Preparation of an Environmentally Friendly Formulation of the

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Preparation of an environmentally friendly formulation of the insecticide nicotine hydrochloride through encapsulation in chitosan/tripolyphosphate nanoparticles Ying Yang, Jiagao Cheng, Vasil M Garamus, Na Li, and aihua Zou J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b04147 • Publication Date (Web): 04 Jan 2018 Downloaded from http://pubs.acs.org on January 4, 2018

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Journal of Agricultural and Food Chemistry

Preparation of an environmentally friendly formulation of the insecticide

nicotine

hydrochloride

through

encapsulation

in

chitosan/tripolyphosphate nanoparticles

Ying Yanga, Jiagao Chengb, Vasil M. Garamusc, Na Lid, Aihua Zoua∗

a

State Key Laboratory of Bioreactor Engineering and Institute of Applied

Chemistry, Shanghai Key Laboratory of Functional Materials Chemistry, School of Chemistry and Molecular Engineering, East China University of Science and Technology, Shanghai 200237, PR China b

School of Pharmacy, East China University of Science and Technology, Shanghai

200237, PR China c

Helmholtz-Zentrum Geesthacht, Centre for Materials and Coastal Research,

D-21502 Geesthacht, Germany d

National Center for Protein Science Shanghai and Shanghai Institute of

Biochemistry and Cell Biology, Shanghai 200237, P. R. China

∗

Corresponding author

Aihua Zou School of Chemistry and Molecular Engineering East China University of Science and Technology Meilong Road 130 200237 Shanghai, China E-mail: [email protected] 1

ACS Paragon Plus Environment


Tel: +86 64252231

2


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1

Abstract

2

Insecticide nicotine hydrochloride (NCT) was formulated as nanoparticles

3

composed of chitosan (CS) and sodium tripolyphosphate (TPP) to undermine its

4

adverse impacts on human health and reinforce its physicochemical stability. The

5

study investigated the preparation and characterization of chitosan /tripolyphosphate

6

nanoparticles (CS/TPP NPs) with good encapsulation efficiency (55%), uniform

7

morphology, physicochemical stability (45d) through dynamic light scattering (DLS),

8

transmission electron microscopy (TEM) and small-angle X-ray scattering (SAXS)

9

measurements. A bioassay against Musca domestica NCT CS/TPP NPs exhibited

10

good bioactivity and thermal stability. The monovalent salt’s (NaCl) effect on

11

manipulating the formation and size distribution of ionically cross-linked

12

nanoparticles was demonstrated as well. The formulation of NCT CS/TPP NPs could

13

be a utility candidate in public health and agriculture.

14 15

Keywords: nicotine; chitosan; sodium tripolyphosphate; ionic gelification;

16

monovalent salt effect; environmentally safe formulations

17 18 19 20 3



21 22

1. Introduction

23

Tobacco is deeply rooted in almost 120 countries and territories; 1 while smoking

24

tobacco answers for approximately 6 million deaths per year globally and also have

25

deprived the lives of more than 600,000 nonsmokers.2 Nearly a quarter of waste

26

tobacco are casted aside in China, generating a great waste of resources.3 Owing to

27

the maturing technology of extracting nicotine from waste tobacco,4 both the market

28

of tobacco industry and nicotine are confronted with a new and further opportunity.

29

Nicotine ((S)-3-(1-methyl-2-pyrroli-dinyl) pyridine), is an alkaloid that mostly

30

exists in solanaceaeplant.5 It’s also the crucial ingredient of tobacco. Since 1690, the

31

insecticidal activity of nicotine has been exploited by Aboriginal Americans. By virtue

32

of its short environmental persistence and target pest selectivity, the botanical

33

insecticide nicotine is accepted as an alternative for conventional insecticide for crop

34

protection and public health extensively.6

35

Notwithstanding its superior performance as a botanical insecticide, the high

36

mammalian toxicity of nicotine (half lethal dose, LD50= 50 mg/kg) has restricted its

37

application.7 Nicotine exposure during pregnancy affects the brainstem α7 nicotinic

38

acetylcholine receptor expression, magnifying the risk of sudden unexplained

39

perinatal death.8 It is also confirmed nicotine absorbed through the skin invokes the

40

characteristic green tobacco sickness (GTS), an occupational illness reported by

4


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tobacco workers.9 Furthermore, NCT is easily oxidized when exposed to atmospheric

42

air and light.

43

Different formulations of nicotine have consequently been explored: nicotine oleate

44

formulations,10

45

nanoparticles.12 These formulations protect nicotine from external factors and

46

minimize its side effects, while most of them were applied in drug delivery rather than

47

pesticide delivery.

nicotine

carboxylate

emulsions,11

liposome

and

polymeric

48

Among those formulations, nanoparticles (NPs) have attracted considerable interest

49

for diverse biomedical applications including the development of drug/pesticide

50

delivery systems,13,14 by avoiding the problems of active ingredients toxicity and

51

degradation.15,16 Chitosan, a versatile biomaterial derived from chitin (essentially

52

poly(β-1,4-N-acetyl-D-glucose-2-amine)), is one of the most abundant natural

53

polysaccharides. On account of its biocompatibility, low cost, low toxicity and

54

biodegradability, chitosan is widely utilized for the encapsulation of bioactive

55

compounds.17 It is not just a natural antimicrobial agent in agriculture; its derivatives

56

can also fertilize the soil and thus boosts crop yields.18 Amongst various procedures of

57

chitosan nanoparticles,19-21 ionic gelification using sodium tripolyphosphate (TPP) is

58

most attractive. 22,23

59

In this study, a NCT carrier system was fabricated with chitosan and

60

tripolyphosphate. The activity of the insecticide was assessed using Musca domestica.

5



61

The morphology, encapsulation efficiency, as well as the physicochemical stability

62

were evaluated equally. To obtain abundant uniform particles, the effects of

63

monovalent salt on the formation and size distribution of ionically cross-linked

64

nanoparticles were examined.

65

2. Materials and Methods

66

2.1. Materials

67

Nicotine ( ≥ 99%) was obtained from Sigma-Aldrich; chitosan (CS) (MW:

68

100-300kDa) was from J&K Chemical. Sodium tripolyphosphate (TPP), sodium

69

chloride (NaCl) and acetonitrile (chemical used in HPLC mobile phase) were obtained

70

from Sinopharm Chemical Reagent. Acetic acid and sodium 1-heptanesulfonate

71

(chemical used in HPLC mobile phase) were purchased from Ling Feng Chemical

72

Reagent. The degree of chitosan deacetylation was estimated at 50-60% by pH

73

titration.24 All other chemicals were of analytical grade without further purification.

74

2.2 Preparation of chitosan /tripolyphosphate nanoparticles

75

CS/TPP NPs were formed using ionic gelification method that is firstly described

76

by Grillo et al.25 5mL 0.1% TPP aqueous solution was dropwise added to 20mL 0.1%

77

chitosan in 0.2% acetic acid slowly, under magnetic agitation for 15min, to get

78

nanoparticles. 0.1mL 10g/L nicotine hydrochloride was incorporated into the chitosan

79

solution prior to nanoparticles formation. Additionally, in order to obtain much

80

narrower particle size distributions, a certain amount of NaCl was added separately in

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corresponding concentrations (25-125mM) to chitosan and TPP solutions, ahead of

82

the dropwise addition process. Nicotine nanoparticles were stored in amber flasks at

83

ambient temperature.

84

2.3 Structure characterization of delivery systems

85

2.3.1Dynamic light scattering(DLS) measurement

86

The mean particle size, polydispersity index (PDI) and ζ-potential of CS/TPP NPs

87

(with and without nicotine hydrochloride) were measured by DLS at 25℃ using

88

Nano-ZS90 system with a fixed angle of 90◦.

89

2.3.2 Transmission electron microscopy (TEM)

90

Nicotine nanoparticles (with and without NaCl) were imaged using JEM-1400

91

electron microscope. Samples were prepared by placing a drop of freshly prepared

92

nicotine nanoparticle suspensions with and without NaCl onto a copper grid and

93

air-dried overnight. To prevent NaCl crystal formation on the copper grid, the

94

suspensions with NaCl were dialyzed against an excess of NaCl-free acetic solution

95

prior to being observed.

96

2.3.3 Small-angle X-ray scattering (SAXS)

97

SAXS were performed at beamline BL19U2 of the National Center for Protein

98

Science Shanghai at Shanghai Synchrotron Radiation Facility. Scattered X-ray

99

intensities were measured by a Pilatus 1 M detector (DECTRIS Ltd). The

100

sample-to-detector distance was set such that the detecting the range of momentum

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transfer q （q = 4π sin θ/λ, where 2θ is the scattering angle）of the SAXS experiments

102

was 0.01-0.5 Å−1. A flow cell made of a cylindrical quartz capillary with a diameter of

103

1.5 mm and a wall of 10 µm was used to diminish the radiation damage. The exposure

104

time was set to 1-2 s. The X-ray beam had a size of 0.40 × 0.15 (H × V) mm2 and was

105

adjusted to pass through the center of the capillaries for every measurement.

106

2.4 Encapsulation efficiency (EE)

107

Encapsulation efficiency was determined by ultrafiltration/centrifugation method. A

108

desired amount of nanoparticle suspension was centrifuged for 30min at 8000 rpm at

109

4℃ to remove un-encapsulated pesticide from samples, and the amount of nicotine

110

hydrochloride in the filtrate was quantified by UV-vis spectrophotometer. The total

111

amount of pesticide in the NP suspensions was calculated by adding methanol with

112

10-min sonication to destroy the structure of NPs and then it was quantified by using

113

UV-vis spectrophotometer. Encapsulation efficiency (EE) was calculated as follows:

114 115

EE% =

× 100%

(1)

2.5 In vitro release of nicotine

116

10mL nicotine hydrochloride nanoparticles (with and without NaCl) were put into

117

pre-swelled dialysis bags (8-14 kDa MW (molecular weight) cutoff) respectively,

118

which were then placed in 100 mL 0.1% TPP solution and gently shaken in a

119

thermostated shaker bath at 25°C. Samples were removed at appropriate intervals, and

120

the same volume (3 mL) fresh medium was added to each sample. The amount of

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released nicotine hydrochloride was quantified by high performance liquid

122

chromatography (HPLC) method: Diamond C18 column (5µm, 150 mm × 4.6 mm),

123

mobile phase: 1L 0.109% potassium phosphate monobasic aqueous solution with

124

0.0082g sodium 1-heptanesulfonate mixed with acetonitrile (9:1, v/v), flow rate 1

125

mL/min, and wavelength 260 nm. All experiments were conducted at room

126

temperature (25℃).

127

2.6 Insecticidal activity assays

128

A bioassay of nicotine hydrochloride-loaded nanoparticles against Musca

129

domestica was evaluated as the following procedure.26 A glass plate (200mm ×

130

200mm) with a nonabsorbent surface was smeared with 0.5mL insecticide

131

formulation and dried by airing. A total of 20 individuals were introduced into a glass

132

forced-exposure device after a mild anesthesia with ethyl ether; the device was then

133

put on the glass plate when the individuals resumed normal activities. The number of

134

dead individuals was recorded every minute until the completion of 20minutes. Three

135

replicates for the formulation were carried out. Evaluation was made on a dead/alive

136

basis, and toxicity regression equations, half knock-down time (KT50), and confidence

137

limits were calculated by Origin software.27

138

3. Results and discussion

139

3.1 Optimization of NCT CS/TPP NPs formulation

140

To verify the conditions for the formation of CS/TPP NPs, CS and TPP solution

9



141

with different concentrations were studied as shown in Table 1. There were four

142

phenomena after the addition of TPP to CS solution: clear solution, aggregates with

143

clear solution on the top layer，opalescent suspension, and aggregates with opalescent

144

suspension on the top layer (Table 1). Clear solution represented few nanoparticles

145

formed when CS and TPP were both at low concentrations; aggregates with clear

146

solution on the top layer implied that some micro-particles were formed and gathered

147

at higher TPP concentrations. It can be inferred that only the phenomenon of

148

opalescent suspension signified the presence of stable nanoscale particles. Aggregates

149

with opalescent suspension on the top layer may correspond to the unstable

150

coexistence of microparticles-nanoscale particles.

151

The results in Table 1 implied that CS/TPP NPs could be formed when the

152

concentration of CS and TPP were in the range of 0.1 to 0.2 (wt.%). Calvo et al. found

153

that the construction of CS/TPP NPs was only possible for some specific

154

concentration range of CS and TPP.28 In acetic acid solution, there exist hydrogen

155

bonding interactions and electrostatic repulsion (resulting from the protonated amino

156

groups of CS). Only in some specific concentration range, the two interactions could

157

be in equilibrium to form stable CS/TPP nanoparticles with TPP.29 Below this

158

concentration, the electrostatic repulsion overwhelms the hydrogen bonding

159

interactions, and it is accordingly difficult for chitosan molecules to approach each

160

other, leading to few molecules involved in the cross-linking process. Above this, it is

10


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possible to fabricate nanoparticles which usually develop into aggregates in view of

162

the strong hydrogen bonding attractions. The mean particle size and PDI for seven

163

samples in Table 1 were determined by DLS (Table 2). The diameters of the

164

nanoparticles were around 300 nm when TPP concentration was 0.10% and CS

165

concentration was 0.05% and 0.10% (NP-2 and NP-3), and then was increased rapidly

166

when either of them was above 0.10%. When TPP was increased from 0.10% (NP-3)

167

to 0.15% (NP-6), nanoparticles swelled as they bore a high net charge; however, when

168

TPP increased to 0.20%, TPP: glucosamine (chitosan monomer) molar ratio was near

169

or exceeded the binding site saturation point, the size of nanoparticles decreased

170

slightly which may be ascribed to the increased ionic cross-link density within the

171

nanoparticles and reduction in nanoparticles charge (both of which diminish

172

swelling).

173

The encapsulation efficiency of NCT CS/TPP NPs was investigated (Supporting

174

Information). EE% was increased slowly as the concentration of nicotine

175

hydrochloride went down, but the low nicotine hydrochloride concentration may

176

inhibit its pesticide activity to some extent. Taking both of the encapsulation

177

efficiency and pesticide activity into consideration, we chose 40mg/L as the nicotine

178

hydrochloride concentration of NCT CS/TPP NPs. Then the encapsulation efficiency

179

of nicotine hydrochloride has been further conducted as a semi-combinatorial

180

optimization of the preparative process. EE values for four formulations NP-2, NP-3,

11



181

NP-4, NP-5 are 42.1%, 54.1%, 56.3%, and 59.7%, respectively. Considering both the

182

particle size and EE value, NP-3 was chosen for further research.

183

3.2 Physicochemical stability of NCT CS/TPP NPs

184

The hydrodynamic diameter, polydispersity, ζ-potential, and encapsulation

185

efficiency for both NCT CS/TPP NPs and blank CS/TPP NPs (Figure 1) were

186

evaluated as a function of time (0, 15, 30 and 45 days) to determine the sample

187

physicochemical stability.

188

CS/TPP NPs was around 270nm with a high PDI value of 0.3. The Zeta potential of

189

blank CS/TPP NPs was 50mV (positive) due to the cationic charge on the chitosan

190

molecules, which was thought to be stable in suspension.

191

NPs virtually stayed the same for the duration time. Compared with blank NPs, size of

192

NCT CS/TPP NPs became larger with loaded nicotine, whereas ζ-potential went down

193

a bit probably due to more anionic TPP offset of the cation of NPs.29 For NCT

194

CS/TPP NPs, the diameter was maintained around 300nm with a PDI value of 0.3 and

195

a ζ-potential value of 45mV during 45 days. The EE value of NCT CS/TPP NPs was

196

approximately 55% and did not change during 45 days keeping. Therefore, all the

197

results showed the formulation of NCT CS/TPP NPs was physicochemical stable for

198

at least 45 days at ambient temperature (25℃), which was similar with the blank

199

CS/TPP NPs.

200

From Figure 1, it can be seen that the size of blank

So, parameters for blank

SAXS was used to determine the mean or global features of sample structure,

12


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Figure 2 exhibited SAXS curves of blank CS/TPP NPs and NCT CS/TPP NPs. The

202

SAXS curves were investigated in a slope approximation, i.e., I(q) vs qα to study the

203

microscopic structure of the samples.30,31 The slopes at the low q range were -2.10 and

204

-2.48 for blank CS/TPP NPs and NCT CS/TPP NPs, respectively. This result indicated

205

that blank CS/TPP NPs and NCT CS/TPP NPs may be oblate (disc like). The slopes at

206

large q range were around -3.10 and -3.14 for blank CS/TPP NPs and NCT CS/TPP

207

NPs, respectively, which showed that the interface was rough (surface fractal

208

like).32,33 In approximation of volume fractals, the slope at low q showed the

209

connection between length of aggregates and volume. It was evident that the addition

210

of nicotine revealed increase of α (the absolute value of slope) meaning aggregates

211

with nicotine was more compact.

212

The structure of NCT CS/TPP NPs was confirmed by TEM imaging (Figure 3a).

213

Figure 3a revealed that NCT CS/TPP NPs has a round and flat shape ranging from

214

100 to 300nm in diameter, which was in good agreement with SAXS results.

215

3.3 Monovalent salt effect on NCT CS/TPP NPs

216

To achieve more homogenous particles and bring down the high PDI value of NCT

217

CS/TPP NPs, various amounts of NaCl (25-125mM) were added in sample NP-3. As

218

presented in Table 3, low PDI value of NCT CS/TPP NPs can be attained through

219

NaCl addition, especially when its concentration was above 100mM. Furthermore,

220

there was a subtle minimum in both size and PDI at approximately 100mM NaCl

13



221

addition. This stemed from amplified colloidal stability by monovalent salt which

222

inhibited the bridging of NCT CS/TPP nanoparticles by TPP first described by

223

Yakovetal.34,35 They interpreted NaCl-induced structural rearrangement also played a

224

part in the reduced PDI.

225

Physicochemical stability of NCT CS/TPP NPs with 100mM NaCl was evaluated

226

in Figure 4 as a function of time (0, 15, 30 and 45 days). The size of fresh NCT

227

CS/TPP NPs with NaCl was smaller compared with fresh sample without NaCl in

228

Figure 1. The size of the nanoparticles was manipulated by the number of polymer

229

chains within each particle and the extent of swelling,36 while the latter depended on

230

temperature, pH or ionic strength of the medium. Thus, the diameter of CS/TPP NPs

231

shrank as the result from the reduced swelling due to the weakening osmolality

232

disparity between nanoparticles and supernatant fluid at high ionic strengths with

233

added NaCl (Figure 5).37

234

The ζ- potential of fresh NCT CS/TPP NPs with NaCl was lower than fresh samples

235

without NaCl in Figure 1, while the PDI vaule of NCT CS/TPP NPs descent from 0.3

236

to 0.2 after the addition of 100 Mm NaCl. As shown in Figure 5, the high PDI of

237

NaCl-free system could be attributed to the interparticle cross-linking among NCT

238

CS/TPP NPs. With NaCl addition, the interparticle cross-linking among the NCT

239

CS/TPP NPs was weaker,

240

rate of coagulation and kept the newly formed nanoparticles stably dispersed. This

38-40

so the rate of CS and TPP mixing exceeded the slow

14


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difference conducted by monovalent salt predominantly inferred a kinetic effect

242

managed by their short-term colloidal stability during nanoparticles preparation.

243

However, it should be emphasized that both of the ζ- potential values and

244

encapsulation efficiency of NCT CS/TPP NPs formed with 100mM NaCl went down

245

a bit during physicochemical stability assay. As the long-term stability of the system

246

mainly counts on the electrical state of the nanoparticles,41 the salt-free system

247

exhibited superior long-term stability considering its high ζ- potential.

248

Figure 6 described SAXS curve of NCT CS/TPP NPs with 100mM NaCl. The slope

249

at low q range for of NCT CS/TPP NPs with NaCl was -2.34; while the slope of NCT

250

CS/TPP NPs without NaCl was -2.48, which exhibited that the addition of NaCl

251

caused a minor decreasing of α (the absolute value of slope). The slope at large q

252

range for NCT CS/TPP NPs samples with 100mM NaCl changed to -4.02, pointing

253

that the interface was smooth and sharp.32,33 Therefore, it was clear that NCT CS/TPP

254

NPs with NaCl showed sharp and smooth interface compared with NCT NPs without

255

NaCl. From Figure 3b, it seemed that TEM image and SAXS spectra were well in

256

agreement.

257

3.4 In vitro release of nicotine hydrochloride from CS/TPP NPs

258

Profiles of in vitro release of nicotine hydrochloride from NPs were studied in 0.1%

259

TPP solution at 25℃ by using the dialysis method. TPP solution can keep CS/TPP NPs

260

from swelling and disintegration in the sustained release assay36. The release of the

15



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261

free nicotine hydrochloride was also investigated as a control. As can be seen from

262

Figure 7, more than 90% of free nicotine hydrochloride was released in the medium

263

after 24h under experimental conditions; however, for the pesticide associated with

264

CS/TPP NPs (with and without NaCl), only approximately 10% nicotine

265

hydrochloride was released during the first 30 minutes, and less than 20% of the

266

pesticide was released within 24h. Evidently, the release of the pesticide nicotine was

267

significantly underdeveloped in nanoparticles, especially in nanoparticles with NaCl.

268

The restricted release of nicotine from nanoparticles could be ascribed to the capture

269

of the active principle by the reticulated network of CS/TPP NPs as a result of

270

electrostatic forces.42 As for those from CS/TPP nanoparticles with monovalent salt,

271

the slower release is mainly derived from the much more uniform particles and raised

272

ionic cross-link density.43,44

273

3.5 Insecticidal activity assays

274

With the purpose to demonstrate the practicality of the nanoparticles as a favorable

275

formulation

276

hydrochloride-loaded nanoparticles against Musca domestica was conducted.

277

Casanova et al.12,13 have organized the bioassay tests of two nicotine formulations

278

against adults of Drosophila melanogaster. The suspo-emulsion based on nicotine

279

oleate showed lethal time 50 (LT50) is 11min at the beginning of the evaluation period

280

and then it was increased to 13min after 7 days. For nicotine carboxylate insecticide

281

emulsions, the capric acid emulsion showed the highest encapsulation of nicotine but

in

public

hygiene

and

agriculture,

16


a

bioassay

of

nicotine

Page 17 of 35


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the lowest bioactivity (i.e., the highest LT50 14min). Compared with the above

283

formulations, as emphasized in Figure 8, NCT CS/TPP NPs without NaCl had a

284

slightly lower KT50 (half knock-down time）value and a mildly higher 24h mortality

285

than the system with NaCl in consideration of the different release profiles of

286

pesticide within 24 hours.45 Both samples of NCT CS/TPP NPs with and without

287

NaCl were kept for 30 days, then were treated against Musca domestica. The

288

experiments showed that 24h mortality of NCT CS/TPP NPs was 95%, and the one of

289

NCT CS/TPP NPs with NaCl was 85%; while the KT50 values for NCT CS/TPP NPs

290

without and with NaCl were 7.24min and 9.22min, respectively. Therefore, both of

291

these two formulations have effective duration of more than 30 days. The high

292

efficacy should be explained by the sustained release and so the inhibition of active

293

ingredient’s degradation with CS/TPP NPs .46,47 In addition, nanosized particles can

294

heighten the adhesion and penetrability of pesticide on surface of pests, subsequently

295

lowering the leaking loss of pesticide during spraying process.48,49

296

Integrating with the physicochemical stability results of NCT CS/TPP NPs, it

297

implicated that NCT CS/TPP NPs with NaCl were much more homogeneous owing to

298

the short-term colloidal stability during their formation; and NCT CS/TPP NPs free of

299

NaCl exhibited long-term stability which relied on electrical properties.37 These

300

results were in consistence with literatures on this subject.30,33

301

formulations would provide further guidelines for the pesticide carrier system on

17


Though both of the


Page 18 of 35

302

varying demand side, the formulation formed in salt-free system, is considered as a

303

promising candidate in public health & agriculture; and it may offer insights into the

304

pursuit of an environmentally friendly formulation.

305

Abbreviations Used

306

CS, chitosan; TPP, tripolyphosphate; NCT, nicotine hydrochloride; NCT CS/TPP

307

NPs,

308

transmission electron microscopy; SAXS, small-angle X-ray scattering; GTS, green

309

tobacco sickness; LD50, half lethal dose; EE, encapsulation efficiency; PDI,

310

polydispersity index; KT50, half knock-down time; HPLC, high performance liquid

311

chromatography; DLS, dynamic light scattering; conc., concentration. LT50, half lethal

312

time.

313

Acknowledgement

nicotine

hydrochloride

chitosan/tripolyphosphate

nanoparticles;

TEM,

314

The corresponding author designed the study and revised the manuscript. All of the

315

authors carried out this research under the guidance of the corresponding author. The

316

final version was approved by all authors.

317

Also thanks a lot for the support and help from Xuhong Qian, Wei Jia, Zaihong

318

Long and Yawen Li for this article.

319

Funding

320

The present study was supported by grants from the National Key Research and

321

Development Plan (No. 2017YFD0200306), the National Natural Science Foundation

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322

of China (No. 31200617), Shanghai Natural Science Foundation (Grant No.

323

15ZR1409900), and Knowledge Innovation Program of CAS (Grant No.

324

2013KIP103).

325

Supporting Information Description

326

Additional information on optimization of NCT NPs formulation. The alteration of

327

the encapsulation efficiency of nicotine hydrochloride in CS/TPP nanoparticles along

328

with different nicotine hydrochloride concentration.

329

References

330

(1) Shi, H.; Wang, R.; Bush, L. P.; Zhou, J.; Yang, H.; Fannin, N., et al. Changes in

331

tsna contents during tobacco storage and the effect of temperature and nitrate level on

332

TSNA formation. J. Agric. Food Chem. 2013, 61, 11588-11594.

333

(2) Organization W H. WHO Report on the Global Tobacco Epidemic 2011: Warning

334

about the dangers of tobacco. J. Gen. Info. 2011, 17, 83.

335

(3) Xie, Z.; Whent, M.; Lutterodt, H.; Niu, Y.; Slavin, M.; Kratochvil, R.; Yu, L. L.

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Phytochemical, antioxidant, and antiproliferative properties of seed oil and flour

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extracts of Maryland-grown tobacco cultivars. J. Agric. Food Chem. 2011, 59,

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9877-84.

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(4) Lee, J. G.; Lee, C. G.; Kwag, J. J.; Rhee, M. S.; Buglass, A. J.; Lee, G. H. Fast

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Table 1 Four phenomena after the addition of TPP to CS solution CS (wt.%) TPP（wt.%）

0.01

0.02

0.05

0.10

0.15

0.20

0.05

＼

＼

＼

＼

＼

√

0.10

↘

↘

√

√

√

√

0.15

↘

↘

↘

√

↙

↙

0.20

↘

↘

↘

√

↙

↙

＼indicates clear solution; ↘indicates aggregates and clear solution on the top layer; √indicates opalescent suspension;↙indicates aggregates and opalescent suspension on the top layer.

Table 2 Particle size and PDI of NPs along with CS and TPP mass concentration alteration formulation

TPP

CS

diameter

（wt%）

（wt%）

（nm）

PDI

NP-1

0.05

0.20

1619.3

0.36

NP-2

0.10

0.05

249.9

0.32

NP-3

0.10

0.10

325.2

0.31

NP-4

0.10

0.15

812.7

0.32

NP-5

0.10

0.20

1376.0

0.37

NP-6

0.15

0.10

1051.3

0.35

NP-7

0.20

0.10

859.7

0.34

Table 3 Alteration of size and PDI of CS/TPP NPs along with NaCl concentration increasing conc./mM

25

50

75

100

125

150

diameter/nm

202.5

367.4

476.4

259.7

393.3

427.3

PDI

0.28

0.28

0.26

0.22

0.22

0.20

27



Figure 1 Physicochemical stability of blank CS/TPP NPs and NCT CS/TPP NPs as a function of time (0, 15, 30 and 45 days)

28


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Figure 2 SAXS curves of blank CS/TPP NPs and NCT CS/TPP NPs

29



Figure 3 TEM images of NCT CS/TPP NPs (a) and NCT CS/TPP NPs with 100mM NaCl (b) a

30


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b

Figure 4 Physicochemical stability of NCT CS/TPP NPs with100mM NaCl as a function of time (0, 15, 30 and 45 days) 31



Figure 5 Schematic representation of short-term colloidal stability during nanoparticles preparation in NaCl system due to the prevention of interparticle 32


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cross-linking.

Figure 6 SAXS curves of NCT CS/TPP NPs with 100mM NaCl

Figure 7 Release profiles of free nicotine, nicotine from CS/TPP NPs and CS/TPP 33



NPs with 100mM NaCl. Data are presented as the mean ± standard deviation (n = 3)

Figure 8 The KT50 values and 24h mortality of the CS/TPP NPs and CS/TPP NPs with 100mM NaCl

34


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TOC Graphic

35


Preparation of an Environmentally Friendly Formulation of the

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