparticles

513

prepared by mPEG (Mw: 5kDa)–PLGA (Mw: 45kDa); E, rice seedlings incubated with micro-sized

514

particles prepared by mPEG (Mw: 5kDa)–PLGA (Mw: 95kDa)

515 516

Figure 7. Herbicide activity of MNPs, L-MNPs and MMPs. (A) Herbicide activity on Oryza sativa.

517

(B) Oryza sativa under metolachlor concentration 0.1 mg/L. (C) Herbicide activity on Digitaria

518

sanguinalis. (D) Digitaria sanguinalis under metolachlor concentration 16 mg/L. (E) Herbicide

519

activity on Arabidopsis thaliana. (F) Arabidopsis thaliana under metolachlor concentration 0.02

520

mol/L.

521 23 ACS Paragon Plus Environment


Page 24 of 34

522

Figure 8. Cytotoxicity of MNPs and metolachlor-tech at different concentration. a, MNPs; b,

523

metolachlor technical.

524 525

Table 1 The effect of copolymer/pesticide weight ratio on the encapsulation efficiency,

526

pesticide loading and particle size distribution. Sample Copolymer Metolachlor

Average

PDI a

EE b [%]

PL c [%]

[mg mL-1]

[mg mL-1]

1

20

20

125.6±3.3 0.100±0.011 86.47±3.12 46.37±3.39

2

20

10

121.3±0.8 0.149±0.005 77.92±6.43 28.04±2.58

3

20

5

97.87±5.5 0.128±0.020 70.31±3.05 14.95±1.20

4

20

2.5

90.90±2.1 0.121±0.031 68.58±4.91

7.80±1.04

5

30

3.75

116.3±3.7 0.208±0.016 64.44±5.25

7.45±2.11

6

30

2.5

128.7±5.0 0.127±0.004 36.55±6.16

2.96±0.86

7

10

2.5

90.49±4.8 0.209±0.005 21.05±2.84

4.50±1.19

8

10

1.25

91.03±5.3 0.212±0.033 24.26±4.11

2.94±2.41

Size [nm]

527

a

Polydispersity Index.

528

b

Encapsulation Efficiency.

529

c

Pesticide Loading.

530

Table 2. The constants of fitting Korsmeyer–Peppas model to the release of metolachlor from

531

MNPs under different conditions Medium

n

R2

lgk


Page 25 of 34


MS

0.1947

7.2655

0.9314

10% acetonitrile MS

0.2409

5.6484

0.9458

0.1% SDS MS

0.2054

7.3238

0.9251

532



Figure 1 The preparation procedure of metolachlor-loaded nanoparticles. 84x33mm (300 x 300 DPI)

ACS Paragon Plus Environment

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Page 27 of 34


Figure 2. The TEM images (A) and DLS size distribution (B) of metolachlor-loaded nanoparticles. 84x34mm (300 x 300 DPI)



Figure 3. The FTIR spectra of metolachlor, mPEG-PLGA and metolachlor-loaded mPEG-PLGA nanoparticles (MNPs). 84x75mm (300 x 300 DPI)


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Figure 4. The in vitro release profile of metolachlor from MNPs in different mediums. 59x42mm (300 x 300 DPI)



Figure 5. The stability of metolachlor-loaded nanoparticles (MNPs) in deionized water, MS medium and DMEM. 62x45mm (300 x 300 DPI)


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Figure 6 Plant root cross section imaging using confocal laser scanning microscope. The left rank was recorded at 633 nm by confocal microscope. The left rank was recorded at 633 nm by confocal microscope. The middle rank was in 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-labelled nanoparticles prepared by mPEG (Mw: 5kDa)–PLGA (Mw: 8kDa); D, rice seedlings incubated with nanoparticles prepared by mPEG (Mw: 5kDa)–PLGA (Mw: 45kDa); E, rice seedlings incubated with micro-sized particles prepared by mPEG (Mw: 5kDa)–PLGA (Mw: 95kDa) 84x145mm (300 x 300 DPI)



Figure 7. Herbicide activity of MNPs, L-MNPs and MMPs. (A) Herbicide activity on Oryza sativa. (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. 177x209mm (300 x 300 DPI)


Page 32 of 34

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Figure 8 Cytotoxicity of MNPs and metolachlor-tech at different concentration. a, MNPs; b, metolachlor technical. 64x49mm (300 x 300 DPI)



TOC Graph 84x33mm (300 x 300 DPI)


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Polymeric Nanoparticles as a Metolachlor Carrier: Water-Based

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