Tripolyphosphate Nanoparticles: Efficient Carriers for

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Agricultural and Environmental Chemistry

Pectin/Chitosan/Tripolyphosphate Nanoparticles: Efficient Carriers for Reducing Soil Sorption, Cytotoxicity and Mutagenicity of Paraquat and Enhancement of its Herbicide Activity Marzieh Rashidipour, Afshin Maleki, Sajad Kordi, Mehdi Birjandi, Naser Pajohi, Ebrahim Mohamadi, Rouhollah Heydari, Reza Rezaee, Bahram Rasoulian, and Behroz Davari J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b01106 • Publication Date (Web): 01 May 2019 Downloaded from http://pubs.acs.org on May 1, 2019

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Journal of Agricultural and Food Chemistry

ACS Paragon Plus Environment


1

Pectin/Chitosan/Tripolyphosphate Nanoparticles: Efficient Carriers for

2

Reducing Soil Sorption, Cytotoxicity and Mutagenicity of Paraquat and

3

Enhancement of its Herbicide Activity

4

Marzieh Rashidipour1, Afshin Maleki1, Sajad Kordi2, Mehdi Birjandi3, Naser Pajouhi3, Ebrahim

5

Mohammadi1, Rouhollah Heydari3, Reza Rezaee1, Bahram Rasoulian3*, Behroz Davari1,4*

6 7

1

8

University of Medical Sciences, Sanandaj, Iran

9

2

Environmental Health Research Center, Research Institute for Health Development, Kurdistan

Young Researchers and Elite Club, Khorramabad Branch, Islamic Azad University,

10

Khorramabad, Iran

11

3

12

Khorramabad, Iran

13

4

14

Sciences, Hamadan, Iran

Razi Herbal Medicines Research Center, Lorestan University of Medical Sciences,

Department of Medical Entomology, School of medicine, Hamadan University of Medical

15 16 17

Corresponding authors:

18

*Bahram Rasoulian, Razi Herbal Medicines Research Center, Lorestan University of Medical

19

Sciences, Khorramabad, Iran

20

E-mail: [email protected] and [email protected]; TelFax: +98 66-33204007

21

*Behroz Davari, Environmental Health Research Center, Research Institute for Health

22

Development, Kurdistan University of Medical Sciences, Sanandaj, Iran

23

E-mail: [email protected]; TelFax: +988733661817


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Table of contents

25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42



43

Abstract

44

As a potent herbicide capable of contaminating water and soil environments, paraquat which is

45

still widely used worldwide is toxic to mammals, algae, aquatic animals, etc. Paraquat was loaded

46

on novel nanoparticles composed of pectin, chitosan, and sodium tripolyphosphate (PEC/CS/TPP).

47

The size, polydispersity index and zeta potential of nanoparticles were characterized. Further

48

assessments were carried out by SEM, AFM, FT-IR and DSC. The encapsulation was highly

49

efficient and there was a delayed release pattern of paraquat. The encapsulated herbicide was less

50

toxic to alveolar and mouth cell lines. Moreover, the mutagenicity of the formulation was

51

significantly lower than pure or commercial forms of paraquat in a Salmonella typhimurium strain

52

model. The soil sorption of paraquat and the deep soil penetration of the nanoparticle-associated

53

herbicide were also decreased. The herbicidal activity of paraquat for maize or mustard was not

54

only preserved but also enhanced after encapsulation. It was concluded that paraquat encapsulation

55

with PEC/CS/TPP nanoparticles is highly efficient and the formulation have significant herbicide

56

activity. It is less toxic to human environment and cells as was evidenced by less soil sorbtion,

57

cytotoxicity and mutagenicity. Hence, paraquat-loaded PEC/CS/TPP nanoparticles have potential

58

advantages for future usage in agriculture.

59 60

Keywords: Paraquat; Nanoparticle; Pectin; Cytotoxicity; Mutagenicity

61 62 63 64 65


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66

Introduction

67

Application of nano-encapsulation for targeted delivery has been efficient in the production of new

68

formulations of effective herbicides with lower contamination of human environment and

69

agricultural products. The main purpose of such delivery systems is using lower doses of toxins

70

and the controlled release of them. In some elegant studies, chitosan, as a non-toxic and

71

biodegradable carrier system, has been used to design new formulations of herbicides 1.

72

Paraquat (PQ) is a non-selective contact herbicide. It has been used as a herbicide for agricultural

73

purposes in more than 100 countries for pre-planting weed control in the fields of tobacco, cotton,

74

rice, etc. 2. Paraquat can enter into the water network and even low level of it is considered as a

75

threat to human health. It is classified as a highly toxic herbicide with a permissive limit of 0.03

76

mg/L in water supplies. Paraquat is not only highly toxic to mammals but also has toxic effects on

77

algae, fish and other aquatic animals 3, 4, 5. This herbicide strongly bounds to soil, and its half-life

78

in soil varies from 1.3 to 13 years. Residual paraquat up to about 70 mg/kg has been detected in

79

fruits and vegetables 6. Paraquat poisoning in mammals causes severe lung 7, 8 and renal injuries 9.

80

The toxicity is associated with the dysfunction of mitochondrial and microsomal oxidation-

81

reduction systems and the production of reactive oxygen species. Hydrogen peroxide and

82

superoxide anions are the main toxicants. These free radicals cause lipid peroxidation,

83

mitochondrial membrane dysfunction and damage to DNA and Proteins 6, 10, 11.

84

Chitosan is a linear polymer and the deacetylated form of chitin that is produced from the shells

85

of crustaceans. This compound is biodegradable, non-toxic, and is cheap and accessible. It is easily

86

absorbed in the plants through the leaves and stems. Hence, when chitosan is used as an herbicide

87

carrier, the contact area at plant level increases and the absorption time of toxin shortens 1. The



88

use of compounds such as tripolyphosphate (TPP) as a crosslinking agent in the synthesis of

89

chitosan-based nanoparticles obviates the need to use organic solvents in the synthesis process 7.

90

Chitosan/TPP nanoparticles were used as carriers for paraquat. Although this formulation is

91

surprisingly effective in reducing soil sorption and also cytotoxicity and genotoxicity of paraquat,

92

the herbicide activity was preserved but not enhanced by the formulation 7.

93

Pectin is a polysaccharide that exists in the plant cell wall and is used in gel, film and nanoparticle

94

formulations because of its biocompatibility and non-toxicity

95

plant-based material increases the absorption of the toxin by herbs. Existence of de-esterified

96

galacturonase in pectins enables them to have a major role in the binding of cations on the plant

97

cell walls 13. Consequently, they are good candidates for binding to paraquat as a divalent cation

98

14.

99

polymer's erosion time, resulting in delayed release of the toxin from the formulation 42.

12.

It is expected that this natural

Also, it seems that, due to the high water absorption capacity of pectin, it could increase the

100

Accordingly, we added pectin to the nano-formulation. The physico-chemical properties of this

101

new nanogel and the herbicide release kinetics were assessed. Moreover, herbicide activity, soil

102

sorption kinetics, cytotoxicity and mutagenicity of these paraquat-containing nanoparticles were

103

assessed by various models.

104 105 106 107


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108

Experimental Procedures

109

Materials

110

Chitosan (MW: 27 kDa, degree of deacetylation: 75–85%), paraquat dichloride, sodium

111

tripolyphosphate (TPP), pectin (≥74.0 %), sodium 1-hexanesulfonate and acetic acid were

112

obtained from Sigma-Aldrich (the United States). Methanol, histidine, biotin, sodium azide, top

113

agar, hydrochloric acid, acetonitrile, sodium phosphate and calcium chloride were purchased from

114

Merck Chemical Co. (Germany). Membrane filters were purchased from Millipore (the United

115

States). The plant seeds were acquired from Pakan Bazr Co. (Iran).

116

Alveolar epithelial cells (A549, adenocarcinomic human alveolar basal epithelial cells) and

117

carcinoma of the mouth cells (KB) were prepared from the cell bank of Pasteur Institute in Iran.

118

The cells were incubated in a 50- mL flask containing 20 mL of the RPMI culture medium (BIO-

119

IDEA) with 10% FBS (Sigma Corp.) in the CO2 incubator (Memert Corp., Germany) at 37 ◦C and

120

with 5% of CO2.

121

Bacteria strain (Salmonella typhimurium tester strains TA100) was obtained from Razi Herbal

122

Medicines Research Center (Iran).

123

Preparation of the Nanoparticles

124

Preparation method of nanoparticles was based on the ionic gelification technique described by P.

125

Calvo 15 with some essential modifications: a solution of chitosan (0.04% in 0.2% acetic acid) was

126

prepared at pH of 4.7 under magnetic stirring for 12h. An aqueous TPP solution (0.01%) was also

127

prepared. To remove any insoluble or aggregated material, solutions were filtered via a membrane

128

(0.45 µm). Subsequently, the TPP solution (5 mL) was added to 20 mL of the chitosan solution

129

dropwise under agitation with magnetic stirring and the solution was stirred for another 10 min.

130

Then, 2 mL of the pectin solution (0.08%) was added to the chitosan/TPP solution under agitation.



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131

The resulting “PEC/CS/TPP” nanoparticles were stored at ambient temperature (25◦C). In the cases

132

of paraquat loading, prior to TPP and pectin addition, 0.5 mL of paraquat solution (10000 µg/mL)

133

was combined with the chitosan solution. Due to the high solubility of paraquat in water,

134

nanoparticles prepared after centrifugation were washed with distilled water to remove unloaded

135

paraquat.

136

Encapsulation Efficiency

137

After the disintegration of the nanoparticles by dilution in acidic distilled water followed by

138

filtration through a 0.22 µm membrane, total paraquat amounts in suspensions were determined

139

by HPLC (Shimadzu, Japan) consisting of a quaternary pump, UV-Vis detector, vacuum degasser,

140

and system controller that was used as described previously 15. A CN (Cyano) analytical column

141

(MZ, 150×4.6 mm id, 5μm particle size; Germany) was also used. One gram of sodium 1-

142

hexanesulfonate was added to 1 L of phosphate buffer (pH 3.7) and was used as the mobile phase.

143

The flow rate of 0.8 mL/min, detector wavelength of 257 nm and oven temperature of 30◦C were

144

applied. The encapsulation efficiency (EE) was calculated from Eq. 1:

 mass of loaded paraquat   Encapsulation Efficiency(%)  100    mass of initial paraquat 

Eq. [1]

145

Physicochemical Characterization of the Particles

146

Size, Polydispersity Index and Zeta Potential

147

Size distribution of the nanoparticles and their zeta potential were evaluated via dynamic light

148

scattering (DLS) and zeta potential analysis using a Malvern Zetasizer Nano range instrument

149

(Malvern Instruments Ltd., Malvern, UK). The colloidal emulsions were diluted in deionized water

150

(1:1000, v/v) and analyzed in triplicate, at 25 ◦C, using this instrument with a fixed angle of 90◦.

151

Scanning Electron Microscopy (SEM)


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152

To carry out SEM imaging, freeze-dried nanoparticles (at -50 ◦C for 24h) were placed on a stub,

153

sputter coated with gold and evaluated at 30 kV using a 6300 field emission scanning electron

154

microscope (Hitachi model S-4160).

155

Atomic Force Microscopy Analysis (AFM)

156

The atomic force microscopy (AFM, model Full plus, Ara-AFM Iran) was used to characterize

157

nanoscale objects as described by Grillo et al 7.

158

Fourier-Transform Infrared Spectroscopy (FT-IR)

159

The infrared absorption spectra of the samples including CS, TPP, PEC, PQ, NPs and NP:PQ were

160

investigated using FT-IR (BRUKER, model TENSOR 27, Germany) analysis.

161

Differential Scanning Calorimetry (DSC)

162

The differential scanning calorimetry curves were obtained by differential scanning calorimeter

163

(DuPont Instrument, Model DSC 910S) in the temperature range of 50–400 ◦C using an aluminum

164

crucible at heating rate (10◦C/min) under helium atmosphere with a flow rate of 50 mL/min. The

165

freeze-dried sample mass was about 3.00 mg.

166

Release Kinetics

167

The release kinetics were assessed using a system containing a dialysis bag (as donor

168

compartment) and surrounding acceptor compartment separated by membrane of dialysis bag as

169

described previously

170

dialysis bag membrane was 1kDa. Samples (PQ or NP:PQ) were placed in the dialysis bag and

171

aliquots were collected from the acceptor compartment at certain intervals under agitation and

172

analyzed by HPLC.

173

The Korsmeyer–Peppas model 17 was used to analyze the kind of paraquat release. The model is

174

described as follows:

16

and briefly shown in Figure 4. The molecular exclusion pore size of the



𝑀𝑡 𝑀∞

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Eq. [2]

= 𝐾𝑡𝑛

175 176

Mt is the quantity of paraquat released in time t, M∞ is the quantity of paraquat release at infinite

177

time, k represents the kinetic release constant, and n is the release exponent.

178

Soil Column Experiments

179

Soil column experiments were conducted based on the method described by Pereira et al.18, though

180

minor modifications were made. By joining 5 PVC rings, a 25 cm high column was made. After

181

closing one end of the column with a filter paper, it was filled with soil. The soil was constructed

182

by mixing silt (2.8%), clay (11.1%), sand (83%) and organic fertilizer 3.1 (%). Subsequently, PQ,

183

commercial PQ or NP:PQ samples with an equivalent concentration of 2.5 kg/ha of PQ, were

184

placed on the soil column top, and after 24 and 48 h, 25 mL of water (equivalent to 70 mm) was

185

added. The columns were then divided into individual rings. Finally, paraquat concentration was

186

quantified in each ring by HPLC.

187

The amount of paraquat was determined at 0–5, 5–10, 10–15, 15–20 and 20-25 cm depths of the

188

soil column for different formulations (Figure 5). The sorption kinetics was evaluated using the

189

pseudo-second order mathematical model 18.

190

Soil Sorption Assays

191

The herbicide soil sorption assays were carried out in triplicate as described by Grillo et al.7 using

192

herbicide-free soil from Khorramabad, Iran. Briefly, 15 mL of 0.01 M CaCl2 solution containing

193

PQ, commercial PQ or NP:PQ was placed in an Erlenmeyer flask together with 1 g of soil. The

194

mixture was maintained under continuous magnetic stirring at 25 ◦C. One mL aliquots was

195

periodically removed and centrifuged. Subsequently, the supernatant was filtered through a 0.22

196

µm membrane and the paraquat content was quantified by HPLC. Throughout the 180-min period


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197

of the study, the system volume was maintained by adding CaCl2 solution. Regarding the fact that

198

paraquat is hydrolyzed in alkaline pH and is stable in neutral and acid pH, the pH of the medium

199

was adjusted to 4.5 using buffering solution. The sorption was calculated as the herbicide

200

concentration difference between the initial solution and the solution after equilibrium

201

achievement with the soil. The soil sorption kinetic curves were assessed using pseudo-first and

202

pseudo-second order mathematical models. The data were best matched using pseudo-second

203

order model which are reported in (Figure 6 and Figure SI5).

204

Herbicidal Activity Assays

205

The herbicidal activity of free or commercial paraquat (500g/ha) and paraquat loaded on

206

nanoparticles (with three different amounts of 125, 250 and 500 g/ha) were investigated using Zea

207

mays as a monocotyledon and Brassica sp. as a dicotyledon plant. Distilled water (DW) and NPs

208

were used as control. Paraquat employment rate was equivalent to 500g/ha, similar to application

209

rates in grain fields (i.e. 500-600 g/ha, equal to 2.5-3 L of a 20% solution of commercial paraquat)

210

19, 20.

211

Herbicide activity was assayed based on the procedure described by Grillo et al. 7, though minor

212

modifications were made. Briefly, maize or mustard seeds were planted in plastic pots (13 cm

213

high, 16 and 13 cm upper and lower diameters, respectively) containing 2000 g of plant substrate.

214

Ten seeds were planted in each vase. There were 7 groups, 4 replications per group, resulting in a

215

fully randomized 7×4 experimental design. Herbicide solutions or related control solutions were

216

applied 20 days after planting the seeds. The pots were photographed 48 h later and the plants were

217

removed, washed, dried and finally weighted. Dry masses (g) were reported and compared.

218

Cytotoxicity



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219

The cytotoxicity of the samples was determined by MTT test using 7 different concentrations

220

(based on 3.75 to 500 µg/mL of paraquat) as was described previously 21. A549 and KB cell lines

221

were used. ELISA reader was used for MTT test (BioTek, USA).

222

Salmonella Mutagenicity Test

223

The mutagenic effects of PQ, NPs, NP:PQ and commercial PQ were evaluated on S. typhimurium

224

strain TA100 with and without rat liver microsomal fraction (S9) activation as described by Ames

225

22.

226

control by its mutagenic index (MI) value 23 that was calculated by the following formula:

The number of histidine-positive revertants in each sample was compared to the negative

  Number of his‫ ‏‬ revertants induced in the samples  Mutagenic index (MI)  100    Number of his  ‫ ‏‬revertants induced in the negative control 

Eq. [3]

227

S9 was prepared as described by Garner et al 24.

228

Statistical Analysis

229

IBM SPSS software for windows, version 22 was used to carry out the statistical analysis. Data

230

were expressed as Mean±SD. Analyses of variance (ANOVA) with Tukey or LSD post-hoc tests

231

were used for comparing mean values. P 0.999 indicating that calculated maximum qe data are quite close to the

514

experimental values. Pseudo-second-order kinetic model is a suitable model for the adsorption of

515

dyes, oils, metal ions, organic substances and herbicides from aqueous solutions

516

by materials with heterogeneous surfaces such as the soil 40.

517 518 519 520 521 522 523 524


39

and sorption

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525 526

Figure 6. Soil sorption of paraquat, NP:PQ (A), PQ (B) and Commercial PQ (C), at 25◦C and pH

527

4.5 (n=3). Application of the pseudo-second order mathematical model (Aa-Cc) where qt is amount

528

of solute sorbate at any time t (mg/g).

529 530 531 532 533 534 535 536 537 538 539 540 541 542



543

Evaluation of Herbicidal Activity

544

All forms of paraquat indicated herbicidal activity against both plants within 48 h, as was

545

characterized by leaf necrosis and significantly lower dry plants’ weights compared to DW groups

546

(Figure 7). It has been determined that the herbicide activity of paraquat is lower in the case of Z.

547

mays probably due to having P-450S glutathione-S-transferase enzyme 18. Similarly, in the present

548

research, it was determined based on the observed faster leaf necrosis, that the herbicidal activities

549

of all paraquat forms were more significant on Brassica sp. than Z. mays.

550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581


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582

583 584

Figure 7. Herbicidal activity of paraquat in the cultivations of maize and mustard after 48 h and

585

dry weights of the plants after PQ application and drying. a: no significant difference vs. DW

586

group; b: significant difference vs. control group and no significant difference between each other.

587 588 589 590 591 592 593 594 595 596 597 598



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599

Cytotoxicity Assays

600

The results of the cytotoxicity assays on A549 and KB cell lines are shown in Figure 8 and Table

601

SI 1 & SI2. The toxicity of NPs on A549 was relatively low in the majority of doses. This toxicity

602

was nearly more on KB cells, which may be a reflection of antitumoral activity of pectin

603

Paraquat had comparatively more intense cytotoxic effects, particularly on A549 cells. The

604

mortality was lowered when the paraquat was loaded on nanoparticles. This reduction was more

605

evidenced in A549 cells. Lungs are the major target organ of paraquat toxicity

606

lowering cytotoxic effects of paraquat on pulmonary cells, which is achieved by loading them on

607

PEC/CS/TPP nanoparticles, is a considerable phenomenon referred to in our findings that should

608

be further investigated in in-vivo models.

609 610 611 612 613 614 615 616 617 618 619 620 621


43;

42.

hence, the

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622 623

Figure 8. Toxicity of NPs, PQ, NP:PQ and commercial PQ on two cell lines (A: A549, B:KB).

624

A549: Adenocarcinomic human alveolar basal epithelial cells, KB: human epidermoid mouth

625

carcinoma cell line.

626 627 628 629 630 631 632 633 634 635 636 637 638 639 640 641



642

Mutagenicity Assays

643

The results of the present study showed that encapsulation of paraquat reduces its mutagenesis

644

compared to its free and commercial forms (Table 3). Materials with mutagenic index (MI) of

645

greater than 2 are considered as mutagens 23. The mutagenic index of sodium azide, as a positive

646

control mutagen, was 1.54-3.55 (data not shown) which is slightly higher than commercial

647

paraquat.

648 649 650 651 652 653 654 655 656 657 658 659 660 661 662 663 664


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Table 3. Mutagenicity of NPs, PQ, NP:PQ or commercial paraquat in Salmonella typhimurium

666

tester strains (TA100). Dose (µg/plate)

665

Mutagenic index (MI) NPs

NP:PQ

PQ

Commercial paraquat

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

1

0.28±0.06

0.29±0.08

0.55±0.10

0.40±0.14

0.73±0.16

1.13±0.19*

1.87±0.20 a

1.53±0.28

5

0.07±0.06

0.02±0.01

0.67±0.05

0.57±0.20

0.89±0.17

1.17±0.19*

2.40±0.17 a

2.07±0.11 a

10

0.09±0.10

0.26±0.14

0.81±0.02

0.67±0.31

1.07±0.18

1.32±0.07*

2.58±0.06 a

3.07±0.06 a

30

0.29±0.04

0.34±0.07

0.88±0.18

0.93±0.10

0.97±0.08

1.48±0.06*

2.20±0.56 a

2.65±0.30 a

50

0.39±0.00

0.40±0.09

0.97±0.13

0.92±0.26

1.24±0.34

1.73±0.25*

2.55±0.15 a

2.49±0.11 a

70

0.54±0.06

0.25±0.02

0.91±0.20

0.98±0.10

1.72±0.04*

1.68±0.19*

2.35±0.33 a

2.27±0.26 a

90

0.62±0.08

0.17±0.04

1.29±0.06

0.96±0.13

2.01±0.10*

1.94±0.09*

2.76±0.20 a

2.95±0.18 a

100

0.77±0.07

0.84±0.11

1.50±0.09

0.79±0.07

2.18±0.29*

2.04±0.30*

2.72±0.19 a

3.21±0.15 a

667 668

*: significantly different from the NP:PQ group (-S9 data are compared to -S9 and +S9 data are compared

669

to +S9).

670

a: significantly different from the PQ group (-S9 data are compared to -S9 and +S9 data are compared to

671

+S9).

672 673 674 675 676 677 678



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679

Discussion

680

The efficiency of encapsulation in nanoparticles which was 89.41±5.70% indicates better affinity

681

between the components than previous CS/TPP-based paraquat-containing nanoparticles with

682

about 62% encapsulation7. The affinity between the components is thought to be a result of

683

electrostatic forces. Chitosan and paraquat have positive charges7. The only negatively charged

684

component in CS/TPP nanoparticles is TPP. Adding pectin anions as a new negatively charged

685

component might lead to higher encapsulation efficacy 25.

686

Increasing the particle sizes after paraquat loading can be related to the lowering surface charge

687

and reducing the electrostatic repulsion between the particles. By decreasing the electrostatic

688

repulsion, the particle accumulation will increase and consequently, the size will be larger

689

Increased particle size of NPs or (NP:PQ) after 60 days, could be due to the presence of pectin in

690

our formulation as a water-absorbent polysaccharide 26. No significant change was observed in the

691

particle size of CS/TPP after a sixty-day period 7.

692

The values of the zeta potential, as a measure of the surface charge of particles, were negative. It

693

is substantially due to the presence of the anionic pectin polymer and TPP. This negative zeta

694

potential increased about -10 millivolts after 60 days which may result in the improved stability of

695

nanoparticles due to the greater charge repulsion between particles and the inhibition of their

696

aggregation 27. As another stability parameter 28, pH approximately increased from 5.6 to about

697

7.5-7.7 after 60 days (table 1 & Figure SI2). This neutralization that occurs over time is probably

698

due to the release of the hydroxyl groups from TPP or pectin. CS/TPP nanoparticles had an acidic

699

pH that gradually changed to a more acidic one (about 3.5) 7. Nearly neutral pH of PEC/CS/TPP

700

nanoparticle could be considered as an advantage because the acidic pH of the soil in the fields

701

decreases the production of nutrients required by the plants 29.


49,50.

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702

The release pattern of a material from nanoparticles is related to some factors like the shape and

703

size of the particles, the polymer’s molecular weight, the nature of the reagents, and the type of

704

interactions 33.

705

Surprisingly there was a kind of delayed release of paraquat from PEC/CS/TPP nanoparticles.

706

There was almost no release until 30 min. This delayed release could be considered as an advantage

707

when famers are spraying the herbicide, and could reduce direct human exposure to paraquat. In

708

fact, matrices incorporating pectin have been employed to prolong the release of many drugs in

709

pharmaceutical industry. After contact with aqueous media, pectin rapidly forms viscous solutions

710

and gels and is widely used as a carrier in the sustained/controlled-release of oral drugs. Not only

711

do the actions of pectin regulate the swelling degree through osmotic effects but also they increase

712

the cross-linking density and could regulate the release rate

713

herbicide could also be explained by affinity-controlled mechanisms which occur by means of

714

ionic interactions between the pectin and the oppositely charged paraquat 25.

715

Since paraquat exerts its herbicidal activity through surface plant absorption

716

infiltrations in various formulations could not have any direct influence on this main effect of the

717

herbicide. Although less soil infiltration of paraquat after its encapsulation might be considered as

718

an advantage in terms of underground water sources pollution, the main constituents of the

719

formulation are biodegradable

720

priority in this regard in long term. Nevertheless, maintaining the paraquat in more superficial

721

layers of the soil may increase its biodegradation by microbiological and photochemical processes

722

before its infiltration to deeper soils and contaminating groundwater resources 37. In comparison

723

to other formulations like poly (epsilon-caprolactone) nanoparticles containing atrazine

724

reduction in soil penetration in present formulation was considerable quantitatively.

36.

34.

Here, the delayed release of

35,

different soil

Hence, this encapsulation could not be considered as a major


18,

the


725

In the present study, less sorption of paraquat was achieved when it was nanoparticle-associated.

726

Less sorption of paraquat in case of being nanoparticle-associated has been considered as an

727

advantage in CS/TPP formulation because it might reduce the relatively deep soil sorption of

728

paraquat and leave more concentrations of the herbicide available to the intended weeds 7. Less

729

paraquat sorption in case of being nanoparticle-associated could be explained by the repulsion of

730

the negative surface charge of PEC/CS/TPP nanoparticles by the negatively-charged clay particles

731

41.

732

weights between NPs and DW groups. Also, there was not any significant difference between dry

733

weights in all forms of applied herbicide. In the case of Z. mays, better herbicide activity in all

734

NP:PQ groups, compared to free or commercial PQ groups, were not statistically significant.

735

Overall, lower doses of loaded paraquat on CS/TPP/PEC nanoparticles had at least similar

736

herbicide activity in Brassica sp. weed control with no further toxicity to Z. mays. The greater

737

efficacy of paraquat when loaded in CS/TPP/PEC nanoparticles could be explained by the better

738

adhesion of paraquat to plant surfaces due to the presence of chitosan and pectin. Pectin, as a

739

natural polysaccharide in plant cells' walls, and chitosan are both easily absorbed to plant surfaces,

740

and this could increase the effective concentration of the loaded target for acting on the plant

741

surfaces 23. In should be noted once more that pectin/chitosan-based encapsulation could lead to

742

the fact that paraquat, in concentrations lower than the normal rate (125, 250 g PQ/ha), is able to

743

eliminate weeds (Figure 7). The major mechanism of paraquat toxicity is related to free radical

744

production. It has been proposed in other models that prior exposure of cells to sub-lethal doses of

745

free radicals preconditions them against lethal doses and reduces subsequent injury due to the

746

upregulation of innate defense mechanisms against reactive oxygen species 44, 45, 46.

Nanoparticles had no toxic effects on both plants as evidenced by not significantly different dry


Page 36 of 46

Page 37 of 46


747

Similarly, the sustained delayed release of paraquat might produce sub-lethal doses of free radicals

748

as a trigger factor for the induction of cellular antioxidants and other defense mechanisms. This

749

phenomenon could explain the lower overall toxicity of paraquat when loaded on PEC/CS/TPP

750

nanoparticles 42, 43. It should be noted that commercial paraquat had more cytotoxicity on KB cells

751

and less cytotoxicity on A549 cells than pure paraquat. Paraquat causes genetic damaging to both

752

prokaryotic (e.g. Salmonella typhimurium) and eukaryote (e.g. A. nidulans) microorganisms. The

753

damage is further in Salmonella typhimurium strain 47. The results of the present study indicated

754

that encapsulation of paraquat could reduce its mutagenesis compared to the both free and

755

commercial forms of this herbicide. The commercial form of paraquat could serve as a mutagen

756

in relatively all the concentrations that were used but the encapsulated form of paraquat was not a

757

mutagen even in high concentrations. The results emphasize the significance of developing new

758

and safer herbicide formulations, such as the present encapsulated paraquat, to be used in

759

agricultural fields. Paraquat encapsulation in PEC/CS/TPP nanoparticles is relatively more

760

efficient than some previous formulations and there is a delayed release pattern of paraquat which

761

seems at least partially responsible for lower cytotoxicity of this new formulation. Moreover, the

762

mutagenicity of the formulation is considerably lower than pure or especially commercial forms

763

of paraquat. The soil sorption of paraquat was reduced by encapsulation too. There was a tendency

764

for less deep penetration of paraquat by using this formulation which may help its faster

765

biodegradation. The enhanced herbicide activity of this novel formulation could also be considered

766

as one of the major findings of the research. It seems that the new formulation is less toxic to

767

human environments and cells than some previous formulations and has potential advantages for

768

future usage in agriculture. Further research is required for more practical assessments especially

769

in animal models and humans.



770

Supporting Information

771

The supporting information include:

772

Figure SI1: Optimal amount determination of ingredients (chitosan, pectin, TPP and paraquat) for

773

nanoparticle formation.

774

Figure SI2: The pH of particles as a function of time (0-60 days).

775

Figure SI3: Analysis of the samples using Fourier transform infrared spectroscopy (FTIR).

776

Figure SI4: Differential scanning calorimetry (DSC) thermograms.

777

Figure SI5: Soil sorption of paraquat.

778

Table SI 1: Toxicity of NPs, PQ, NP:PQ and commercial PQ on cell line (Adenocarcinomic human

779

alveolar basal epithelial cells (A549).

780

Table SI 2: Toxicity of NPs, PQ, NP:PQ and commercial PQ on cell line (Carcinoma of the

781

mouth cells (KB)

782

Acknowledgements

783

The authors gratefully appreciate the support of the Environmental Health Research Center of

784

Kurdistan University of Medical Sciences (grant number: IR.MUK.1395.73) and Razi Herbal

785

Medicines Research Center of Lorestan University of Medical Sciences (grant number: 1980).

786

This study was financially supported by grant No: 970503 of the Biotechnology Development

787

Council of the Islamic Republic of Iran. We also thank Dr. Azadeh Alinaghi, Dr. Behjat

788

Sheikholeslami, Behnam Ashrafi, Dr. Sajad Sohrabi, Dr. Mohsen Sohrabi, Dr. Claudio Martin

789

Jonsson, Dr. Siamak Beiranvand, Dr. Asghar Sepahvand, Dr. Neda Eslami, Mojgan Zarei Venovel

790

and Samaneh Hadavand for their contributions to the accomplishment of this research.

791

Disclosure Statement

792

The authors report no conflicts of interest.

793

Abbreviations

794

PEC, pectin; CS, chitosan; TPP, tripolyphosphate; PQ, paraquat; NP(s), nanopraticle(s)


Page 38 of 46

Page 39 of 46

795


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