Gum Arabic Nanoparticles: A

Subscriber access provided by Kaohsiung Medical University

Agricultural and Environmental Chemistry

Geraniol encapsulated in chitosan/gum arabic nanoparticles: a promising system for pest management in sustainable agriculture Jhones Luiz de Oliveira, Estefania Vangelie Ramos Campos, Anderson E. S Pereira, Lucas E. S. Nunes, Camila C. L. da Silva, Tatiane Pasquoto, Renata Lima, Giovani Smaniotto, Ricardo Antonio Polanczyk, and Leonardo F. Fraceto J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b00331 • Publication Date (Web): 07 May 2018 Downloaded from http://pubs.acs.org on May 8, 2018

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 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 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.

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 32

Journal of Agricultural and Food Chemistry

Geraniol encapsulated in chitosan/gum arabic nanoparticles: a promising system for pest management in sustainable agriculture Jhones Luiz de Oliveira1, Estefânia Vangelie Ramos Campos2, Anderson E. S. Pereira2, Lucas E. S. Nunes1, Camila C. L. da Silva1, Tatiane Pasquoto3, Renata Lima3, Giovani Smaniotto4, Ricardo Antonio Polanczyk4 and Leonardo F. Fraceto*1

1

São Paulo State University (UNESP), Institute of Science and Technology, Avenida

Três de Março 511, Alto da Boa Vista, Sorocaba, São Paulo, 18087-180, Brazil 2

Department of Biochemistry and Tissue Biology, Institute of Biology, State University

of Campinas (UNICAMP), Campinas, São Paulo, 13083-862, Brazil 3

LABiToN – Laboratory for Evaluation of Bioactivity and Toxicology of

Nanomaterials, University of Sorocaba, Rodovia Raposo Tavares, km 92.5, Sorocaba, São Paulo, 18023-000, Brazil 4

São Paulo State University (UNESP), Department of Plant Protection, Faculty of

Agronomy and Veterinary Sciences, Jaboticabal, São Paulo,14884-900, Brazil

*

Corresponding author: [email protected] (L.F.F.)

ACS Paragon Plus Environment

1


Page 2 of 32

1

Abstract

2

The nanoencapsulation of botanical compounds (such as geraniol) is an important

3

strategy that can be used to increase the stability and efficiency of these substances in

4

integrated pest management. In this study, chitosan/gum arabic nanoparticles containing

5

geraniol were prepared and characterized. In addition, evaluation was made of the

6

biological activity of geraniol encapsulated in chitosan/gum arabic nanoparticles

7

towards whitefly (Bemisia tabaci). The optimized formulation showed a high

8

encapsulation efficiency (>90%) and remained stable for about 120 days. The

9

formulation protected the geraniol against degradation by UV radiation, and the in vitro

10

release was according to a diffusion mechanism that was influenced by temperature. An

11

attraction effect was observed for Bemisia tabaci, indicating the potential of this type of

12

system for use in pest management, especially in trap devices.

13 14

Keywords: Botanical active agent, environmentally friendly, toxicity.

15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33


2

Page 3 of 32


34 35

Introduction

36

Geraniol (2E-dimethylocta-2,6-dien-1-ol) (GRL) is an acyclic monoterpene alcohol

37

that presents low water solubility (100 mg/L) but is soluble in most organic solvents. It

38

is exhaled from the flowers of various plant species and is also found in the tissues of

39

certain herbs. It is a common component of several essential oils including ninde oil

40

(~66.0%), rose oil (~44%), palmarosa oil (~53%), and citronella oil (~25%) 1. Geraniol

41

is widely used as a chemical component of cosmetic fragrances and for other home

42

uses. It has various biochemical and pharmacological activities, including antimicrobial

43

2

44

repellent and attraction properties

45

concentration of the compound and the insect type 9.

46

, antioxidant 3, anti-inflammatory 4, and antitumor 7,8

5,6

. Geraniol also presents insect

. These effects are mainly dependent on the

Due to their biological activities towards insects, essential oils and their main active 10,11

47

components have been identified as potential agents in pest management

48

advantages of these compounds of botanical origin, rather than the more commonly

49

used synthetic compounds, include their low persistence in the field and that fact that

50

they generally exhibit strong selectivity and complexity (which can delay the

51

development of resistance in target organisms)

52

compounds in agriculture is limited by their high sensitivity to light, humidity,

53

temperature, and degradation by microorganisms. Hence, there is a need to develop

54

formulations able to improve the stability and effectiveness of these natural compounds

55

in various different applications 13.

. The

12

. However, the application of these

56

One technique that has shown considerable promise is the use of nanotechnological

57

carrier systems capable of protecting the active agents against premature degradation,

58

while also increasing their water solubility and promoting slower release

59

nanostructured systems can be produced from various matrices, including biodegradable

60

polymers such as chitosan. This is a polymer derived from chitin, found in the

61

exoskeletons of crustaceans, and in terms of use and distribution is considered the

62

second most widespread biomaterial (after cellulose). It is obtained by the deacetylation

63

of chitin in an alkaline medium 14.

10

. These

64

A variety of methods have been described in the literature for the preparation of

65

nanoparticles based on chitosan, the most important being ionic gelation, coacervation,

66

co-precipitation, solvent evaporation, and microemulsion 15. Some of these methods use

67

a crosslinking process in which interlinking of chains is induced by reaction with a


3


Page 4 of 32

68

bifunctional substance capable of generating a three-dimensional polymer network.

69

Various agents can be used for ionic crosslinking of chitosan in order to produce

70

polyelectrolyte

complexation

71

tripolyphosphate

16

72

biocompatible and biodegradable polysaccharide. The carboxyl groups present in the

73

molecule are largely dissociated at neutral pH, with the molecule having an open, highly

74

charged, and expanded structure. These features mean that compared to other

75

polysaccharides, gum arabic presents greater quantities of interaction sites and negative

76

charges capable of associating with polycationic chitosan

77

described the use of such nanoparticles for the encapsulation of natural compounds 20,21.

78

The aim of this study was to develop systems based on chitosan/gum arabic

79

nanoparticles for loading with the botanical active agent geraniol. In addition to

80

preparation and characterization of the nanoparticles, their photostability and release

81

properties were evaluated in vitro at different temperatures. The biological effects of the

82

formulation were investigated in whitefly (Bemisia tabaci). This study opens

83

perspectives for the use of nanoparticles containing geraniol in pest management for

84

sustainable agriculture.

, alginate

nanoparticles,

the

main

ones

17

being

sodium

18,19

, and different types of gums

. Gum arabic is a

19

. Several studies have

85 86

Materials and Methods

87

Materials

88

Chitosan (molecular weight: 27 kDa; degree of deacetylation: 75-85%) gum

89

arabic (molecular weight ~25x105 Da), Tween 80 (average micellar molecular weight

90

79 kDa), and the geraniol active agent were obtained from Sigma-Aldrich. Acetonitrile

91

(HPLC grade) was obtained from J. T. Baker. The seeds were from ISLA Sementes and

92

the substrate was from Hortaliça Mix.

93 94

Optimization of preparation of the formulation: 23 factorial design

95

The effects of the amounts of some of the components used to prepare the

96

chitosan/gum arabic formulations were evaluated using a 23 factorial design performed

97

with Statgraphics Plus statistical software. This approach is often adopted for

98

optimization of preparation processes, since it enables evaluation of the effects of the

99

variables using only a few experiments. For these nanoparticle formulations containing

100

geraniol, the experimental design was expected to lead to the production of


4

Page 5 of 32


101

nanoparticles with average diameter of 200-300 nm, polydispersity index below 0.25,

102

and high zeta potential, in addition to high encapsulation efficiency.

103

The factors studied were the concentrations of chitosan, gum arabic, and Tween

104

80, while the concentration of geraniol was kept constant at 5 mg/mL. The polymer

105

concentrations were defined after initial preparation tests. The concentrations were

106

studied at three levels: 0.15, 1, and 1.5% (g/g) of chitosan; 0.3, 1, and 3% (g/g) of gum

107

arabic; and 0.5, 1, and 1.5% (g/g) of Tween 80. The dependent variables were the mean

108

nanoparticle diameter (MD), polydispersity index (PI), zeta potential (ZP), and

109

encapsulation efficiency (EE).

110

The nanoparticles were prepared based on the method described previously

22

,

111

with some modifications. The procedure consisted of two main steps: emulsification,

112

followed by a process of ionic gelation between the positive charges of the chitosan (pH

113

3) and the negative charges of the gum arabic (pH 7). The low pH of the chitosan

114

solution was due the solubilization in acid solution (acetic acid 0.5%). Firstly, solutions

115

of chitosan and gum arabic were prepared at different concentrations, under agitation

116

overnight at ambient temperature, with the chitosan diluted in 0.5% acetic acid solution.

117

The solutions were filtered through a 0.45 µm syringe filter (Millipore) and the final

118

concentrations were checked after filtration (pH 3 for chitosan and pH 7 for gum

119

arabic). In the next step, the geraniol active agent and the Tween 80 surfactant were

120

added to 16 mL of the chitosan solution, which was stirred at 10,000 rpm (UltraTurrax)

121

for 5 min. The emulsion formed was then agitated slowly for around 10 min, followed

122

by slow dropwise addition of 6 mL of the aqueous gum arabic solution to the chitosan

123

solution. The formulation was stirred for a further 10 min and stored at 25 ºC.

124 125

Characterization of the nanoparticles

126 127

Mean diameter (using dynamic light scattering and nanoparticle tracking analysis)

128

and zeta potential

129

Dynamic light scattering (DLS) and nanoparticle tracking analysis (NTA) are

130

complementary techniques that provide important information about the mean particle

131

diameter of nanoparticle formulations, as well as the particle concentration. In this

132

study, it was decided to submit the samples to analysis by the two techniques, in order

133

to improve the accuracy of information about the nanoparticle formation process and the

134

stability of the suspension. DLS and microelectrophoresis (zeta potential) analyses were


5


Page 6 of 32

135

performed using a ZetaSizer Nano ZS90 particle analyzer (Malvern Instruments) at a

136

fixed angle of 90º and temperature of 25 ºC. For the NTA measurements, the data were

137

collected using a NanoSight LM 10 cell (532 nm laser) and a sCMOS camera,

138

controlled by NanoSight v. 2.3 software. The nanoparticle stability was investigated

139

over 120 days. The stability evaluation over time is performed in order to observe

140

eventual changes in formulations physical-chemical parameters over the storage time.

141

Also, this is important in special for market issues as well as the shelf life of the

142

product.

143 144

Quantification of geraniol and determination of encapsulation efficiency

145

Geraniol was quantified by high performance liquid chromatography, using an

146

UltiMate 300 system (Thermo Scientific) controlled with Chromeleon v. 7.2 software.

147

The chromatographic conditions are described in the Supplementary Material.

148

Concentrations from 5 to 10 µg/mL were used to obtain the analytical curve. The

149

samples were diluted in the mobile phase (acetonitrile:water, 60:40, v/v). Geraniol

150

encapsulated in the chitosan/gum arabic nanoparticles was quantified by an

151

ultrafiltration/centrifugation method employing regenerated cellulose ultrafiltration

152

devices (Microcon 10 kDa, Millipore). This enabled quantification of the amount of

153

non-encapsulated active agent, so the encapsulation efficiency (EE) could be

154

determined by difference, considering the total amount added. The total amounts of

155

geraniol (100%) present in the formulations were calculated taking into account the total

156

amount added, as well as the losses during the preparation process.

157 158

Evaluation of interaction of geraniol with the nanoparticles using infrared

159

spectroscopy (FTIR) and differential scanning calorimetry (DSC)

160

FTIR and DSC analyses were used to investigate possible interactions between

161

geraniol and the chitosan/gum arabic nanoparticles 23. Sample pellets were obtained by

162

centrifugation at 8000 x g for 15 min. The FTIR analyses employed an Agilent

163

spectrophotometer equipped with an attenuated total reflectance (ATR) accessory and

164

operated in the range from 400 to 4000 cm-1, with 64 scans and 8 cm-1 resolution. The

165

DSC analyses were performed using a DSC Q20 system (TA Instruments), under a flow

166

of nitrogen at 50 mL/min, with heating from 0 to 300 °C at a rate of 10 °C/min.

167


6

Page 7 of 32


168

Morphological analysis of the nanoparticles

169

Atomic force microscopy (AFM) was used to analyze the morphology of the

170

chitosan/gum arabic nanoparticles containing the geraniol active ingredient. For this, an

171

Easyscan 2 microscope (Nanosurf, Switzerland) was operated in contactless mode, with

172

TapAl-G cantilevers (BudgetSensors, Bulgaria), at a scan rate of 90 Hz. Gwyddion

173

software was used to produce images in TIFF format (256 x 256 pixels).

174 175 176

Release assays and release mechanisms The release assays were performed as described previously

20

, with some

177

modifications. The nanoparticles containing geraniol were placed in dialysis membrane

178

bags (1 kDa exclusion pore size, Spectra/Pore) and immersed in 5% (w/v) Tween 80

179

solution. Aliquots were periodically collected and the compound was quantified as

180

described above. In order to avoid any losses by evaporation, the vessels were closed

181

and were only opened during sampling (performed in triplicate). The release assays

182

were performed at different temperatures (20, 25, and 30 °C) in order to investigate the

183

influence of this parameter on the profile of release of geraniol from the chitosan/gum

184

arabic nanoparticles. The release data were evaluated using the zero order, first order,

185

Higuchi, and Korsmeyer-Peppas models 34.

186 187

Photostability assays

188

The photostability assays were performed as described previously 24, with some

189

modifications. The formulations were subjected to ultraviolet light (365 nm) in a dark

190

chamber and aliquots were periodically collected for determination of the amount of

191

geraniol present in the solution. The nanoparticle formulations (100 µL) were extracted

192

using acetonitrile (400 µL), followed by quantification of the compound using HPLC.

193

The zero order, first order, pseudo-first order, and pseudo-second order kinetic models

194

were applied to the photodegradation curves in order to obtain further information about

195

the geraniol degradation process.

196 197

Biological effect on whitefly

198

The biological activity assays were performed with whitefly (Bemisia tabaci). The

199

insects were reared on tomato plants (Solanum lycopersicum) in a greenhouse. For the

200

bioassays, the insects were collected using a manual sucker. The attraction/repellency

201

tests were conducted with a four-way olfactometer, similar to the one described


7


Page 8 of 32

202

previously 25, which enabled recording of the insect responses to different odors, offered

203

simultaneously. The insects were exposed to the four fields, two with only filtered air,

204

and two with nanoparticle formulations and/or the botanical active agent. The

205

treatments (odors) were positioned randomly in the olfactometer fields and the insects

206

were individually introduced into the central arena of the olfactometer, where the odors

207

derived from the four fields were mixed and the insects could move freely. The air

208

flows passing through the odor fields were controlled at 200 mL/min with four

209

flowmeters. For each treatment, 10 insects (repetitions) were observed during 10 min.

210

The nanoparticles containing geraniol and geraniol emulsified with Tween 80 were

211

tested at a geraniol concentration of 5 mg/mL. A response to the formulation was

212

considered when the insect crossed the dotted line in the center of the arena, noting the

213

time that it remained in such a position. This was performed every time that the insect

214

moved to a different region. After each repetition, the olfactometer was rotated by 90°

215

in order to avoid conditioning the insects to the laboratory environment.

216 217

Results and Discussion

218 219

Preparation and optimization

220

From the experimental design, it was expected to obtain a formulation with

221

nanoparticles with size in the range 200-300 nm, polydispersity index below 0.25, high

222

absolute zeta potential, and high efficiency of encapsulation of the botanical active

223

ingredient. A total of 9 formulations were performed, based on the 23 factorial design.

224

The effects of three factors were evaluated: chitosan concentration (X1), gum arabic

225

concentration (X2), and Tween 80 concentration (X3), each at three different levels

226

(coded as -1, 0, and +1). The dependent variables were the mean nanoparticle size

227

(MD), polydispersity index (PI), zeta potential (ZP), and encapsulation efficiency (EE).

228

Table 1 provides the absolute results obtained with the factorial design applied to

229

preparation of the chitosan/gum arabic nanoparticles containing geraniol. The ranges of

230

values obtained were 140 - 750 nm (MD), 0.21 - 0.78 (PI), -21 - 35 mV (ZP), and 91-

231

98% (EE).

232 233

[Table 1]

234 235

Pareto charts (Figure 1) were used to identify the factors that significantly (p*


8

Page 9 of 32


236

Gum Arabic Nanoparticles: A

Recommend Documents