Preparation and Characterization of Controlled-Release Avermectin

Article

Preparation and Characterization of the Controlled Release Avermectin/Castor Oil-based Polyurethane Nanoemulsions Hong Zhang, He Qin, Lingxiao Li, Xiaoteng Zhou, Wei Wang, and Chengyou Kan J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 31 May 2017 Downloaded from http://pubs.acs.org on June 1, 2017

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Journal of Agricultural and Food Chemistry

Preparation and Characterization of the Controlled Release Avermectin/Castor Oil-based Polyurethane Nanoemulsions Hong Zhang, He Qin, Lingxiao Li, Xiaoteng Zhou, Wei Wang, Chengyou Kan* Department of Chemical Engineering and Key Laboratory of Advanced Materials of Ministry of Education, Tsinghua University, Beijing 100084, P. R. China *Corresponding author: Email: [email protected]; Tel: +86-10-62773456; Fax: +86-10-62794191

ACS Paragon Plus Environment


1

Abstract

2

Avermectin (AVM) is a low-toxic and high-active bio-pesticide, but it can be easily

3

degraded by the UV light. In this article, biodegradable castor oil-based polyurethanes

4

(CO-PU) are synthesized and used as carriers to fabricate a new kind of AVM/CO-PU

5

nanoemulsions through an emulsion solvent evaporation method, and the chemical

6

structure, the colloidal property, the AVM loading capacity, the controlled release

7

behavior, the foliar adhesion and the photostability of the AVM/CO-PU drug delivery

8

systems are investigated. Results show that AVM is physically encapsulated in the

9

CO-PU carrier nanospheres, and the diameter of the AVM/CO-PU nanoparticles is

10

less than 50 nm and the AVM/CO-PU films are flat and smooth without any AVM

11

aggregate. The drug loading capacity is up to 42.3 wt% with a high encapsulation

12

efficiency of above 85%. The release profiles indicate that the release rate is relatively

13

high at earlier stage and then slowdown, which can be adjusted by loaded AVM

14

content, temperature and pH of release medium. The foliar pesticide retention of the

15

AVM/CO-PU nanoemulsions is improved, and the photolysis rate of AVM in the

16

AVM/CO-PU nanoparticles is significantly slower than that of the free AVM. A

17

release mechanism of the AVM/CO-PU nanoemulsions is proposed, which is

18

controlled by both diffusion and matrix erosion.

19

Keywords

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Avermectin; Castor oil-based polyurethane; Drug-loaded nanoemulsion; Controlled

21

release; Photostability

22


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1. Introduction

24

Pesticides are very important agrochemicals and largely used in agriculture to

25

maintain high crop yields and sufficient food supplies, but most of the pesticides are

26

lost or decomposed in application 1, and only about 0.1% can finally affect harmful

27

organisms 2. Without doubt, the low bioavailability and overdosing will bring about

28

the problems of environmental pollution and human health

29

drawbacks of conventional pesticides, pesticide delivery system has been investigated

30

with the development of nanotechnology over the past few years

31

advanced pesticide delivery system can provide a sustained long effect through

32

maintaining a stable release rate and an appropriate effective concentration of active

33

ingredient over a specified period of time, an appropriate controlled release

34

formulation is contributed to the decrease of the waste and harm of pesticide, as well

35

as the improvement of bioavailability 7-9.

3, 4

. To overcome these

5, 6

. Since an

36

Avermectin (AVM), an alternative of high-toxic pesticides, is recognized as a

37

nuisanceless biological pesticide with a broad insecticidal spectrum and high activity.

38

However, its disadvantages are also obvious, which leads to not only overdose and

39

high cost but also harm to environment and human. On one hand, a large amount of

40

organic solvents have to be used in the main AVM emulsifiable concentrate

41

formulation for its water-insolubility. On the other hand, AVM is easily

42

photo-oxidized and degraded under the irradiation of UV light, resulting in the poor

43

photostability and short half-life

44

have been concerned and some AVM delivery systems with continuous release and

10-13

. Thus, controlled release AVM formulations



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UV degradation resistance have been investigated, in which some inorganic materials

46

14-17

47

silica nanoparticles (PHSN) as carriers to prepare AVM/PHSN systems by a simple

48

immersion loading method or supercritical fluid technology, which showed good

49

controlled release behavior and UV-shielding property

50

polydopamine (PDA) microcapsule to encapsulate AVM, and they found that the

51

AVM/PDA system could not only supply the controlled-release and UV-shielding

52

properties, but also prolong the foliar pesticide retention by adjusting the adhesion

53

property of the microcapsule surface

54

microcapsules with the organic-inorganic composite of silica-glutaraldehyde-chitosan

55

as the carrier was also prepared, and better controlled release longevity was obtained

56

in comparison with single-shelled microcapsules 27. However, the sizes of most AVM

57

drug-loaded particles are not in the range of 1-100 nm, which does not conform to the

58

rigid definition of nanotechnology 28. Taking advantage of the benefits of materials at

59

nanoscale to prepare nanoscale pesticide delivery systems would improve their

60

performances in agricultural application, for the nanoscale pesticide delivery system

61

has smaller size and larger surface area, which is beneficial to spread uniformly over

62

leaves and improve the foliar pesticide deposition and retention 6, 29.

and polymers

18-22

have been used to load AVM. Wen et al. used porous hollow

23, 24

. Jia et al. used

25, 26

. In addition, a novel double-shelled AVM

63

As is known, polyurethane (PU) is the polymer with a variety of building blocks

64

and alternating soft and hard segments, which can be designed and adjusted to meet

65

different requirements, and has been applied in biomedical devices and drug delivery

66

systems 30-33. The polymers prepared using renewable sources such as vegetable oil as


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starting material have attracted widespread attention for the economic and

68

environmental concerns recent years

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castor oil-based PU (CO-PU) has been synthesized due to its low cost, low toxicity

70

and renewability

71

pesticide delivery systems, especially for the systems at nanoscale

72

CO-PU is considered to be a suitable material for the pesticide delivery system due to

73

its biodegradability, optimized size at nanoscale and other adjustable physical

74

properties.

34, 35

. Specifically, as a biodegradable material,

36, 37

. However, there were barely no research on PU-containing 38, 39

. Hence, the

75

In this article, waterborne CO-PU was chosen as a novel carrier to load AVM, and a

76

new kind of AVM/CO-PU drug-loaded nanoemulsions were prepared through an

77

emulsion solvent evaporation method. The colloidal property, the chemical structure,

78

and the performances of the AVM/CO-PU drug-loaded systems were investigated.

79

2. Materials and Methods

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2.1 Materials

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Avermectin (96%) was kindly supplied by the Chinese Academy of Agriculture

82

Science. Isophorone diisocyanate (IPDI), polyether diol N220 (Mn, 2000) and

83

dimethylolpropionic acid (DMPA) were supplied by Linshi Chem Co. Ltd (Beijing,

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China). Castor oil (CO, the average hydroxyl functionality, 2.7) and 1,4-butanediol

85

(BDO) were purchased from Tianjin Bodi Chemical Co. Ltd (Tianjin, China).

86

Dibutyltin dilaurate (DBTDL) was purchased from Tianjin Guangfu Fine Chemical

87

Research Institute (Tianjin, China). Triethylamine (TEA), acetone and ethanol were of

88

reagent grade and purchased from Beijing Chemical Works (Beijing, China).



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Deionized water was used throughout the research.

90

2.2 Preparation of CO-PU emulsion

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The CO-PU emulsion was prepared by the pre-polymer dispersion method

92

follows. IPDI (18.8 mmol, 4.17 g), N220 (1.6 mmol, 3.20 g) and two drops of DBTDL

93

were firstly added into a 100 ml three-necked round bottom flask equipped with an

94

electric mechanical stirrer and a reflux condenser, and the mixture was stirred for 2 h

95

at 80 °C with the stirring speed of 200 rpm. Then 1.21 g of CO (1.3 mmol) was added

96

into the system and the reaction continued for 2 h. After that, 0.54 g of DMPA (4.0

97

mmol) and 2 mL of acetone were charged into the flask, and after 2 h of reaction, 0.27

98

g of BDO (3.0 mmol) and 2 mL of acetone was charged and the reaction continued for

99

about 2 h. The reaction between –NCO and –OH was monitored by the 41

40

as

100

di-n-butylamine titration method

to determine when the reaction was completed.

101

After that, the reaction system was cooled down to 40 °C, and 2 mL of acetone was

102

poured into the system to reduce the viscosity, and 0.41 g of TEA (4.0 mmol) was

103

then dropwise added to neutralize the system for 30 min. Finally, the reaction system

104

was cooled down to room temperature, and a certain amount of water, which was

105

determined by the solid content, was subsequently added into the system with the

106

stirring speed of 1000 rpm for 40 min to obtain the CO-PU emulsion.

107

2.3 Preparation of AVM/CO-PU nanoemulsions

108

The AVM/CO-PU drug-loaded nanoemulsions were prepared by emulsion solvent

109

evaporation method as follows. According to the recipes listed in Table 1, AVM was

110

first dissolved in acetone to get AVM acetone solution, and the CO-PU emulsion was


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111

then dropwise added into the AVM acetone solution under the magnetic stirring to

112

obtain a homogenous oil-water mixture. Then, the mixture was emulsified by two

113

different methods: one is the ultrasonic method (USM) using an ultrasonic processor

114

(Sonics, VC 105PB, USA) in an ice-water bath for 10 min, and the other is the high

115

speed dispersing method (HSDM) using a high-speed dispersion machine (IKA, T25

116

basic, Germany) at 11000 rpm for 10 min. After that, the acetone was removed by

117

vacuum rotary evaporation at 40 °C for about 10 min, and 5 mL of deionized water

118

was added into the system to reduce the viscosity and carry out the redispersion in the

119

solvent evaporation process. Finally, vacuum rotary evaporation was continued until

120

the acetone was removed completely to obtain the AVM/CO-PU nanoemulsion

121

2.4 Characterization

122

Hydrodynamic diameter (Dh), polydispersity index (P.I.) and zeta potential (ζ) of the

123

nanoparticles were determined on a Zetasizer 3000HS (Malvern, UK) at 25 °C, and

124

the samples were prepared by diluting the emulsions with water to the solid content

125

about 0.1 wt%.

126

FTIR spectra were recorded on a FTIR spectrometer (Thermo Fisher Scientific,

127

Nicolet 560, USA) using the free AVM solid powder in KBr or the thin latex films of

128

the nanoemulsions.

129

UV-Vis spectra were recorded on an UV-Vis spectrophotometer (Pgeneral, T6,

130

China) and the absorbance was determined under the maximum absorption

131

wavelength (λmax) of 245 nm. The AVM concentration of the samples was less than 50

132

mg/L.



133

Morphology and number average diameter (Dp) of the dried nanoparticles were

134

characterized by a transmission electron microscope (TEM, Hitachi, H-7650B, Japan)

135

with the accelerating voltage of 80 kV, and the samples were prepared by mounting

136

and drying the diluted nanoemulsions on the carbon-coated copper grids.

137

Morphology of the latex films were observed on a scanning electron microscope

138

(SEM, JEOL, JSM 7401F, Japan) with the accelerating voltage of 3 kV, and the

139

samples were prepared by mounting the latex films onto a sample stage using

140

conductive adhesive tape and followed by spraying a thin layer of gold on the

141

samples.

142

Glass transition temperature (Tg) was examined by differential scanning calorimeter

143

(DSC, TA Instruments, Q5000IR, America) under nitrogen atmosphere. About 5 mg

144

of the latex film was heated from 20 °C to 120 °C at a rate of 20 °C min-1 and held at

145

120 °C for 10 min to eliminate thermal history, and then cooled down to -90 °C at

146

20 °C min-1 and held at -90 °C for 3 min, and finally heated to 100 °C at a 10 °C

147

min-1.

148

2.5 Drug loading capacity and encapsulation efficiency of the AVM/CO-PU

149

nanoemulsions

150

At first, a certain amount of AVM/CO-PU nanoemulsion was demulsified with 5 wt%

151

CaCl2 solution, and the precipitate was then collected by centrifuging at 6000 rpm for

152

15 min followed by three times washing-centrifuging. After that, the AVM/CO-PU

153

powder was obtained by freeze-drying the resultant precipitate.

154

Then, a certain amount of AVM/CO-PU powder and 10 mL of ethanol was charged


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into in a 50 mL centrifuge tube, which was placed in an ultrasonic bath for 1 h to

156

make AVM completely dissolved from AVM/CO-PU powder. Subsequently, the

157

mixture was centrifuged at the speed of 12000 rpm for 15 min and the supernatant

158

was collected and diluted to determine the AVM amount by UV-Vis spectrometer

159

under the detection wavelength of 245 nm, where the maximum in the UV-Vis spectra

160

of AVM was located.

161

The drug loading capacity (LCexp) is defined as the mass percentage of the loaded

162

AVM to the CO-PU carrier, which was calculated according to the equation (1). The

163

encapsulation efficiency (EE) is defined as the percentage of the AVM loaded in the

164

CO-PU carrier to the total AVM used in the preparation process, which was calculated

165

according to the equation (2).

166

LCexp (wt %) =

167

EE (%) =

m × 100% M −m

(1)

m ×100% m0

(2)

168

where m is the mass of AVM loaded in the AVM/CO-PU powder, M is the mass of

169

AVM/CO-PU powder, and m0 is the mass of AVM added in the AVM/CO-PU

170

preparation.

171

2.6 Controlled release behavior of AVM/CO-PU nanoemulsions

172

Different AVM/CO-PU nanoemulsions containing 8 mg of AVM were first diluted

173

with 1 mL of ethanol/water mixture (2 : 1, v/v) and added into the dialysis bags

174

(cutting Mw = 3500 Da). The dialysis bags were immersed into the 200 mL

175

ethanol/water (2 : 1, v/v) release mediums in 250 mL brown jars, and the jars were

176

then placed into an incubator shaker (Hualida, HZ-9210K, China) under a designed



177

temperature with a shaking speed of 150 rpm. Then, 3 mL of the release medium was

178

taken from the jar at the pre-designed interval (the release medium was put back to

179

the tester after the measurement), and released amount and cumulative release rate of

180

AVM were obtained by means of the UV-Vis spectroscopy as mentioned above. Three

181

replicates were performed at each interval to obtain the AVM release curve.

182

The controlled release behavior of AVM/CO-PU nanoemulsions in the

183

ethanol/water (2 : 1, v/v) release mediums with different pH was conducted using the

184

same method as above. The pH value of the mediums was adjusted with sulfuric acid

185

aqueous solution (1 mol/L) or sodium hydroxide aqueous solution (1 mol/L). The

186

release medium without adding acid or alkali was regarded as the blank control and its

187

pH was 7.2.

188

2.7 Adhesion property of the AVM/CO-PU nanoemulsions

189

The fresh corn leaves were washed with 200 mL deionized water, and the water of the

190

surface was then dried with a piece of filter paper. The AVM/CO-PU nanoemulsions

191

(sample H1 and H4) were diluted to a certain concentration of AVM (0.1 wt%) with

192

deionized water, and as a comparison, a free AVM aqueous suspension with the same

193

AVM concentration was prepared through ultrasonic dispersion for 0.5 h. Then, the

194

clean leaves were immersed into the different liquids for 2 min, and subsequently put

195

into the glass culture dishes. After naturally dried in air, each of the leaves were

196

divided into two halves. One half was washed with deionized water for 0.5 h and then

197

dried in the air, and the other half maintained its original. The surface of the leaves

198

with or without washing was characterized by SEM (Tescan, Vega3, Czech) with the


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199

accelerating voltage of 20 kV.

200

2.8 Photostability of AVM/CO-PU drug-loaded system

201

Photostability of the AVM/CO-PU drug-loaded system was evaluated as follows: 10

202

mg of free AVM was dissolved in 10 mL of ethanol and the AVM/CO-PU

203

nanoemulsion (sample H2) containing the same amount of AVM was diluted with

204

water to the same concentration of AVM. Then two solutions were respectively

205

poured into the glass culture dishes with diameter of 7 cm. After natural drying in an

206

airing chamber in the dark conditions, the samples in the culture dishes were

207

irradiated under a 1000 W UV lamp at a distance of 30 cm. The irradiated samples

208

were collected at 1, 3, 5, 7, 10, 15 min and extracted with 10 mL of ethanol,

209

respectively. After centrifuging at the speed of 12000 rpm for 15 min, the obtained

210

supernatant was diluted with ethanol to determine the amount of the undecomposed

211

AVM by the UV-Vis spectroscopy as above.

212

3. Results and Discussion

213

3.1 Preparation of CO-PU nanoemulsion

214

In comparison with conversional petroleum-based PU, the introduction of inedible

215

and naturally renewable CO in the PU preparation not only saves fossil resources but

216

also makes the PU biodegradable, which is benefit to the environment when the

217

CO-PU is used as carrier in the pesticide delivery system 36, 37. The synthesis route of

218

the CO-PU emulsion is shown in Scheme 1. Besides, as a blank control, the linear PU

219

emulsion without using CO was prepared in the similar way. As shown in Table 2, the

220

PU nanoemulsions with narrow size distribution were both obtained with or without



221

the cross-linker CO. The size of the CO-PU nanoparticles was larger than that of the

222

linear PU nanoparticles because of the crosslinked network structure of the molecular

223

chains, which resulted in a larger aggregate in the phase inversion process.

224

As indicated in Figure 1 (a), the CO-PU nanoparticles exhibited uniform spheres

225

with the number average diameter (Dp) of about 40 nm, which was close to its

226

hydrodynamic diameter (Dh) in Table 2. The DSC analysis indicated that CO-PU had

227

two glass transition temperatures, the Tg of soft segments was at -46.7 °C while the Tg

228

at -13.3 °C was ascribed to other CO-based constitutional units 42. Apparently, a lower

229

Tg would be favorable for the nanoemulsion to form a smooth and flat film for its

230

lower minimum film forming temperature (Figure 1 (d)). In addition, a lower Tg

231

makes the polymer spread on the leaf surface more easily and has a higher adhesion

232

due to the greater mobility of the polymer chains 43. Moreover, since there are many

233

-NH- groups in the CO-PU chains, the greater polymer chain mobility is also

234

beneficial to form the hydrogen bond between the -NH- groups in the CO-PU and the

235

-OH, -COOH or -CHO groups on the surface of the leaves 44, which favors the longer

236

foliar pesticide retention of the AVM/CO-PU drug-loaded system.

237

3.2 Preparation and characterization of AVM/CO-PU nanoemulsions

238

The ultrasonic method (USM) and the high speed dispersing method (HSDM) were

239

compared in the preparation of AVM/CO-PU nanoemulsions. Experiments showed

240

that although the resulted AVM/CO-PU nanoemulsions were clear without any AVM

241

solid powder suspended for both methods, the USM not only consumed a lot of

242

energy and resulted in the rise of temperature, but also led to the formation of a small


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243

amount of gel. In comparison, the HSDM was relatively mild without temperature

244

rising but needed more acetone as solvent. Considering the acetone can be received in

245

the rotary evaporation process and reused, the HSDM was adopted and the schematic

246

illustration of the AVM/CO-PU nanoemulsion preparation is shown in Figure 2. It was

247

found that in order to ensure the stability of solvent evaporation process,

248

acetone/water ratio should be increased with the increase of AVM/CO-PU ratio as

249

shown in Table 1, which ascribed to the greater solubility of AVM in the higher

250

concentration of acetone aqueous solution.

251

As listed in Table 2, the Dh of AVM/CO-PU nanoemulsions increased slightly with

252

the increase of AVM/CO-PU, and all of the particle diameters were in the range of

253

40-50 nm, which was larger than that of the CO-PU nanoparticles because of the

254

encapsulation of AVM. Since acetone is a good solvent for PU and AVM, the

255

crosslinked network structure of CO-PU made the nanoparticles swell in the acetone

256

aqueous solution, and oil-soluble AVM molecules dissolved in acetone could be easily

257

penetrated into the swelled CO-PU nanoparticles during emulsification process. In the

258

following solvent evaporation process, the AVM molecules remained and restricted

259

inside the nanoparticles due to a good compatibility between AVM and PU matrix and

260

cross-linked network of CO-PU, and thus a stable drug-loaded nanoemulsion was

261

obtained. In addition, with the increase of the AVM content, the absolute value of the

262

zeta potential (ζ) decreased and the size distribution (P.I.) of the AVM/CO-PU

263

nanoparticles became boarder, since the introduction of water-insoluble AVM would

264

decrease the stability of emulsions.



265

As a comparison, the linear PU nanoemulsion was used to prepare AVM/linear PU

266

nanoemulsion with the same method as above. Results showed that large amount of

267

solid AVM was precipitated in the solvent evaporation procedure no matter how much

268

acetone was used, and the PU particles even gradually dissolved in the acetone

269

aqueous solution, indicating the necessity of cross linker CO in the preparation of

270

CO-PU nanoparticles.

271

As the TEM images indicated, the AVM/CO-PU nanoparticles (Figure 1 (b) and (c))

272

were approximate uniform, but the regularity of the nanoparticles became worse with

273

the increase of AVM/CO-PU, and deformed morphology were even observed for the

274

AVM/CO-PU 50% nanoemulsion (sample H4), which might be due to the effect of the

275

large amount of acetone used in its preparation, because acetone would make the

276

nanoparticles softer and make them easier to deform at a high speed stirring. The Dp

277

of dried AVM/CO-PU nanoparticles estimated from TEM images were in the range of

278

40-50 nm, which were agreed with the corresponding Dh in Table 2. Note that, the

279

diameter of the AVM/CO-PU nanoparticles was larger than that of CO-PU carrier, and

280

Dp of the AVM/CO-PU nanoparticles was consistent with its theoretical value, which

281

was estimated according to the density, drug loading capacity and the diameter of the

282

CO-PU nanoparticles.

283

The transparent films could be easily obtained from all the nanoemulsions

284

including CO-PU and AVM/CO-PU systems at ambient temperature. As illustrated in

285

Figure 1 (d), the AVM/CO-PU film was smooth and flat without any AVM powder on

286

the surface or domain inside the film, indicating the water insoluble AVM molecules


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287

were uniformly encapsulated in the CO-PU nanoparticles, and not aggregated during

288

the film-forming process of AVM/CO-PU nanoemulsions.

289

Fourier transform infrared spectroscopy was used to investigate the chemical

290

composition and structure of the AVM/CO-PU drug-loaded systems. As shown in

291

Figure 3, the peak at 985 cm-1 was attributed to C-H out-of-plane blending vibration

292

in –C=C– in the FTIR spectrum of AVM (Figure 3 (a)), and the peaks at 1699 cm-1

293

and 1531 cm-1 were attributed to –NH-COO– in the FTIR spectrum of CO-PU carrier

294

(Figure 3 (b)) 45. It is clear that the characteristic peaks of both CO-PU carrier and the

295

AVM appeared in the FTIR spectra of AVM/CO-PU drug-loaded systems (Figure 3 (c)

296

and (d)), and no any new peak appeared in comparison to the FTIR spectra of AVM

297

and CO-PU carrier. Furthermore, the intensity of the absorption peak at 985 cm-1

298

increased significantly with the increase of AVM content in the AVM/CO-PU

299

drug-loaded systems. These results demonstrated that AVM was successfully

300

encapsulated in the CO-PU nanoparticles without any chemical reaction between

301

AVM and CO-PU carrier 46.

302

3.3 Drug loading capacity and encapsulation efficiency of the AVM/CO-PU

303

nanoemulsions

304

The loading capacity and encapsulation efficiency are both important for a drug

305

loaded release system. As indicated in Table 3, with the increasing AVM content from

306

20 wt% to 50 wt%, the measured AVM loading capacity (LCexp) increased from 18.3

307

wt% to 42.3 wt%, and the encapsulation efficiency (EE) was above 85% for all of

308

AVM/CO-PU nanoemulsions despite a slightly decrease, indicating that the loss of the



309

AVM in the preparation of AVM/CO-PU nanoemulsions was a little and most of the

310

AVM could be effectively encapsulated in CO-PU nanoparticles. It is worth pointing

311

out that the encapsulation of AVM became difficult with the increase of AVM amount,

312

and even a very small amount of AVM was precipitated in sample H4 after one month

313

storage, resulting in a slight decrease of the encapsulation efficiency from 92% to

314

85%.

315

3.4 Controlled release behavior of the AVM/CO-PU nanoemulsions

316

Effect of the AVM content on the controlled release behavior of the AVM/CO-PU

317

nanoemulsions is shown in Figure 4. The release rate was rapid at the beginning of

318

about 40 h and then slowdown for all of the samples with the increase of release time.

319

The initial fast release of AVM might be attributed to the non-uniform distribution of

320

AVM in the AVM/PU nanoparticles, which meant that some of AVM molecules

321

including absorbed onto the particle surface and closed to the surface inside the

322

particles would be released fast with respect to the AVM loaded more deeply inside

323

the nanoparticles. In addition, because of the greater difference between internal and

324

external concentration and the faster diffusion process with higher AVM content, the

325

release rate of AVM was slightly increased with the increase of AVM content.

326

Environment conditions including temperature and pH will certainly influence the

327

release rate of a drug loaded release system 23. As an example, the release behavior of

328

the AVM/CO-PU nanoemulsion (sample H1) was investigated at different temperature

329

with the same medium pH 7.2. As Figure 5 indicated, although the release rate of

330

AVM exhibited “fast followed by slow” trend at any temperature, the release rate was


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331

significantly increased as temperature rose. For instance, when the temperature was

332

set at 35 °C or above, all of AVM in the nanoparticles released completely during 72 h,

333

but the cumulative release rate was only 54% when the temperature was 25 °C, and

334

even though the release time was prolonged to 180 h, about 20% AVM was still

335

remained in the nanoparticles. No doubt, this accelerating release phenomenon

336

ascribed to the accelerated molecular thermal motion and increased drug solubility at

337

a higher temperature.

338

Effect of pH value of the release medium on the controlled release behavior of the

339

AVM/CO-PU nanoemulsion (sample H2) was investigated at 25 °C. As illustrated in

340

Figure 6, the AVM/CO-PU nanoparticles were pH-responsive. Both the acidic and

341

alkali conditions could accelerate the AVM release, and the acceleration was faster in

342

acidic medium. For example, within 120 h of release, the cumulative release rate

343

reached 99.6 % and 94.1% at pH 4.0 and 10.0, respectively, but this value was only

344

71.7% for the blank control experiment (pH 7.2). Since the neutralization of carboxyl

345

groups was designed to be 100% in the CO-PU carrier preparation, the dispersion of

346

CO-PU in water was stabilized by the anionic COO- groups on the corona, and the pH

347

of the resulted CO-PU nanoemulsion was around 7.7. In this case, any change of the

348

release medium pH would break the electrical equilibrium of the AVM/CO-PU

349

nanoparticles established in its preparation process, and as a result, the electrical

350

double layer of the nanoparticles thinned and the nanoemulsion became unstable,

351

which facilitated the release of AVM from the AVM/CO-PU nanoparticles into the

352

release medium.



Page 18 of 42

353

In order to understand the release mechanism of the AVM/CO-PU nanoemulsions,

354

the exponential relation proposed by Ritger and Peppas was adopted to analyze the

355

release profiles 47:

356

Mt = kt n M∞

357

where Mt is the mass of AVM released at time t, M∞ is the mass of AVM released as

358

time approaches infinity, k is a constant, and n is the diffusional exponent

359

characteristic of the release mechanism.

(3)

360

The fitting results of the release profiles were given in Table 4. The correlation

361

coefficients (r2) were higher than 0.94, indicating that the release behavior of AVM

362

from the AVM/CO-PU nanoemulsions was in good correlation with the

363

Ritger-Peppas empirical equation. According to the literature 47, since the values of

364

n in all of the AVM/CO-PU release profiles were between 0.45 and 0.55, the release

365

mechanism of the AVM/CO-PU nanoemulsions belonged to non-Fickian transport,

366

and the release of AVM from the AVM/CO-PU nanoemulsions was controlled by

367

both diffusion and matrix erosion 2, 21.

368

3.5 Adhesion property of the AVM/CO-PU nanoemulsions

369

In order to prove the AVM/CO-PU drug-loaded system could prolong the foliar

370

pesticide retention, the measurements of adhesion property were conducted according

371

to the literature 25. Results indicated that for the AVM/CO-PU nanoemulsions, many

372

small particles deposited on the corn leaves (Figure 7 (a) and (b)), and most of these

373

particles retained on the leaves after washing (Figure 7 (a’) and (b’)). However, for

374

the free AVM, although some larger particles deposited on the corn leaf at first, most


Page 19 of 42


375

of them were washed away from the leaf (Figure 7 (c) and (c’)). It is clear that the

376

AVM/CO-PU nanoemulsions had better adhesion property and could improve the

377

foliar pesticide retention.

378

3.6 Photostability of the AVM/CO-PU drug-loaded system

379

Since AVM is easily degraded by UV irradiation, the protection of AVM from

380

photolysis is important for an AVM drug delivery system. Here, an acceleration test

381

under the irradiation of 1000 W UV lamp was used to investigate the photostability of

382

AVM in the AVM/CO-PU nanoemulsion (sample H1), and results were plotted in

383

Figure 8. Apparently, the decomposition rate of AVM in the AVM/CO-PU drug-loaded

384

system was slower than that of the free AVM, which was attributed to the

385

UV-shielding and protective effect of the CO-PU carrier on AVM.

386

As shown in the Figure 8, the irradiation time for the free AVM to decompose to 50%

387

was 3.5 min, while for the AVM in the AVM/CO-PU drug-loaded system prolonged to

388

11.5 min, indicating that the AVM/CO-PU drug-loaded system had a better

389

photostability. It should be noted that since the decomposition rate of the AVM was

390

accelerated greatly under the irradiation of a 1000 W UV lamp in this work, the actual

391

decomposition rate will be much slower in the condition of normal sunlight. Thus, the

392

AVM/CO-PU drug-loaded system had a better photostability and would be helpful to

393

reduce the loss of AVM caused by photolysis in the agricultural application.

394

In summary, a new kind of AVM/CO-PU nanoemulsions with biodegradable

395

CO-based polyurethane as the carrier was successfully prepared by means of

396

emulsion solvent evaporation method, and the AVM loading capacity was up to 42.3



397

wt% with a high encapsulation efficiency above 85%. The diameter of all the

398

AVM/CO-PU nanoparticles was less than 50 nm, which was in favor of their

399

deposition on the leaf surface as well as the foliar pesticide retention. The release rate

400

of AVM from the AVM/CO-PU nanoemulsions increased slightly with the increase of

401

AVM content and speed up significantly with the rise of temperature, and it was also

402

accelerated in acidic or alkaline medium. Meanwhile, as compared to the free AVM,

403

the CO-PU carrier provided AVM with the UV-shielding and protection to make the

404

drug in the AVM/CO-PU drug-loaded system with a better photostability. Based on

405

the results, a release mechanism of the AVM/CO-PU drug-loaded nanoemulsions was

406

proposed, which was controlled by both diffusion and matrix erosion. Using this

407

facile method, the water-insoluble AVM was successfully transferred into a stable

408

waterborne system, and the resultant AVM/CO-PU nanoemulsions could significantly

409

improve the bioavailability and decrease the waste and harm of AVM.

410

Funding

411

This work is supported by grants from the National Basic Research Program of China

412

(nos. 2014CB932202).

413


Page 20 of 42

Page 21 of 42


414

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415

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polyurethane intravaginal rings for the sustained combined delivery of antiretroviral

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37-42.



Figure Captions Scheme 1. Synthesis route of the CO-PU nanoemulsions Figure 1. TEM images of the nanoparticles: (a) CO-PU, (b) AVM/CO-PU (sample H1), and (c) AVM/CO-PU (sample H4); (d) SEM image of AVM/CO-PU (sample H4) film Figure 2. Schematic illustration of the synthesis of AVM/CO-PU nanoemulsions Figure 3. FTIR spectra of (a) AVM, (b) CO-PU carrier, and AVM/CO-PU drug-loaded system (c) sample H2, (d) sample H4 Figure 4. Release curves of the different AVM/CO-PU nanoemulsions at 25 °C and pH 7.2 Figure 5. Release curves of the AVM/CO-PU nanoemulsion (sample H1) at different temperature with the same pH 7.2 Figure 6. Release curves of the AVM/CO-PU nanoemulsion (sample H2) under different pH at 25 °C Figure 7. SEM images of the different samples on the corn leaves: AVM/CO-PU nanoemulsion (sample H1) before (a) and after (a’) washing; AVM/CO-PU nanoemulsion (sample H4) before (b) and after (b’) washing; free AVM before (c) and after (c’) washing Figure 8. Photolysis curves of the AVM in the AVM/CO-PU drug-loaded system (sample H1) and the free AVM under UV irradiation Figure 9. For Table of Contents Only


Page 28 of 42

Page 29 of 42


Table 1. Recipes for the preparation of AVM/CO-PU nanoemulsions CO-PU emulsion

AVM/CO-

Amt of

Amt of

PU (wt%)

AVM (g)

acetone (mL)

Amt (g)

Solid cont. (wt%)

U1

20

0.80

20

13.74

29.1

U2

30

1.20

20

13.74

29.1

U3

50

2.00

30

13.74

29.1

H1

20

1.00

35

18.90

26.5

H2

30

1.50

35

18.90

26.5

H3

40

2.00

42

18.90

26.5

H4

50

2.50

49

18.90

26.5

Sample

USM a

HSDM b

a

The ultrasonic method;

b

The high speed dispersing method



Page 30 of 42

Table 2. Colloidal properties of the linear PU, CO-PU and AVM/CO-PU (sample H1, H2, H3, H4) nanoemulsions Nanoemulsion

Dh (nm)

P.I.

ζ (mV)

Dp (nm)

Linear PU

32.0

0.161

-40.2

30.4

CO-PU

40.2

0.132

-38.2

39.2

H1

45.0

0.215

-34.3

43.8

H2

46.6

0.246

-33.2

45.1

H3

47.7

0.271

-32.0

46.1

H4

48.8

0.311

-29.7

47.1


Page 31 of 42


Table 3. AVM loading capacity and encapsulation efficiency of the AVM/CO-PU (sample H1, H2, H3, H4) nanoemulsions

a

Sample

LCthero a (wt%)

LCexp (wt%)

EE (%)

H1

20

18.3

92

H2

30

26.0

87

H3

40

34.7

87

H4

50

42.3

85

The theoretical value of the loading capacity



Page 32 of 42

Table 4 Fitting results of the AVM/CO-PU release profiles by Ritger-Peppas equation Sample

AVM cont. a (wt%)

Temp. (°C)

pH

k

n

r2

H1

20

25

7.2

7.60

0.46

0.994

H2

30

25

7.2

8.38

0.45

0.994

H3

40

25

7.2

6.93

0.51

0.999

H1

20

35

7.2

10.07

0.52

0.987

H1

20

40

7.2

13.37

0.48

0.946

H2

30

25

4.0

10.41

0.50

0.947

H2

30

25

6.8

9.09

0.51

0.986

H2

30

25

9.0

8.38

0.49

0.999

H2

30

25

10.0

7.21

0.55

0.991

a

The mass percentage of the AVM to the CO-PU carrier used in the preparation of the

AVM/CO-PU nanoemulsion


Page 33 of 42


Scheme 1. Synthesis route of the CO-PU nanoemulsions 108x98mm (600 x 600 DPI)



Figure 1. TEM images of the nanoparticles: (a) CO-PU, (b) AVM/CO-PU (sample H1), and (c) AVM/CO-PU (sample H4); (d) SEM image of AVM/CO-PU (sample H4) film 101x101mm (300 x 300 DPI)


Page 34 of 42

Page 35 of 42


Figure 2. Schematic illustration of the synthesis of AVM/CO-PU nanoemulsions 150x63mm (300 x 300 DPI)



Figure 3. FTIR spectra of (a) AVM, (b) CO-PU carrier, and AVM/CO-PU drug-loaded system (c) sample H2, (d) sample H4 65x53mm (600 x 600 DPI)


Page 36 of 42

Page 37 of 42


Figure 4. Release curves of the different AVM/CO-PU nanoemulsions at 25 °C and pH 7.2 60x46mm (600 x 600 DPI)



Figure 5. Release curves of the AVM/CO-PU nanoemulsion (sample H1) at different temperature with the same pH 7.2 60x46mm (600 x 600 DPI)


Page 38 of 42

Page 39 of 42


Figure 6. Release curves of the AVM/CO-PU nanoemulsion (sample H2) under different pH at 25 °C 60x46mm (600 x 600 DPI)



Figure 7. SEM images of the different samples on the corn leaves: AVM/CO-PU nanoemulsion (sample H1) before (a) and after (a’) washing; AVM/CO-PU nanoemulsion (sample H4) before (b) and after (b’) washing; free AVM before (c) and after (c’) washing 151x228mm (300 x 300 DPI)


Page 40 of 42

Page 41 of 42


Figure 8. Photolysis curves of the AVM in the AVM/CO-PU drug-loaded system (sample H1) and the free AVM under UV irradiation 61x47mm (600 x 600 DPI)



Figure 9. For Table of Contents Only 53x44mm (300 x 300 DPI)


Page 42 of 42

Preparation and Characterization of Controlled-Release Avermectin

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