Fabrication of Novel Avermectin Nanoemulsion Using a Polyurethane

Subscriber access provided by AUSTRALIAN NATIONAL UNIV

Article

Fabrication of novel avermectin nanoemulsion using polyurethane emulsifier with cleavable disulfide bonds Wenxun Guan, Wenxiang Zhang, Liming Tang, Yan Wang, and Haixin Cui J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b01427 • Publication Date (Web): 12 Jul 2017 Downloaded from http://pubs.acs.org on July 17, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 34

Journal of Agricultural and Food Chemistry

Fabrication of novel avermectin nanoemulsion using polyurethane emulsifier with cleavable disulfide bonds Wenxun Guan a, Wenxiang Zhang a, Liming Tang a,*, Yan Wang b, Haixin Cui b [a]

Key Laboratory of Advanced Materials of Ministry of Education of China Department of Chemical Engineering, Tsinghua

University, Beijing 100084, China [b]

Institute of Environment and Sustainable Development in Agriculture, Chinese Academic of Agriculture Sciences, Beijing

100081, China

ABSTRACT: 1

In this study, a polyurethane emulsifer with various functional groups was prepared from isophorone

2

diisocyanate (IPDI), avermectin, 2,2-dimethylol propionic acid (DMPA) and bis(2-hydroxyethyl) disulfide

3

(HEDS). The chemical structure of the polymer was confirmed by FT-IR, 1H NMR and element analysis. The

4

polymer exhibited adequate emulsification ability for avermectin after neutralization with triethylamine. A

5

satisfaying nanoemulsion was obtained, in which avermectin was encapsulated in nanoparticles with 50 wt% drug

6

loading, low organic solvent content and high stability under dilution and centrifuging treatment in addition to low

7

surface tension, high affinity to crop leaf and improved avermectin photostability. The resulting nanoparticles

8

showed degradability in the presence of DL-dithiothreitol (DTT) or inside the insect due to the disulfide bonds,

9

promoting the release of avermectin. As a result, the avermectin nanoparticles showed higher insecticidal ability

10

compared with both the avermectin nanoparticles without disulfide group and the avermectin emulsifiable

11

concentrate (EC).

12

KEYWORDS:

13

polyurethane emulsifier; avermectin nanoemulsion; disulfide group; insecticidal ability.

14

ACS Paragon Plus Environment


15 16

1. INTRODUCTION

17

Pesticides play an important role in defending against biological disasters and improving

18

agricultural production efficiency, saving more than 30% of the total crop production worldwide

19

according to the data from the United Nations Food and Agriculture Organization (FAO).1,2 More

20

than 4.6 million tons pesticides per year, involving more than 400 chemical compounds, are

21

applied worldwide in controlling agricultural pests.3 Traditional pesticides are usually utilized in

22

the form of emulsifiable concentrate (EC) or wettable powder, consisting of a large amount of

23

organic solvent or other additives,4,5 and suffering from problems such as coarse particles, poor

24

nanoemulsion, low bioactivity and slow degradation.6,7 As a result, the vast majority of pesticides

25

would get into the environment, triggering a series of problems in food safety and ecological

26

environment.

27

The application of nanotechnology in agricultural products gives rise to the development in

28

new type of pesticides.8-13 It has been proved that nano-pesticides, featuring high surface area and

29

appropriate affinity to leaves, may minimize pesticide loss by reducing runoff and modulating the

30

release rate, prevent premature degradation of the active ingredients, enhance uptake into crops,

31

and avoid the use of hazardous organic (co)-solvents.14 However, the use of nano-pesticides in

32

plant protection may bring unforeseeable risks because of a higher input of nanoparticles to the

33

environment and bioaccumulation of nanoparticles through the food chain. So the environmental

34

risk assessment for nano-pesticides must be carried out to guarantee their safe use.15-16

35

During the past decade, various nano-pesticides have been developed using lipids,17-18

36

polymer,19-21 emulsion,22-23 titanium dioxide,24-25 silver,26-27 silica,28-30 aluminium,31-32 zinc


Page 2 of 34

Page 3 of 34


37

oxide,33-34 copper oxide,35 and carbon nanotubes.36-37 Some of these nanomaterials are used as

38

carriers or stabilizers of active ingredients, and the others are active ingredients themselves.

39

However, most of the preparation methods were complicated and the properties of the products

40

needed to be improved, especially the drug releasing profile.

41

Avermectin is a highly effective bio-pesticide and has been widely used for decades. It is a

42

16-membered ring lactone compound produced by Streptomyces avermitilis with good insecticidal

43

and acaricidal activity.38-40 Avermectin and its derivatives have a wide range of applications in the

44

areas of agriculture, animal husbandry, and human parasitic diseases.41-42 However, avermectin has

45

two major shortcomings limiting its application, including extremely low solubility (about 7.8

46

µg/mL)43 in water and poor photostability. Although various avermectin nano-pesticides have been

47

fabricated in literatures,44-48 their properties are not satisfactory in terms of drug loading, organic

48

solvent content, stability and so forth. Therefore, an effective and multifunctional

49

nano-formulation of avermectin is still desirable.

50

In this study, a multifunctional polyurethane emulsifier was designed and prepared from

51

isophorone diisocyanate (IPDI), avermectin, 2, 2-dimethylol propionic acid (DMPA) and

52

bis(2-hydroxyethyl) disulfide (HEDS). After neutralization with triethylamine, the polymer

53

exhibited adequate emulsification ability for avermectin. The resulting nanoemulsion containing

54

nano-sized avermectin particles exhibited a series of desirable properties, including high drug

55

loading, low organic solvent content, good stability, strong affinity to leaves, improved avermectin

56

photostability and insecticidal activity. Besides, the disulfide bonds in the emulsifier molecules

57

can be broken by DTT or the reducing agents in vivo49, which provide a facile approach to

58

promote the pesticide releasing. This study may be helpful for designing and fabricating new



59

emulsifiers of nano-pesticides, considering its convenient preparation process, reduced

60

environmental pollution, and controllable drug releasing profiles.

61 62

2. EXPERIMENTAL SECTION

63

2.1. Materials and instruments

64

Isophorone diisocyanate (Alfa Aesar, 98%), 2, 2-dimethylol propionic acid (Aladdin, 99%),

65

avermectin (MYM Biological Technology Company, 95%), dibutyltin dilaurate (Sinopharm

66

Chemical Reagent Co. Ltd, chemical pure), butanone (Beijing Chemical Works, AR),

67

2-hydroxyethyl disulfide (Aldrich, technical grade), N-methyl-2-pyrrolidone (NMP, Beijing

68

Chemical Works, AR), DL-dithiothreitol (Aladdin, 99%), phosphotungstic acid (Shanghai Macklin

69

Biochemical Co. Ltd, AR), BYK-345 (a polyether modified siloxane surfactant supplied by BYK

70

Additives and Instruments), avermectin EC (Hebei Weiyuan Chemical Co., Ltd., 5wt%).

71

Fourier-transform IR spectrometer (FT-IR) (Nicolet 560), nuclear magnetic resonance

72

spectrometer (JNM-ECA 600), elemental analyzer (Vario EL Ⅲ), Zetasizer (3000HS Malvern),

73

transmission electron microscopy (TEM) (Hitachi H600), surface tension tensiometer (HZ 800,

74

Zibo Boshan Haifen Instrument Plant), UV-vis spectrophotometer (UV-3200, Mapada

75

Instruments).

76

2.2. Synthesis of the polyurethane

77

The synthetic procedure of polyurethane is outlined in Scheme 1. The preparation process of

78

polyurethane precursor of emulsifier No. 3 (see Table 1) was described below in detail as an

79

example.

80

Firstly, avermectin (1.774 g, 2 mmol) and excessive isophorone diisocyanate (IPDI) (4.112 g,

81

18.5 mmol) were dissolved in butanone (15 mL) in a three-necked flask, and dibutyltin dilaurate


Page 4 of 34

Page 5 of 34


82

(DBTDL) (0.05 g) was added as catalyst. The reaction mixture was heated to 75±2℃ for four

83

hours under mechanical stirring, producing a prepolymer with terminal isocyanate groups. Then,

84

the NMP (15 mL) solution of 2, 2-dimethylol propionic acid (DMPA) (1.396 g, 10.5 mmol),

85

2-hydroxyethyl disulfide (HEDS) (0.924 g, 6 mmol) and DBTDL (0.025 g) were added

86

continuously into the reactor. The reaction solution was maintained at 75±2℃ for another two

87

hours. After cooling to room temperature, a solution of the polyurethane containing avermectin

88

units, carboxylic acid groups and disulfide bonds was obtained and used below.

89

2.3. Fabricating avermectin nanoemulsion

90

Firstly, 0.212 g trimethylamine (2.1 mmol) was added into 3.500 g polyurethane solution

91

(with 0.820 g polyurethane containing 1.05 mmol DMPA units) in a glass bottle under stirring for

92

neutralization. Then, 0.820 g avermectin was dissolved into the solution. The solution was added

93

slowly into water (30 mL) under moderate stirring (600 rpm) at room temperature. The avermectin

94

nanoemulsion was obtained with pale yellow.

95

2.4. Characterization of the polyurethane

96

A certain amount of polyurethane solution was added into deionized water under magnetic

97

stirring. The resulting precipitate was filtrated and washed with deionized water twice. After dried

98

under vacuum, the polyurethane was obtained as white solid. FT-IR spectrometer, nuclear

99

magnetic resonance spectrometer and elemental analyzer were used to characterize the chemical

100

structure of polyurethane.

101

2.5. Characterization of the nanoemulsion

102

The size of the particles in the nanoemulsion was evaluated by dynamic light scattering (DLS,

103

3000HS Malvern Zetasizer). A four-sided cuvette was filled with deionized water. Drops of the



104

nanoemulsion were added into the cuvette until the liquid looked blue. Then the cuvette was

105

placed in the instrument and measured.

106

The morphology of the particles was imaged by using TEM. A few drops of the

107

nanoemulsion which was diluted with deionized water for 50 times were deposited on a

108

carbon-coated copper grid. The excess solution was removed with filter paper. The samples were

109

then stained with 2% phosphotungstic acid solution (the pH value was adjusted to 7-8) for 60

110

seconds. The excess dye was removed with filter paper, and the copper grids were dried at room

111

temperature. The sample was imaged using Hitachi H600 TEM operated at 80 KeV.

112

2.6. Analysis the stability and retention on leaves

113

The avermectin nanoemulsion was diluted for 100 times by using deionized water, and the

114

particle size was determined by DLS. The nanoemulsion was divided and transferred into identical

115

centrifuge tubes. After centrifugation under 10000 rpm for 5, 10, 15, 20, 25, 30 minutes, the

116

particle sizes in the supernatant were measured by DLS.

117

The amount of liquid retention on the surface of the plant leaves was measured by the micro

118

weighing method and the dipping method.50 Specifically, the liquid sample and an elongated tip

119

tweezers were placed in the beaker and weighed with a balance. Then, the balance was zeroed.

120

Leaves were cut into pieces, and areas S (cm2) of the pieces were measured. One leaf piece was

121

immersed in the liquid with tweezers for 15 seconds, then quickly pulled out and hung over the

122

liquid, until no liquid drops were dripping. Then, the leaves were placed aside and the tweezers

123

were put back into the beaker, and the balance reading W (g) was recorded. Leaf retention is

124

calculated by 1000×W/S (mg/cm2).

125

2.7. Measurement the surface tension


Page 6 of 34

Page 7 of 34


126

Different concentrations (0.1 g/L, 1 g/L, 10 g/L, 100 g/L, 200 g/L) of methanol solution of

127

the solid polyurethane were prepared. Then an excess of trimethylamine (two times the molar

128

amount of DMPA in the polyurethane) was added to the solution for neutralization. The solution

129

was dropped into deionized water to prepare aqueous solution of emulsifier with different

130

concentrations. The surface tension of the solution was measured by the tensiometer.

131

2.8. Measurement the photostability

132

The ethanol solutions of the emulsifier and pure avermectin were separately prepared at the

133

concentration of 100 mg/L. The absorption curves of the two samples were measured by UV-vis

134

spectrophotometer.

135

The nanoemulsion was treated by acetic acid until precipitate was formed. The precipitate

136

was dried under vacuum to give a solid sample designated as the encapsulated avermectin.

137

Suitable amount of the encapsulated avermectin powder was placed in a watchglass, and placed

138

under a UV lamp (1000 W) and irradiated for a pre-designed time period. After that the sample

139

was dissolved in ethanol and analysed by UV-vis spectrophotometer to determine the

140

concentration of avermectin. The photostability of pure avermectin was measured by the same

141

procedure.

142

2.9. Measurement the releasing profile

143

The drug releasing profile of the nanoemulsion was measured by the dialysis method.51 Ten

144

milliliter nanoemulsion was transferred into a dialysis bag. The bag was sealed and placed into a

145

wide-mouth bottle. The ethanol and water (1:4) mixture (the pH was adjusted to 9 with

146

triethylamine) and 20 mM DL-dithiothreitol (DTT) were added into the bottle. The bottle was kept

147

at room temperature (25℃) with magnetic stirring at 100 rpm. At predesigned time interval, a 200



148

µL solution was withdrawn from the bottle and diluted with ethanol and water (1:4) mixture to 2

149

mL. The diluted sample was analysed by UV-vis spectrophotometer to determine the concentration

150

of avermectin. A control experiment was also carried out by using ethanol and water (1:4) mixture

151

at pH 9.0 as the releasing medium without DTT.

152

2.10. Toxicological experiments of the pesticide

153

The moth used in the toxicological experiment was kept indoors by the Institute of Plant

154

Protection Pesticide Resistance Group of Chinese Academy of Agricultural Sciences. The

155

avermectin EC and measuring samples were diluted with water to 100, 50, 20, 10, 5 and 1 mg/L

156

series. The cabbage leaves were cleaned with water and dried. The suitable sized leaves were

157

immersed in different concentrations of liquid for 10 seconds. After dried at room temperature, the

158

leaves were placed in petri dishes lined with filter paper. The diamondback moth larvae were

159

selected as the testing insects. The moths were carefully placed onto the leaves using a brush with

160

ten moths in each dish. And each treatment was repeated for three times. Pure water was used as

161

the blank control. The dishes were sealed with plastic wrap, which was perforated using a

162

dissecting needle. These samples were incubated at normal indoor feeding condition (temperature

163

was 25 ± 2 ºC, humidity was 75 ± 5%). After 48 hours, the mortality was calculated by counting

164

the dead and live insects. The virulence regression equation, the lethal concentration 50 (LC50) and

165

the confidence limit of the correlation coefficient were calculated by DPS software.

166 167

3. RESULTS AND DISCUSSION

168

3.1. Synthesis and characterization of the polyurethane emulsifier

169

In order to fabricate avermectin nanoemulsion, a polyurethane based emulsifier was designed


Page 8 of 34

Page 9 of 34


170

to possess drug-affinity, hydrophilicity and cleavable linkages. The polyurethane emulsifier was

171

prepared from IPDI, avermectin, DMPA and HEDS by a procedure outlined in Scheme 1.

172

Although there are three hydroxyl groups in each avermectin molecule, only the two secondary

173

hydroxyl groups at C4’’and C5 are able to react with the isocyanate group, while the tertiary

174

hydroxyl at C7 is inactive due to steric hindrance.52 Therefore, avermectin should be regarded as a

175

difunctional monomer in the synthesis of polyurethane. To achieve a sufficient reaction yield,

176

avermectin was reacted first with excessive IPDI for sufficient time to obtain a polyurethane

177

prepolymer. Afterwards, the prepolymer was reacted with DMPA and HEDS to give the final

178

polyurethane. Through modulating the molar ratios of the reactants, a series of polyurethanes were

179

synthesized (see Table 1).

180

The chemical structure of the resulting polyurethane was mostly confirmed by FT-IR

181

spectrum. The FT-IR spectrum of avermectin itself had a peak at 3400-3500 cm-1, representing the

182

stretching vibration of hydroxyl groups (see Figure S1). However, in the FT-IR spectrum of the

183

polyurethane (see Figure 1), no obvious peak at 3400-3500 cm-1 was shown, indicating that most

184

of hydroxyl groups were reacted. The asymmetric stretching vibration peak of isocyanate group at

185

2260-2280 cm-1 also disappeared, indicating no remaining isocyanate groups in the sample. The

186

peaks at 3200-3350, 1670 and 1712 cm-1 represented the stretching vibration of hydrogen bonding

187

of N-H, amine ester carbonyl group and carbonyl group of carboxylic acid group, respectively.

188

The peak at 1530-1540 cm-1 represented the second band of amide group. In addition, the

189

characteristic adsorption peaks of avermectin at 2990-3000, 1057, 1454 and 1383 cm-1 were also

190

found in Figure 1, indicating the existence of avermectin units. The peak at 2565cm-1 proved the

191

existence of disulfide bonds in the sample.



192

Moreover, the existence of disulfide bonds could be confirmed by 1H-NMR and elemental

193

analysis. In the 1H-NMR spectrum of HEDS, the characteristic peaks were appeared at 4.8, 3.6

194

and 2.8 ppm (see Figure S2). The 1H-NMR spectrum of polyurethane (see Figure S3) also has the

195

characteristic peak at 2.8 ppm, which was attributed to the hydrogen atom of methylene group

196

connecting to disulfide bond. In contrast, this peak was not found in the 1H-NMR spectrum of the

197

polyurethane without disulfide bond.48 Moreover, several characteristic peaks of avermectin (0.91

198

ppm, 3.86~3.92 ppm, and 5.59 ppm) could also be found in the spectrum. The content of sulfur

199

element in polyurethane could be obtained by elemental analysis. The result in Table S1 showed

200

that the actual sulfur content was slightly lower than the theoretical one, attributed to the

201

incomplete recovery of polyurethane during the precipitation process. In summary, the above

202

results confirmed that the polyurethane contained all the structure units as designed.

203

3.2. Fabrication avermectin nanoemulsion

204

After neutralized by triethylamine, the polyurethane became a suitable emulsifier for

205

avermectin considering its amphiphilicity and drug-affinity. Avermectin nanoemulsion were

206

formed spontaneously when the solution, containing the emulsifier and avermectin (at weight ratio

207

1:1), was dropped into deionized water under stirring. Driven by hydrophobic interaction, the

208

emulsifier molecules folded and wrapped around avermectin molecules. At the same time there

209

was electrostatic repulsion among the hydrophilic segments thus increasing stability. Therefore the

210

stable nanoemulsion containing avermectin nanoparticles was formed.

211

The feeding ratio of raw materials and the particle sizes of resulting nanoemulsions are

212

summarized in Table 1. From the table, the Z-average particle sizes were in the range of

213

64.4-145.5 nm. Moreover, the particle sizes decreased gradually as increasing mass fraction of


Page 10 of 34

Page 11 of 34


214

DMPA (from emulsifier 1 to emulsifier 5), owing to the decrease in the emulsifier hydrophilicity.

215

We also found if avermectin was not used in the synthesis, the resulting polymer was unable to

216

stabilize avermectin due to the lack of sufficient hydrophobicity. For comparison, a polyurethane

217

without disulfide bonds was prepared from IPDI, avermectin and DMPA (emulsifier No. 6, see

218

Table 1). The corresponding nanoemulsion had a particle size of 56.6±1.7 nm. Unless otherwise

219

indicated, the nanoemulsion formed in present of emulsifier No. 3 was used in the following

220

investigation.

221

Based on the feeding composition, the nanoemulsion had a drug loading of 50 wt% and the

222

concentration of avermectin was 22.9 mg/mL. This nanoemulsion was an environmentally friendly

223

product because it used water as the main solvent for the continuous phase and the organic solvent

224

content could be lowered to 3 wt% after removal butanone by distillation.

225

The morphology of the nanoparticles in the nanoemulsion was observed by TEM. From the

226

results in Figure 2(a, b), the particles were all with spherical shape. Based on TEM images, the

227

particle size and the size distribution were obtained. As shown in Figure 2(c), the diameters of

228

most particles were in the range of 40-100 nm. The average particle size was calculated to be 66.2

229

nm, smaller than the Z-average particle size of 85.8±3.3 nm (see Table 1).

230

3.3. Stability of the nanoemulsion

231

The nanoemulsion stability against dilution should be tested because the concentration of

232

pesticide nanoemulsion is usually rather low in practical use. 53 We found that after diluted for 100

233

folds using deionized water, the nanoemulsion displayed improved transparency with the particle

234

size decreased from 85.8±3.3 nm to 74.5±3.5 nm, as shown in Figure 3(a).

235

The storage stability of the nanoemulsion was evaluated by centrifugal accelerated



236

sedimentation experiment. After centrifugation for different time periods, the appearance and

237

transparency of the nanoemulsion did not change at all, while the particle sizes remained in the

238

range of 80-90 nm, as indicated in Figure 3(b). Actually, the nanoemulsion remained stable even

239

after one month storage. The reason for the high stability of the nanoemulsion could be the strong

240

charge repulsion caused by the large surface area in the nanoemulsion.

241

3.4. Surface tension measurement

242

Low surface tension is a desirable property for the nanoemulsion droplets’ spreading and

243

wetting over the plant leaves, thus preventing the droplets from slipping. The results of surface

244

tension test of the aqueous solution of the emulsifier with different concentrations were shown in

245

Figure 4, which exhibited a typical surface tension curve of surfactants.54 The surface tension of

246

pure deionized water was measured to be 71.0 mN/m at 25 ℃ . In the lower emulsifier

247

concentration range, the surface tension of the liquid decreased very significantly with the increase

248

of emulsifier concentration. After passing through a critical concentration, the surface tension

249

gradually reached to a constant value (35.5 mN/m). Based on this measurement, the critical

250

micelle concentration of the emulsifier was determined to be 1.95 g/L.

251

The rice leaf has a rather low surface tension of 29.9 mN/m55 because it contains a thick wax

252

layer, tomenta, and mastold microstructure. It is a challenge to make the nanoemulsion droplet

253

spreading on the leaf. With the help of the emulsifier, the surface tension of the nanoemulsion was

254

lowered to 35.5 mN/m, which is still larger than that of the rice leaf. So the contact angle of the

255

nanoemulsion droplet measured on the rice leaf was large and the rolling angle was small (see

256

Table S2). When the surfactant BYK-345, a polyether modified polysiloxane, was added into the

257

nanoemulsion, the surface tension was further reduced (see Table S2). At the BYK-345


Page 12 of 34

Page 13 of 34


258

concentration of 0.4 wt%, the surface tension of the nanoemulsion reached 29.8 mN/m, lower than

259

that of the rice leaf. In such case, the nanoemulsion droplet spread completely on the leaf (see

260

Figure S4). Even at BYK-345 concentration of 0.1 wt%, the rolling angle was elevated to more

261

than 90º (see Table S2). High rolling angle is indeed favourable for reducing the loss of pesticide

262

during the spraying process.

263

3.5. Retention on leaves

264

During the spraying process, pesticide is deposited on the crop leaf, and then it goes to other

265

parts of plant for pest poisoning. High affinity of the pesticide liquid to leaf is desired for reducing

266

the waste and extending the efficacy time of a pesticide. Jia and co-workers reported that

267

polydopamine encapsulated avermectin nanoparticles could adhere to the leaf surface, but the

268

preparation process of polydopamine was complicated.56

269

In this study, leaf retention was firstly measured to understand the affinity of pesticide liquid

270

on the leaves. The results in Figure 5 indicated that the retention on hydrophobic cabbage leaves

271

of the nanoemulsion was 1.24 times and 1.92 times of the commercial avermectin EC and

272

deionized water, respectively, while the retention on hydrophilic cucumber leaves was 1.41 times

273

and 1.76 times of the commercial avermectin EC and deionized water, respectively. These results

274

demonstrated that the nanoemulsion had strong affinity on both hydrophilic and hydrophobic

275

leaves, which was likely owing to the lower surface tension of the emulsifier itself.

276

After the solvent evaporated, the pesticide powder should also adhere on the leaf for a long

277

period of time, maintains long-term efficacy and thus reduces the frequency of spraying. The

278

as-prepared and diluted nanoemulsions were separately sprayed on rice leaves. After the solvent

279

evaporated, the leaves were rinsed by water for sufficient time (30 minutes). We noticed that the



280

avermectin residues still existed on the leaves after this treatment. However, when

281

avermectin/ethanol solution was sprayed, the avermectin could be rinsed away almost completely

282

(see Figure S5). In is proved that the high boiling point solvent (NMP) enabled the nanoparticles

283

coalescing together into thin film during evaporation process, similar as the coalescing agent in

284

latex paint. 57 This could make the pesticide powder adhere tightly on the leaf. A film was clearly

285

observed when avermectin nanoemulsion was sprayed and dried on the glass plate (see Figure S6).

286

3.6. Photostability of avermectin in nanoparticles

287

Avermectin is a photosensitive pesticide. The decomposition of avermectin is mainly due to

288

the allyl peroxide reaction on the main ring.58 It is interesting to investigate the photostability of

289

avermectin when it was encapsulated in the nanoparticles. The UV absorption spectra of the

290

emulsifier and avermectin were obtained separately at concentration of 100 mg/L. As indicated in

291

Figure 6(a), the characteristic absorption peaks were almost the same for the two samples, but the

292

absorption intensity of avermectin was much higher than that of the emulsifier (about 13.5 times at

293

244 nm).

294

The encapsulated avermectin powder was obtained by acid treatment of the nanoemulsion.

295

The powders of pure avermectin and the encapsulated avermectin were irradiated by UV light

296

separately. The UV-vis absorption spectra of the solutions of the two treated samples were

297

measured. The remaining percentage of avermectin could be calculated based on the spectra. From

298

the results in Figure 6(b), pure avermectin degraded much faster than the encapsulated one even

299

from the very beginning. After exposed to UV light for 7 minutes, only 42.5±2.2% of pure

300

avermectin remained, while 77.2±6.2% encapsulated avermectin remained. Located at the outer

301

layer of the nanoparticles, the emulsifier molecules absorbed UV light before the interior


Page 14 of 34

Page 15 of 34


302

avermectin molecules since they had similar UV absorption profiles. So the photostability of

303

avermectin could be improved considerably in the encapsulated state.

304

3.7. Drug releasing profile

305

The releasing performances of nano-pesticides were very important for their practical use. If

306

pesticide molecules were released from nanoparticles in a controlled manner or under specific

307

conditions, the efficacy and safety of nano-pesticide would be improved. For this purpose,

308

disulfide groups were deliberately introduced into the emulsifier molecules in this research.

309

To investigate the releasing behavior of the nanoemulsion, the nanoemulsion was incubated

310

in the mixture of water and ethanol (4:1) at pH 9 with or without DTT. As shown in Figure 7, the

311

nanoemulsion released avermectin to the environment quite slowly without DTT, indicating the

312

chemical stability of the emulsifier. However, avermectin was released in an accelerated rate with

313

the help of DTT.

314

In the presence of DTT under basic condition, disulfide bonds were reduced to two mercapto

315

groups through a two-step continuous thiol-disulfide bond exchange reaction.59 As disulfide bonds

316

being cleaved by DTT, the emulsifier molecules degraded into low molecular weight fragments. If

317

all disulfide bonds were cleaved, only one fourth the fragments had avermectin units and the

318

others had no avermectin unit because the stoichiometric ratio of HEDS and avermectin is 3:1 for

319

emulsifier No. 3 (see Table 1). The part of fragments without the avermectin unit was hydrophilic

320

enough to be dissolved in water. As the degradation going on, more and more fragments were

321

dissolved in water. As a result, the particles gradually became unstable in water due to the lack of

322

surface charge stabilization. They aggregated together into large sized particles until precipitates

323

appeared in the system (see Figure S7). As the destruction of the emulsifier, avermectin were



324

released in an accelerated rate.

325

3.8. Toxicological property

326 327

Drug releasing performance of nano-pesticide has great influence on the insecticide effect. The insecticide effect could be improved if the pesticide could be released in a specific manner.

328

Firstly, the litura experiment was applied to determine the virulence of the emulsifier itself.

329

The results indicated that the emulsifier itself was substantially non-toxic compared to avermectin

330

EC (see Table S3). So the toxicity of the nanoemulsion was all due to the avermectin encapsulated

331

in the nanoparticles.

332

The insecticidal effect of a pesticide could be evaluated by the LC50 value.60 The virulence of

333

avermectin nanoparticles with (using emulsifier No. 3) and without disulfide bonds (using

334

emulsifier No. 6, as the control) were determined. As shown in Table 2, the LC50 value of the

335

present nanoparticles was only 52.71 µg/mL, significantly lower than that (385.99 µg/mL) of the

336

control sample. In the same condition, the LC50 value of avermectin EC was 69.56 µg/mL. Based

337

on these results, the insecticidal effect decreased by the order of the present nanoparticles,

338

avermectin EC, the control nanoparticles. The high insecticidal ability of the current nanoparticles

339

should be attributed to the cleavage of disulfide bonds inside the insect. Disulfide bond is a

340

common bond in proteins, it could be cleaved in vivo by high concentrations of glutathione as

341

well.61-62 As a result, high insecticidal property was achieved since avermectin were released easily.

342

The existence of disulfide bonds in the emulsifier molecule provided a facile approach to control

343

the pesticide releasing from practical viewpoint since no additional trigger was required.

344

In summary, our investigation had demonstrated the importance in structural designing of

345

emulsifier. With this emulsifier, the resulting avermectin nanoemulsion exhibited many satisfying


Page 16 of 34

Page 17 of 34


346

performances, including high insecticidal ability. We believe this study would be helpful for

347

designing more advanced emulsifiers of nano-pesticides. The scale-up experiments and

348

application of the nanoparticles are now under investigation in our group.

349 350

ASSOCIATED CONTENT

351

352

Figure S1: FT-IR spectrum of avermectin. Figure S2: 1H-NMR spectrum of 2-hydroxyethyl

353

disulfide. Figure S3: 1H-NMR spectrum of the emulsifier with disulfide bonds. Figure S4: Contact

354

angles measured on rice leaves. Figure S5: Comparison of leaf affinity for various samples. Figure

355

S6: Film formed of the as-prepared nanoemulsion. Figure S7: Nanoemulsion (1) and the system

356

after the addition of DTT (2). Table S1: Elemental analytic result of the emulsifier with disulfide

357

bonds. Table S2: Surface tensions, contact angles and rolling angles measured on rice leaves.

358

Table S3: Toxicity test of spodoptera litura with the emulsifier and avermectin EC.

359

AUTHOUR INFORMATION

360

Corresponding Author

361

* Tel: +86 10 6278 2148. E-mail: [email protected].

362

Notes

363

The authors declare no competing financial interest.

364

ACKNOWLEDGEMENTS

Supporting Information

365

This work was financial supported by the National Basic Research Program of China (973

366

plan, No. 2014CB932202), the National Natural Science Foundation of China (No. 21574074) and

367

the Fund of Key Laboratory of Advanced Materials of Ministry of Education (No. 2017AML08).

368

REFERENCES



369 370 371 372 373 374

(1) International code of conduct on the distribution and use of pesticides. Food and Agriculture Organization of

the United Nations, 2007.

(2) Lamberth, C.; Jeanmart, S.; Luksch, T. Plant, A. Current challenges and trends in the discovery of

agrochemicals. Science 2013, 341, 742–746.

(3) Grube, A.; Donaldson, D.; Kiely, T.; Wu, L. Pesticides industry sales and usage. Environmental Protection

Agency, 2011.

375

(4) Arcella, T.; Hood, G. R.; Powell, T. H. Q.; Sim, S. B.; Yee, W. L.; Schwarz, D.; Egan, S. P.; Goughnour, R.

376

B.; Smith, J. J.; Feder, J. L. Hybridization and the spread of the apple maggot fly, rhagoletis pomonella

377

(Diptera: tephritidae), in the northwestern United States. Evol. Appl., 2015, 8, 834–846.

378

(5) Woodard, S. H.; Lozier, J. D.; Goulson, D.; Williams, P. H.; Strange, J. P.; Jha, S. Molecular tools and

379

bumble bees: revealing hidden details of ecology and ecolution in a model system. Mol. Ecol. 2015, 24,

380

2916–2936.

381

(6) El-Wakeil, N. E. Botanical pesticides and their mode of action. Gesunde Pflanz. 2013, 65, 125–149.

382

(7) Rosenheim, J. A.; Parsa, S.; Forbes, A. A.; Krimmel, W. A.; Law, Y.H.; Segoli, M.; Segoli, M.; Sivakoff,

383

F.S.; Zaviezo, T.; Gross, K. Ecoinformatics for integrated pest management: expanding the applied insect

384

ecologist’s tool-kit. J. Econ. Entomol. 2011, 104, 331–342.

385 386 387 388 389 390

(8) Knauer, K.; Bucheli, T. Nano-materials-the need for research in agriculture. Agrarforschung Schweiz 2009,

16, 390−395.

(9) Ghormade, V.; Deshpande, M. V.; Paknikar, K. M. Perspectives for nano-biotechnology enabled protection

and nutrition of plants. Biotechnol. Adv. 2011, 29, 792−803.

(10) Kah, M.; Beulke, S.; Tiede, K.; Hofmann, T. Nano-pesticides: state of knowledge, environmental fate and

exposure modelling. Crit. Rev. Environ. Sci. Technol. 2013, 43, 1823−1867.


Page 18 of 34

Page 19 of 34


391 392

(11) Mahendra, R.; Avinash, I. Role of nanotechnology in agriculture with special reference to management of

insect pests. Appl. Microbiol. Biotechnol. 2012, 94, 287–293.

393

(12) Servin, A.; Elmer, W.; Mukherjee, A.; Torre-Roche, R. D. L.; Hamdi, H.; White, J. C.; Bindraban, P.;

394

Dimkpa, C. A review of the use of engineered nanomaterials to suppress plant disease and enhance crop yield.

395

J. Nanopart. Res. 2015, 17, 92.

396 397 398 399

(13) Scott, N.; Chen, H. D. Nanoscale science and engineering for agriculture and food Systems. Ind. Biotechnol.

2012, 8, 340–343.

(14) Gogos, A.; Knauer, K.; Bucheli, T. Nanomaterials in plant protection and fertilization: current state, foreseen

applications, and research priorities. J. Agric. Food Chem. 2012, 60, 9781−9792.

400

(15) Kookana, R. S.; Boxall, A. B. A.; Reeves, P. T.; Ashauer, R.; Beulke, S.; Chaudhry, Q.; Cornelis, G.;

401

Fernandes, T. F.; Gan, J.; Kah, M. Nanopesticides: Guiding principles for regulatory evaluation of

402

environmental risks. J. Agric. Food Chem. 2014, 62, 4227–4240.

403 404 405 406

(16) Servin, A. D.; White, J. C. Nanotechnology in agriculture: Next steps for understanding engineered

nanoparticle exposure and risk. NanoImpact. 2016, 1, 9–12.

(17) Vasir, J. K.; Labhasetwar, V. Biodegradable nanoparticles for cytosolic delivery of therapeutics. Adv. Drug

Delivery Rev. 2007, 59, 718−728.

407

(18) Metselaar, J. M.; Bruin, P.; de Boer, L. W. T.; de Vringer, T.; Snel, C.; Oussoren, C.; Wauben, M. H. M.;

408

Crommelin, D. J. A.; Storm, G.; Hennink, W. E. A novel family of L-amino acid-based biodegradable

409

polymer-lipid conjugates for the development of long-circulating liposomes with effective drug-targeting

410

capacity. Bioconjugate Chem. 2003, 14, 1156−1164.

411 412

(19) Shah, R. B.; Schwendeman, S. P. A biomimetic approach to active self-microencapsulation of proteins in

PLGA. J. Control Release, 2014, 196, 60–70.



413

(20) Yu, M. L.; Yao, J. W.; Liang, J.; Zeng, Z. H.; Cui, B.; Zhao, X.; Sun, C. J.; Wang, Y.; Liu, G. Q.; Cui, H. X.

414

Development of functionalized abamectin poly(lactic acid) nanoparticles with regulatable adhesion to

415

enhance foliar retention. RSC Adv. 2017, 7, 11271–11280.

416

(21) Yang, F. L.; Li, X. G.; Zhu, F.; Lei, C. L. Structural Characterization of Nanoparticles Loaded with Garlic

417

Essential Oil and Their Insecticidal Activity against Tribolium castaneum (Herbst) (Coleoptera:

418

Tenebrionidae). J. Agric. Food Chem. 2009, 57, 10156−10162.

419 420 421 422 423 424 425 426 427 428 429 430

(22) Onwulata, C. I. Microencapsulation and functional bioactive foods. J. Food Process. Preserv. 2013, 37, 510–

532.

(23) Elek, N.; Hoffman, R.; Raviv, U.; Resh, R.; Ishaaya, I.; Magdassi, S. Novaluron nanoparticles: Formation and

potential use in controlling agricultural insect pests. Colloids Surf. A 2010, 372, 66−72.

(24) Thomas, J.; Kumar, K.; Praveen, C. Synthesis of Ag doped nano TiO2 as efficient solar photocatalyst for the degradation of endosulfan. Adv. Sci. Lett. 2011, 4, 108−114.

(25) Lu, J. W.; Li, F. B.; Guo, T.; Lin, L. W.; Hou, M. F.; Liu, T. X. TiO2 photocatalytic antifungal technique for crops diseases control. J. Environ. Sci. 2006, 18, 397−401.

(26) Jo, Y. K.; Kim, B. H.; Jung, G. Antifungal activity of silver ions and nanoparticles on phytopathogenic fungi.

Plant Dis. 2009, 93, 1037−1043.

(27) Kim, H.; Kang, H.; Chu, G.; Byun, H. Antifungal effectiveness of nanosilver colloid against rose powdery

mildew in greenhouses. Solid State Phenom. 2008, 135, 15−18.

431

(28) Nandiyanto, A. B. D.; Kim, S. G.; Iskandar, F.; Okuyama, K. Synthesis of spherical mesoporous silica

432

nanoparticles with nanometer-size controllable pores and outer diameters. Micropor Mesopor Mat. 2009, 120,

433

447–453.


Page 20 of 34

Page 21 of 34


434 435 436 437 438 439 440 441 442 443

(29) Pu, H. T.; Liu, L.; Chang, Z. H.; Yuan, J. J. Organic/inorganic composite membranes based on

polybenzimidazole and nano-SiO2. Electrochim. Acta 2009, 54, 7536–7541. (30) Debnath, N.; Das, S.; Seth, D.; Chandra, R.; Bhattacharya, S.; Goswami, A. Entomotoxic effect of silica

nanoparticles against Sitophilus oryzae (L.). J. Pestic. Sci. 2010, 84, 99−105.

(31) Stadler, T.; Buteler, M.; Weaver, D. K. Novel use of nanostructured alumina as an insecticide. Pest Manage.

Sci. 2010, 66, 577−579.

(32) Liang, R.; Liu, M. Z. Preparation of poly(acrylic acid-coacrylamide)/kaolin and release kinetics of urea from

it. J. Appl. Polym. Sci. 2007, 106, 3007−3015.

(33) Wu, J.; Xue, D. Progress of science and technology of ZnO as advanced material. Sci. Adv. Mater., 2011, 3,

127−149.

444

(34) Milani, N.; McLaughlin, M. J.; Stacey, S. P.; Kirby, J. K.; Hettiarachchi, G. M.; Beak, D. G.; Cornelis, G.

445

Dissolution kinetics of macronutrient fertilizers coated with manufactured zinc oxide nanoparticles. J. Agric.

446

Food Chem. 2012, 60, 3991−3998.

447

(35) Schneider, K. H.; Karpov, A.; Voss, H.; Dunker, S.; Merk, M.; Kopf, A.; Kondo, S. Method for treating

448

phytopathogenic microorganisms using surface-modified nanoparticulate copper salts. WO/2011/067186,

449

2011.

450 451

(36) Musameh, M.; Notivoli, M. R.; Hickey, M. Carbon nanotube-web modified electrodes for ultrasensitive

detection of organophosphate pesticides. Electrochim. Acta 2013, 101, 209–215.

452

(37) Khodakovskaya, M.; Dervishi, E.; Mahmood, M.; Xu, Y.; Li, Z. R.; Watanabe, F.; Biris, A. S. Carbon

453

nanotubes are able to penetrate plant seed coat and dramatically affect seed germination and plant growth.

454

ACS Nano 2009, 3, 3221−3227.



455

(38) Chang, X. L.; Zhai, B. P.; Liu, X. D.; Wang, M. Effects of temperature stress and pesticide exposure on

456

fluctuating asymmetry and mortality of copera annulata (selys) (odonata: zygoptera) larvae. Ecotox. Environ.

457

Safe. 2007, 67, 120–127.

458 459

(39) Fiel, C. A.; Saumell, C. A.; Steffan, P. E.; Rodriguez, E. M. Restance of cooperia to ivermectin treatments in

grazing cattle of the humid pampa. Argentina. Vet. Parasitol. 2001, 97, 211–217.

460

(40) Shen, B. A new golden age of natural products drug discovery. Cell 2015, 163, 1297–1300.

461

(41) Li, M.; You, T. Z.; Zhu, W. J.; Qu, J. P.; Liu, C.; Zhao, B.; Xu, S. W.; Li, S. Antioxidant response and

462

histopathological changes in brain tissue of pigeon exposed to avermectin. Ecotoxicology 2013, 22, 1241–

463

1254.

464

(42) Dalzell, A. M.; Mistry, P.; Wright, J.; Williams, F. M.; Brown, C. D. A. Characterization of multidrug

465

transporter-mediated efflux of avermectins in human and mouse neuroblastoma cell lines. Toxicol. Lett. 2015,

466

235,189–198.

467 468

(43) Shoop, W. L.; Mrozik, H.; Fisher, M. H. Structure and activity of avermectins and milbemycins in animal

health. Vet. Parasitol. 1995, 59,139–156.

469

(44) Li, Z. Z.; Xu, S. A.; Wen, L. X.; Liu, F.; Liu, A. Q.; Wang, Q.; Sun, H. Y.; Yu, W.; Chen, J. F. Controlled

470

release of avermectin from porous hollow silica nanoparticles: Influence of shell thickness on loading

471

efficiency, UV-shielding property and release. J. Control Release 2006, 111, 81−88.

472 473 474 475

(45) Guan, H. N.; Chi, D. F.; Yu, J.; Zhang, S. Y. Novel photodegradable insecticide W/TiO2/Avermectin

nanocomposites obtained by polyelectrolytes assembly. Colloids Surf. B 2011, 83, 148−154.

(46) Jia, X.; Sheng, W. B.; Li, W.; Tong, Y. B.; Liu, Z. Y.; Zhou, F. Adhesive polydopamine coated avermectin

microcapsules for prolonging foliar pesticide retention. ACS Appl. Mater. Inter. 2014, 6, 19552–19558.


Page 22 of 34

Page 23 of 34


476 477 478 479

(47) Li, D. Q.; Wu, X. W.; Yu, X. Q.; Huang, Q. C.; Tao, L. M. Synergistic effect of non-ionic surfactants Tween

80 and PEG6000 on cytotoxicity of insecticides. Environ. Toxicol. Pharmacol. 2015, 39, 677–682.

(48) Zhang, W. X.; Mei, H.; Guan, W. X.; Tang, L. M. Preparation, Characterization and stability of avermectin

nano aqueous dispersion, Chem. J. Chinese U. 2015, 36, 1208–1212.

480

(49) Wang, C.; Wesener, S. R.; Zhang, H.; Cheng, Y. Q. An FAD-depend enpyridine nucleotide-disulfide

481

oxidoreductase is involved in disulfide bond formation in FK228 anticancer depsipeptide. Chem. Biol. 2009,

482

16, 585–93.

483 484

(50) Yuan, H. Z.; Qi, S. H.; Yang, D. B. Study on the point of runoff and the maximin retention of spray liquid on

crop leaves. Chin. J. Pestic. Sci. 2000, 2, 66–71.

485

(51) Ren, G.; Liu, D.; Guo, W.; Wang, M.; Wu, C.; Guo, M.; Ai, X. Y.; Wang, Y. J.; He, Z. G. Docetaxel prodrug

486

liposomes for tumor therapy: characterization, in vitro and in vivo evaluation. Drug Deliv. 2016, 23, 1272–

487

1281.

488

(52) Wang, D. Q. Structural modification of avermectins. J. China Agr. Uni. 1994, 20, 431–437.

489

(53) Yuan, H. Z.; Yang, D. B.; Yan, X. J.; Zhang, L. N. Pesticide efficiency and the way to optimize the spray

490 491 492 493 494 495 496

application. Plant Protection 2011, 37, 14–20.

(54) Islam, M. T.; Sarwar, A. K. M. G.; Begum, H. H.; Ito, T. Epidermal features of rice leaf cv. BRRI dhan 29.

Bangl. J. Plant Taxon. 2009, 16, 177–180.

(55) Eastoe, J.; Dalton, J. S. Dynamic surface tension and adsorption mechanisms of surfactants at the air-water

interface. Adv. Colloid Interface Sci. 2000, 85, 103–144.

(56) Liu, W. J.; Yao, J.; Cai, M. M.; Chai, H. K.; Zhang, C.; Sun, J. J.; Radhika, C.; Kanaji, M.; Synthesis of a

novel nanopesticide and its potential toxic effect on soil microbial activity. J. Nanopart. Res. 2014, 16, 2677.



497

(57) Mu, Y. C.; Li, X. C.; Qiu, T.; He, L. F.; Zhang, S. W.; Li, X. Y. The study on the film formation of the

498

core-shell latex by using the coalescing agent, J. B. Uni. Chem. Technol.( Nat. Sci. Ed.) 2011, 38, 44–49.

499

(58) Burkhard, R.; Binz, H.; Roux, C. A.; Brunner, M.; Ruesch, O.; Wyss, P. Environmental fate of emamectin

500 501 502 503 504

benzoate after tree micro injection of horse chestnut trees. Environ. Toxicol. Chem. 2015, 34, 297–302.

(59) Rowan, S. J.; Cantrill, S. J.; Cousins, G. R. L.; Sanders, J. K. M.; Stoddart, J. F. Dynamic covalent chemistry.

Angew. Chem., Int. Ed. 2002, 41, 898–952.

(60) Kees, V. F. Insecticidal activity of Bacillus thuringiensis crystal proteins. J. Invertebr. Pathol., 2009, 101, 1–

16.

505

(61) Arunachalam B, Phan U T, Geuze HJ, Cresswell P. Enzymatic reduction of disulfide bonds in lysosomes:

506

Characterization of a Gamma-interferon-inducible lysoso mal thiol reductase (GILT). Proc. Natl. Acad. Sci.

507

USA. 2000, 97, 745–750.

508 509

(62) Wenzel, T.; Baumeister, W. Conformational constraints in protein-degradation by the 20S proteasome. Nat.

Struct. Biol. 1995, 2, 199–204.


Page 24 of 34

Page 25 of 34


Figures and tables

Scheme 1. Synthetic procedure for the polyurethane emulsifiers



Figure 1. FT-IR spectrum of the emulsifier with disulfide bonds


Page 26 of 34

Page 27 of 34


Figure 2. TEM images (a, b) of the particles and particles size distribution (c) of the nanoemulsion



Figure 3. Change in particle sizes after (a) dilution and (b) centrifugal treatments


Page 28 of 34

Page 29 of 34


Figure 4. Surface tension at different concentration of the emulsifier aqueous solution



Figure 5. Leaf retention of avermectin nanoemulsion, avermectin EC and deionized water


Page 30 of 34

Page 31 of 34


Figure 6. (a) UV-vis spectra of the emulsifier and pure avermectin . (b) Remaining percentage of avermectin at different irradiation time



Figure 7. Avermectin releasing curves for the nanoemulsions with and without DTT


Page 32 of 34

Page 33 of 34


Table 1. Formulation of the emulsifiers and particle size of the nanoemulsions emulsifier

IPDI: Avermectin: HEDS:

Mass fraction of

Z-average particle size of the

DMPA (molar ratio)

DMPA (wt%)

nanoemulsions (nm)

1

16.5 : 2 : 4 : 10.5

18.8

64.4±4.9

2

17.5 :2 : 5 : 10.5

17.9

76.8±3.7

3

18.5 : 2 : 6 : 10.5

17.1

85.8±3.3

4

19.5 : 2 : 7 : 10.5

16.4

94.8±1.9

5

20.5 :2 : 8 : 10.5

15.7

145.5±5.6

6

12.5 : 2 : 0 : 10.5

23.6

56.6±1.7

Table 2. Toxicity test results of different pesticide samples Pesticide samples

Toxicity regression

LC50

95% confidence

equation

(µg/mL)

interval

Present avermectin nanoparticles a

y=2.9576+1.1861x

52.71

40.03~70.38

Control avermectin nanoparticles b

y=1.5174+1.3464x

385.99

202.21~2570.13

Avermectin EC

y=1.6918+1.7956x

69.56

55.32~93.63

a

Using emulsifier No. 3, b using emulsifier No. 6.



Table of content


Page 34 of 34

Fabrication of Novel Avermectin Nanoemulsion Using a Polyurethane

Recommend Documents