Controlled Release of Agrochemicals Using pH and Redox Dual

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Agricultural and Environmental Chemistry

Controlled-release of Agrochemicals Using pH and Redox Dual-responsive Cellulose Nanogels Xiaobang Hou, Yuanfeng Pan, Huining Xiao, and Jie Liu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b00536 • Publication Date (Web): 28 May 2019 Downloaded from http://pubs.acs.org on May 29, 2019

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Journal of Agricultural and Food Chemistry

Controlled-release of Agrochemicals Using pH and Redox Dual-responsive Cellulose Nanogels Xiaobang Hou1,3, Yuanfeng Pan2*, Huining Xiao3* and Jie Liu1

1. Department of Environmental Engineering, North China Electric Power University, 689 Huadian Road, Baoding, Hebei 071003, China. 2. Guangxi Key Lab of Petrochem. Resource Proc. & Process Intensification Tech., School of Chemistry and Chemical Engineering Guangxi University, 100 Daxue Road, Nanning, Guangxi 530004, China. 3. Department of Chemical Engineering, University of New Brunswick, 15 Dineen Dr. Fredericton, Canada E3B 5A3

1

ACS Paragon Plus Environment


1

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ABSTRACT

2

A novel pH and redox dual-responsive cellulose-based nanogel was prepared

3

for the controlled-release of agrochemicals. To synthesize the responsive nanogel,

4

palmitoyl chloride and glyoxal was modified on carboxymethyl cellulose

5

sequentially and 3,3'-dithiobis(propionohydrazide) was used as a crosslinker to

6

assemble nanogel. The morphology, structure, and physical properties of nanogels

7

were characterized with transmission electron microscope (TEM), Fourier transform

8

infrared spectroscopy (FTIR), particle size analysis and zeta-potential measurement.

9

Facing pH and redox stimulation, the nanogel showed reversible sol-gel transitions,

10

indicating good pH- and redox-responsiveness. The nanogel loaded with

11

agrochemicals exhibited high loading capacity and various release behaviors. In

12

addition, the experiment of nanogel on heavy metal ions complexation displayed the

13

potential of improving soil condition while delivering agrochemicals.

14

KEYWORDS:

15

controlled-release

pH-responsive,

redox-responsive,

agrochemicals,

nanogel,

16

2


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17

INTRODUCTION

18

Hydrogels are polymeric materials which have been extensively applied to

19

pharmaceutical, biotechnology and environmental sectors because of their excellent

20

performance on biocompatibility and biodegradability1. Large amounts of water can

21

be restricted in the matrix of hydrogels by the hydrophilic groups meanwhile the 3D

22

structure of hydrogels is maintained. In particular, stimuli-responsive hydrogels have

23

drawn much attention due to their unique transformation behaviors in specific

24

environment. Stimuli-responsive hydrogels can realize shrinkage and expansion or

25

reversible disassembly in response to exogenous stimuli, such as temperature2, 3, pH4,

26

magnetic fields5, enzymes6, light7, 8 and reductive or oxidative chemicals9. The dual-

27

or multi-responsive systems10 have also been established by combining various

28

functional groups. For instance, pH- and thermal-responsive dansyl grafted

29

polyacrylamide

30

N,N'-bis(acryloyl)cystamine

31

magnetic field-, and pH-responsive glutaraldehyde crosslinked carboxymethyl

32

chitosan hydrogels13. These “smart” materials have been widely applicated in drug

33

release14, wound healing15, cell culture16 and sensing17.

hydrogel11,

thermo-,

crosslinked

pH-

cellulose

and

redox-responsive

hydrogels12

and

thermo-,

34

In order to prevent and control various pests and diseases and improve crop

35

yield, agrochemicals are widely used in agricultural production. The applications of

36

conventional

agrochemical

formulas

are inefficient;

and

excessive toxic 3



37

agrochemicals come into the food chain through water cycle, which seriously affects

38

the ecological safety and human health. Stimuli-responsive hydrogels are usually

39

applied to the drug controlled-release in human body due to their complex synthesis

40

process and high cost. However, with the development of the environment science

41

and the increasing of the people's ecological consciousness, the applications of

42

stimuli-responsive hydrogels have been extended to the controlled-release of

43

agrochemicals for plants18,

44

formulas, controlled-release technology can significantly improve the utilization

45

efficiency and reduce the dosage of agrochemicals. Therefore, the design and

46

preparation of the stimuli-responsive hydrogels for the controlled-release of

47

agrochemicals could provide an effective approach to realize the reduction of soil

48

and water contamination.

19

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. Compared with the conventional agrochemical

49

Glutathione (GSH) is a ubiquitous antioxidant in organisms. The concentration

50

of glutathione can reach 1 to 100 mg/g in some plant organs and tissues, such as

51

wheat germ, epidermal cells of root hair and fruits20. When the redox-responsive

52

hydrogels are transported into plants via roots or leaves, the loaded agrochemicals

53

are released at the positions with higher glutathione content so that the targeted

54

delivery could be triggered and achieved. In our previous work21, we proposed a

55

method that releasing agrochemicals controlled by cellulose based redox-responsive

56

hydrogels and we confirmed the hydrogels have good performance on stabilizing

57

heavy metal ions (Cu2+ and Hg2+) afterwards due to the abundant carboxyl and thiol 4


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58

in the hydrogels. However, the hydrophilic properties of hydrogels limit the loading

59

capacity of lipid-soluble agrochemicals and macro-sized hydrogel carriers are not

60

conducive to the diffusion and transportation of agrochemicals from in vitro to in

61

vivo. Moreover, though the single-responsive hydrogels have improved the

62

controlled-release behaviors of agrochemicals in vitro, dual- or multi-responsive

63

carriers need to be designed in order to further realize optimal release and delivery

64

performances.

65

In this work, to address the issues related to the more precise and efficient

66

controlled-release of agrochemicals, dual-responsive nanogels were prepared by

67

crosslinking of carboxymethyl cellulose with hydrophobic branches and

68

3,3'-dithio-bis(propionohydrazide). Further to our previous work21 focusing on

69

redox-responsive behavior exclusively, the acylhydrazone bonds as reversible switch

70

for pH-responsiveness were introduced in the structure of nanogel and constructed a

71

dual-responsive system, which is expected to enhance the controllability and extend

72

the application scope significantly. Nanogels have higher surface energy than

73

macro-sized hydrogels, which can help the adhesion of carriers on plants, thereby

74

enhancing the utilization efficiency and effective duration of agrochemicals, and

75

providing the possibility of releasing agrochemical in vivo. To improve the loading

76

capacity of lipid-soluble agrochemicals, hydrophobic branches were grafted on

77

cellulose network by palmitic chloride. Palmitic chloride is produced from palmitic

78

acid which is extracted from grease and has good biocompatibility and low 5



79

environmental hazards. The linear chain composed of sixteen carbon atoms reduces

80

the hydrophilicity of nanogels to a certain extent and facilitate the capture of

81

lipid-soluble agrochemicals. Salicylic acid is a lipid-soluble phytohormone which

82

can regulate photosynthesis, transpiration and ion uptake in plants. More importantly,

83

it has been confirmed that salicylic acid can induce the sustained expression of the

84

plant defense gene PR-1 (pathogenesis-related gene 1), thus improving plant disease

85

resistance22. Therefore, salicylic acid is an ideal model-agrochemical for precise

86

controlled-release. The swelling and sol-gel transition behaviors of nanogels

87

prepared in this work were studied in sucrose solution and water. The

88

controlled-release behaviors were systematically investigated in the presence of HCl

89

and GSH. To date, the stimuli-responsive (especially redox-responsive) release of

90

agrochemical using hydrogel has been seldom reported. The unique approaches

91

created in this work is of great potential for agriculture application, particularly in

92

the controlled release of agrochemicals and soil remediation simultaneously, which

93

are unachievable with conventional carriers for agrochemicals.

94

MATERIALS AND METHODS

95

Materials

96

Sodium carboxymethyl cellulose (CMC, Mw = 90 kDa, degree of substitution

97

= 0.7), glyoxal solution (40 wt% in H2O), palmitoyl chloride (PCl, 96%), salicylic

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6


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98

acid (SA, 99%), N,N-Dimethylformamide (DMF, 99.9%), phenolphthalein (99%),

99

methyl orange (99%), hydroxyl amine hydrochloride (98%), glutathione (GSH,

100

98%), copper(II) chloride dihydrate, sodium carbonate, sodium hydroxide,

101

potassium hydroxide, sucrose, ethanol absolute (EtOH), hydrogen peroxide (30

102

wt%),

103

5,5'-Dithiobis-(2-nitrobenzoic acid) (DTNB, 99%). All of the chemicals listed above

104

were purchased from Sigma-Aldrich. 3,3'-dithiobis(propionohydrazide) (DTP, 98%)

105

was obtained from J&K Scientific. All of the chemicals were used without further

106

purification.

107

Characterization

hydrochloric

acid

solution

(1

mol/L),

Tris

base

(98%),

108

Transmission electron microscope (TEM) measurements were carried out using

109

a JEM-2010(S) TEM (JEOL, Japan). Images were collected with a Ultrascan camera

110

(Gatan, USA) using digital micrograph. Fourier transform infrared spectroscopy

111

(FTIR) was recorded using a Nicolet NEXUS 470 spectrophotometer (Thermo

112

Instruments, Canada). The mean diameter and zeta potential of the nanogels were

113

measured using a ZetaPlus zeta potential analyzer (Brookhaven Instruments Co.,

114

USA).

115

Synthesis of hydrophobic carboxymethyl cellulose

116

Hydrophobic carboxymethyl cellulose (HCMC) was prepared by esterification 7



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117

of carboxymethyl cellulose and palmitoyl chloride. Typically, 5 g of CMC was

118

dispersed in 15 mL of DMF and the pH was adjusted to 8-10 by sodium carbonate

119

and then PCl (mPCl/mCMC = 0.24-1.21) dissolved in DMF was dripped into the

120

suspension at 50℃ under vivid stirring. After 3 hours reaction, the mixture was

121

washed by ethanol to remove residual PCl. Finally, the product of HCMC was

122

collected by filtration and vacuum drying. The degree of substitution (DS) was

123

qualified by titration. Briefly, 0.1 g of HCMC was dispersed in 50 mL of

124

KOH-EtOH solution and after 2 hours of reflux, the residual KOH was neutralized

125

by standard hydrochloric acid solution in the presence of phenolphthalein. As a

126

control sample, CMC was also treated with above method. The consumptions of

127

hydrochloric acid were recorded and the DS was calculated as follow:

128

𝐷𝑆 =

(𝑉2 −𝑉1 )𝐶𝐻𝐶𝑙 𝑀1 𝑚1

(1)

129

where V1, V2 are the consumption of HCl solution for neutralizing KOH in HCMC

130

and CMC separately; CHCl is the concentration of HCl standard solution; M1 and m1

131

are the molar mass and weight of CMC separately.

132

Grafting of aldehyde groups on HCMC

133

Aldehyde groups were grafted onto HCMC to facilitate the crosslinking

134

reaction. In brief, 1g of HCMC was dissolved in 50 mL of deionized water and then

135

glyoxal (mglyoxal/mHCMC = 0.4-2.0) was added into the solution. The mixture was

136

stirred at 60℃ for 4 hours and then dialyzed (MW cutoff 2000) against water for 3 8


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137

days. Finally, the products (HCMC-a) was obtained as a white porous sponge by

138

lyophilization. To determine the aldehyde content of HCMC-a, 0.2 g of HCMC-a

139

was dissolved in 50 of mL deionized water, followed by adding and 25 mL of

140

hydroxyl amine hydrochloride (0.25 mmol/L)

141

solution was titrated with 0.1 mmol/L NaOH in the presence of methyl orange.

142

Finally, the experiment was repeated with HCMC as the control. Aldehyde content

143

(AC) of HCMC-a was calculated according to equation (2):

144

AC =

23

. After 24 hours of reaction, the

(𝑉4 −𝑉3 )𝐶𝑁𝑎𝑂𝐻 𝑚2

(2)

145

where V3 and V4 are the NaOH consumptions for HCMC and HCMC-a separately;

146

CNaOH and m2 are the concentration of NaOH solution and dry weight of HCMC-a.

147

Preparation of nanogels and salicylic acid loading

148

SA-loaded pH and redox dual-responsive nanogels were prepared by mixing

149

SA, HCMC-a and DTP solutions at predetermined concentrations and weight ratios

150

with continuous stirring at room temperature for 20 minutes. The unloaded SA was

151

removed by centrifugation at 10000 rpm for 10 min. SA-loaded cellulose nanogels

152

(SA-CNG) were obtained and weighted after freeze drying. For comparison,

153

non-hydrophobic modified CMC was used as a component to prepare hydrophilic

154

nanogels after grafting with glyoxal and non-loaded nanogels (CNG) were also be

155

prepared for characterizations. The amount of SA in the supernatant was measured

156

by UV-Visible spectrum (Genesys 10-s, Thermo Electron Corporation) analysis at 9



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157

297 nm based on a calibration curve. The entrapment efficiency (EN%) and loading

158

capacity (LC%) were calculated as follows:

159

𝐸𝑁% =

160

𝐿𝐶% =

𝐶𝑜 −𝐶𝑡 𝐶0 𝐶0 −𝐶𝑡 𝑚𝑑

× 100%

(3)

× 100%

(4)

161

where C0 and Ct are the initial and residual SA in supernatant; md is the dry weight

162

of nanogels. The content of disulfide bonds in nanogels was quantified by Ellman’s

163

test24. Briefly, 0.1 g of dry nanogels were dispersed in 10 mL of Tris-HCl solution

164

(0.25 mol/L, pH=8) and then mixed with 10 mL of Ellman’s reagent (prepared by

165

0.04 g of DTNB dissolved in 1 L of Tris-HCl solution). The disulfide bonds content

166

was calculated according to the UV adsorption at 412 nm after 10 mins reaction

167

based on a standard curve.

168

Swelling test

169

The swelling behaviours and swelling ratio were determined by tracking the

170

diameter changes of nanogels by particle size analysis. Specifically, dry nanogels

171

were dispersed in a batch of sucrose solutions (0 to 0.6 mol/L) and the average

172

diameter (Da) was recorded after 1 hour when nanogels swelled completely. The

173

average diameter of dry nanogels (Dd) was obtained from TEM photos and the

174

swelling ratio was calculated as follow:

175

Swelling ratio =

𝐷𝑎 −𝐷𝑑 𝐷𝑑

× 100%

(5)

10


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176


Dual-responsive sol-gel transitions

177

To demonstrate the pH and redox dual-responsiveness of nanogels, 0.05 g of

178

SA-CNG was suspended in 10 mL of deionized water and the suspension was

179

divided into two vials (#1 and #2) equally. For the transition of gel-to-sol, 1 mL of

180

HCl solution (0.5 mmol/L) and 1 mL of GSH (50 mmol/L) was added to vial #1 and

181

#2 respectively. For the transition of sol-to-gel, 1 mL of NaOH solution (0.5 mmol/L)

182

was added to vial #1 for neutralizing acid and 1 mL of H2O2 (3 wt%) was added to

183

vial #2 for re-oxidizing the sol. Each transition took 24 hours, and each cycle was

184

repeated three times to demonstrate the reversibility.

185

pH and GSH triggered release behaviours of SA from the nanogels in vitro

186

To investigate the release behaviours of SA triggered by pH and GSH, 0.01 g of

187

SA-CNG was suspended in 10 mL of HCl solution (pH was pre-adjusted to 3.5, 5.5

188

and 7). Then the suspensions were loaded into a dialysis bag (Mw cutoff 2000) and

189

immersed in 40 mL of identical HCl solution. GSH was added to the solution

190

outside the dialysis bag. Cumulative release was quantified by UV-visible spectrum

191

analysis based on the solutions collected from external of dialysis bag. Kinetics of

192

SA release was investigated by fitting the release data with various empirical kinetic

193

equations. Regression coefficients (R2) approaching to almost unity were considered

194

to be the best fit model for the system with reference to a particular equation.

11



195

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Complexation of heavy metal ions

196

The tests of heavy metal ions complexation were carried out in simulated soil

197

leachate. 1 kg of paddy mud (collected from Kunming, China) was placed in a glass

198

container and immersed by 1 L of deionized water. Then the container was sealed up

199

to block oxygen for 7 days and then soil leachate was obtained by filtration. CuCl2

200

were dissolved in as-prepared leachate to obtain simulated soil leachate and HCl

201

solution was used to adjust pH for simulating various soil conditions. To test the

202

removal efficiency of Cu2+ by nanogels, CNG were suspended in 20 mL of

203

simulated soil leachate (contained 100 ppm of Cu2+) with mild stirring for 2 hours

204

and the concentrations of Cu2+ were determined by inductive coupled plasma

205

emission spectrometer (ICP-ES, VISTA-MPX CCD, USA) after centrifugation

206

(10000 rpm, 5 min). The removal efficiency of Cu2+ was calculated as follow: Removal efficiency =

207

𝐶1 −𝐶2 𝐶1

× 100%

(6)

208

where C1 and C2 were the initial and residual concentration of Cu2+ in simulated soil

209

leachate, respectively.

210

RESULTS AND DISCUSSION

211

Preparation of SA loaded nanogels

212

To prepare the nanogels, hydrophobic branches were grafted on carboxymethyl

213

cellulose by the esterification with palmitic chloride. Then glyoxal was grafted onto

12


Page 13 of 29


214

the chains of polysaccharide and the exposed aldehyde groups provided connecting

215

sites for crosslinker i.e. 3,3'-dithiobis(propionohydrazide). Aldehyde groups and

216

amino groups on DTP formed reversible acylhydrazone bonds by Schiff base

217

reaction, which crosslinked and folded the chains of CMC and constructed the 3D

218

network of nanogels. Acylhydrazone bond is a dynamic covalent bond which can

219

proceed the reversible disassembly in a weak-acidic environment, meanwhile, the

220

disjunction and conjunction of disulfide bond in DTP can be controlled by reducer

221

and oxidant. SA was embedded in the networks with hydrophobic branches when

222

the nanogel assembled and was released once either acylhydrazone bonds or

223

disulfide bonds were destructed. Figure 1 demonstrate the synthesizing process and

224

responsive behaviours of nanogels.

225 226

Figure 1. Synthesizing process and responsive behaviours of SA-CNG.

13



Page 14 of 29

Aldehyde groups content (mmol/g)

1.0 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.4

227 228

0.8

1.2

1.6

2

Dosage of Glyoxal (mglyoxal/mHCMC-3)

Figure 2. Effect of glyoxal dosage on aldehyde content.

229

Most frequently used agrochemicals are insoluble in water because of the

230

aromatic structures. In order to improve the entrapment efficiency and loading

231

capacity of SA, hydrophobic branches were introduced on CMC chains. Base on the

232

specification of CMC (Mw = 90 kDa, DS = 0.7), 1 g of CMC was reacted with 0.66

233

to 3.3 mmol of palmitoyl chloride, i.e. the feed ratios (mPCl/mCMC) were 0.24

234

(HCMC-1), 0.48 (HCMC-2), 0.72 (HCMC-3), 0.97 (HCMC-4) and 1.21 (HCMC-5),

235

respectively. The degree of substitution and solubility of HCMC are exhibited in

236

Table 1. The amounts of hydrophobic branches grafted on CMC increased with

237

increasing the dosages of PCl, meanwhile the solubility of the corresponding

238

products decreased. Apparently, HCMC-3 exhibited higher DS of hydrophobic

239

branches and good solubility simultaneously, thus facilitating the SA loading and

240

subsequent reactions.

241 242 243 14


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Table 1. Effects of PCl dosages on DS and solubility of HCMC.

244

HCMC-1

HCMC-2

HCMC-3

HCMC-4

HCMC-5

DS

0.056

0.127

0.178

0.213

0.262

solubility

soluble

soluble

soluble

soluble (slowly)

insoluble

245

Glyoxal was used to graft aldehyde groups on as-prepared HCMC-3. In the

246

papermaking industry, dialdehydes are widely used in cross-linking cellulose as

247

intensifier25. Compared with glutaraldehyde, the carbon-chain length of glyoxal is

248

shorter and the efficiency of cross-linking reaction declines due to steric effect.

249

Therefore, more free aldehydes can be exposed for providing reaction sites for

250

self-assembling with amino groups. To obtain the higher aldehyde contents on

251

cellulose chains, excessive glyoxal (mglyoxal/mHCMC = 0.4-2.0, equal to 6.9 to 34.5

252

mmol/g) was added for grafting. Based on the results shown in Figure 2, the

253

optimum dosage (mglyoxal/mHCMC-3) of glyoxal was about 1.6. Excessive glyoxal did

254

not increase the aldehyde content of the products significantly because of the

255

self-polymerization of glyoxal at high concentration.

15



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5000 5 wt% HCMC-a 10 wt% HCMC-a

Diameter (nm)

4000 3000 2000 1000 0

20:1

10:1

5:1

2:1

1:1

1:2

mHCMC-a/mDTP

256 257

Figure 3. Effects of concentrations and mixing ratio of HCMC-a and DTP on the diameter of nanogels.

258

CMC is a water-soluble and highly anionic polysaccharide; and the average

259

zeta potential of HCMC-a coils in aqueous solution was -35.7 mV. Since the

260

crosslinking reaction exclusively occurred between HCMC-a and DTP, the

261

morphology and size of resulting nanogels were strongly influenced by the solution

262

concentrations and weight ratios of HCMC-a to DTP.

263

The effects of concentrations and mixing ratio of HCMC-a and DTP on the

264

diameter of nanogels are shown in Figure 3. The diameter of nanogels grew

265

significantly with increasing the concentration of mixed solutions, which is owing to

266

the rapid crosslinking reaction caused by the increase of collision probability of

267

HCMC-a chains and DTP molecules. Moreover, the results indicated that nanogel

268

could not form with the crosslinker at a low concentration (5 wt% HCMC-a,

269

mHCMC-a/mDTP=20:1). Therefore, crosslinking reaction can be manipulated via

270

adjusting the solution concentration in an attempt to control the nanogel size. The

271

most stable nanogels with the minimum diameter were obtained by mixing 5 wt% of 16


Page 17 of 29

HCMC-a solution and 0.5 wt% of DTP solution.

Particle size (nm)

800

-100

SA-CNG Ultrasonic treated SA-CNG Zeta-potential

-90 -80

600

-70 -60

400

-50 -40 200 -30

Zeta-potential (mV)

272


-20 0

0

5

10 Time (min)

15

20

273 274

Figure 4. Changes of particle size and zeta-potential in gelation process.

275

To prepare the SA loaded nanogels, 10 mg of SA was dissolved in 10 mL of 5

276

wt% HCMC-a solution and then mixed with 10 mL of 0.5 wt% DTP solution. The

277

particle size changes of nanogels during gelation were recorded; and the

278

zeta-potential was used as an indicator of the stability of colloidal systems. As

279

shown in Figure 4, the diameter of nanogels increased rapidly after 3 minutes

280

because of the forming, growing and aggregating of nanogels; and the

281

zeta-potentials were changed momentarily. From 10-15 minutes, the growth and

282

agglomeration of nanogels are carried out in suspension simultaneously until

283

forming stable aggregates (zeta-potential=-51.4±1.1 mV, Da=716±50 nm). The Da of

284

individual nanogel particle after ultrasonic treatment was 443±34 nm, which

285

facilitates nanogels to be absorbed by roots and leaves of plants and the

286

transportation in vessels26.

17



Page 18 of 29

(b)

2849 1664 1644

2918

CMC HCMC HCMC-a CNG 3500

3000

2500

2000

1500

1000

500

Wavenumber (cm-1)

287 288

Figure 5. (a) TEM image of CNGs, (b) FTIR spectra of CMC, HCMC, HCMC-a

289

and CNG.

290

The morphology characterization of nanogels was carried out by TEM analysis;

291

and the image is exhibited in Figure 5a. Dehydrated CNGs are uniform

292

microspheres with the diameter of 116±42 nm (based on an analysis of 50 particles).

293

FTIR was used to characterize the structure of nanogels; and the results are shown in

294

Figure 5b. The peaks at 2918 cm-1 and 2849 cm-1 are the characteristic absorptions

295

of methylene on HCMC, which can also be observed in the spectra of HCMC-a and

296

nanogels. The new peaks appeared at 1644 cm-1 and 1664 cm-1 can be characterized

297

as the stretching vibrations of imine (C=N) and carbonyl (C=O) of acylhydrazone

298

bonds respectively, demonstrating

299

disulfide bonds content of nanogels was 0.69 mmol/g, determined by Ellman’s test.

the success of Schiff Base reaction27. The

300

The entrapment efficiency (EN%) and loading capacity (LC%) are important

301

parameters for characterizing the loading properties of carriers. In this work, SA was

302

loaded on CNG at various pH ranging from 6 to 10. The control samples

303

(hydrophilic nanogels) were prepared by mixing glyoxal-grafted CMC without 18


Page 19 of 29


304

hydrophobic modification, SA solution and DTP solution with the same formula.

305

The results (Figure 6) showed that the hydrophobic modification improved the

306

entrapment efficiency and loading capacity significantly compared with hydrophilic

307

nanogels. The EN% and LC% of nanogels increased from 34.5% and 9.67% to 74.5%

308

and 38.5% respectively in neutral environment. Hydrophobic branches in nanogel

309

networks firmly adsorb and immobilize salicylic acid without changing the chemical

310

property of SA owing to their similar polarity. This is another feature of our

311

nanogels in addition to the dual-responsiveness compared to other drug delivery

312

systems. The effects of pH on EN% and LC% are also exhibited in Figure 6. In

313

alkaline solution, EN% and LC% of hydrophilic hydrogels decrease, while the

314

performance of CNG is less affected. This is due to the fact that SA is mainly loaded

315

on the hydrophilic nanogels by the Van der Waals' force, while the negative charges

316

in the alkaline solution increase the repulsive force of the carboxyl groups in

317

nanogel and SA. 80 70

EN% of SA-hydrophilic nanogel LC% of SA-hydrophilic nanogel EN% of SA-CNG LC% of SA-CNG

60

%

50 40 30 20 10 6

318

8

10

pH

319

Figure 6. Entrapment efficiency (EN%) and loading capacity (LC%) of SA-CNG

320

and SA-hydrophilic nanogels. 19



321

Page 20 of 29

Swelling test

322

The stability of carriers has a significant influence on the transport, distribution

323

and release of agrochemicals28, which could be evaluated by swelling tests in a

324

hyperosmotic solution. Compared with NaCl solution (usually used in human tissue),

325

sucrose solution is more suitable for the release environment of carrier in plants. The

326

osmotic concentration of cell fluid is not completely equal, depending on the types

327

of plant cells. In plant tissue culture, 2-8% of sucrose solution (i.e., 58-234 mmol/L)

328

is usually added to provide nutrition and maintain osmotic pressure. Therefore, the

329

swelling test of nanogel was carried out in 0-300 mmol/L of sucrose solution; and

330

600 mmol/L of sucrose solution was used to test the stability of nanogels in high

331

osmotic pressure environment. As shown in Figure 7, nanogels are completely

332

swollen in pure water and the diameter of nanogels is 440 nm. Compared with the

333

diameter of dehydrated nanogels, the swelling ratio is calculated to be 282%.

334

Nanogels shrink in a high osmotic pressure environment. With the concentration of

335

sucrose increasing to 0.3 mol/L, the diameter of nanogels decrease to 338 nm and

336

the swelling ratio decrease to 192%. When the concentration of sucrose continues to

337

increase to 0.6 mol/L, the diameter of nanogels is no longer changed. The results

338

suggested that the particle size of nanogels can be maintained steadily in isotonic

339

solution of plant cell29 (0.3 mol/L of sucrose) or more concentrated conditions,

340

which contributes to keep steady properties of nanogels in plants and high salinity 20


Page 21 of 29

soils.

Diameter of nanogels (nm)

(b)

480 460 440 420 400 380 360 340 320 300 280 260 240

400 380 360 340 320 300 280 260 240 220 200 180 0.0

342 343

344

Swelling ratio (%)

341


0.1

0.2

0.3

0.4

0.5

0.6

Concentration of sucrose (mol/L)

Figure 7. Swelling behaviours of SA-CNG in sucrose solution.

pH and GSH triggered release behaviours of SA from the nanogels in vitro

345

Nanogel prepared in this work contain both acylhydrazone and disulfide bonds

346

which are well known to be pH and redox responsive30, respectively. Figure 8

347

summarizes the relationship between cumulative release and time at different pH

348

values and GSH concentrations. As we expected, HCl and GSH promote the release

349

of salicylic acid remarkably. The forming of nanogel and the loading of SA were

350

combined as an “one-step” process; and most of SA was captured by the

351

hydrophobic branches on cellulose chains and embedded in the 3D network of

352

nanogel with the gelation process. However, some SA molecules were attached on

353

the carrier with unstable connection, such as electrostatic association. In addition,

354

the concentration gradient of SA existed between surface layer of nanogel and

355

external solution. As a result, 23% of SA loaded in surface layer could be released

356

by diffusion at neutral environment within 12 hours. With pH decreasing to 5.5, the 21



Page 22 of 29

357

amount of accumulative release of SA from the nanogels increases significantly due

358

to the dynamic breakage and conjunction of acylhydrazone bonds. At pH 3.5, SA is

359

completely released within 8 hours with the dissolution of nanogels, and 80 % is

360

released in first 4 hours. On the other hand, GSH solutions used for simulating the in

361

vivo environment can also accelerate the release of SA evidently. With the

362

concentration of GSH increasing from 10 to 20 mmol/L, the time for SA to be fully

363

released from nanogels has been reduced by half from 12 to 6 hours, indicating that

364

the disassembly of nanogels induced by disconnection of disulfide bonds promote

365

the release of SA. These results demonstrate that the release of SA from hydrogels is

366

controllable. This design of dual-responsive nanogels provides the potential for the

367

controlled-release both in vitro (acid triggered) and in vivo (GSH triggered), which

368

is more flexible and controllable than single-responsive carriers.

Cumulative release (%)

100

pH=7 pH=5.5 pH=3.5 5 mmol/L GSH 10 mmol/L GSH 20 mmol/L GSH pH=5.5 + 10 mmol/L GSH

80 60 40 20 0 0

369

2

4

6

8

10

12

Time (hour)

370

Figure 8. Cumulative release of SA from nanogels at different pH values and GSH

371

concentrations.

372

The adsorption kinetics was investigated in terms of the release curves at 22


Page 23 of 29


373

pH=5.5 and 10 mmol/L GSH; and the results were fitted with various models31. The

374

fitting parameters of the kinetic models are shown in Table 2. According to the data,

375

the profile of SA release from nanogels fit the diffusion-erosion kinetic model best;

376

and the high linearity of the plots was achieved (R2 > 0.99). Diffusion-erosion model

377

is often used to describe Fick diffusion process affected by carrier erosion; whereas

378

the Kopcha model is the special form of diffusion-erosion model when the last two

379

terms are ignored31. The rapid release of SA in the initial stage is dominated by the

380

Fick diffusion of SA at the surface layer of the carrier. Subsequently, SA was

381

released at a relatively uniform speed under the combined action of diffusion path

382

growth and nanogel erosion. In addition, by comparing the values of ka/kb, it can be

383

concluded that the erosion effect in GSH environment has a greater impact on the

384

release of SA than that in acid environment.

385

Table 2. Parameters of release models Equations Zero Order Qt=k0t First Order ln(1-Qt)=k1t Higuchi Qt=kHt0.5 Korsmeyer-Peppas Qt=kKPtn Kopcha Qt=At0.5+Bt Diffusion-Erosion Qt=kat0.5+kbt+kct2+kdt3

2

R 0.718 R2 0.831 R2 0.93 R2 0.855 R2 0.979 R2 0.998

pH=5.5 k0 0.049 k1 0.08 kH 0.196 kKP 0.258 A 0.299 ka 0.153 kc -0.013

10 mmol/L GSH R k0 0.661 0.069 R2 k1 0.970 0.257 R2 kH 0.904 0.284 R2 kKP n 0.964 0.466 0.323 R2 A B 0.993 0.539 -0.076 R2 ka kb 0.993 0.464 0.002 kc kd -0.010 0.005 2

n 0.392 B -0.033 kb 0.093 kd 0.001

23



386

Page 24 of 29

Complexation of heavy metal ions

387

Thiol and carboxyl groups as complexing functional groups are widely used in

388

the removal of heavy metal ions in researches of environment engineering32, 33.

389

Abundant carboxyl and thiol functional groups in nanogel structure contribute to the

390

complexation of heavy metal ions, which is conducive to the soil remediation and

391

the safety of agriculture foods. Agrochemicals are usually sprayed or smeared on

392

plants in agricultural production. Some agrochemicals cannot directly act on plants,

393

but first release in the soil and then be absorbed by plants, thus carriers have access

394

to heavy metal ions in the soil. Therefore, according to our previous research21, the

395

complexation ability of nanogels to copper (II) was evaluated in simulated soil

396

leachate. Figure 9 shows the removal efficiency of Cu2+ by the complexion of

397

nanogels. With increasing the dosage of nanogels from 1 to 5 mg/mL, the removal

398

efficiency of Cu2+ increases from 25% to 89% in neutral solution, demonstrating that

399

the nanogels can capture and stabilize the heavy metal ions in solution. The

400

combination mechanism of thiol group and heavy metal ions is the formation of

401

precipitation with low solubility34. For carboxyl groups, coordination and

402

electrostatic force are the mainly factors of adsorption35. Thus, the removal

403

efficiency of cationic heavy metals decreases with the presence of protons or

404

relatively low pH in the solution. Moreover, the stabilized heavy metal ions will not

405

be absorbed by plants, thus protecting plant growth from toxic contaminants.

406

Meanwhile, the biodegradable cellulose as carriers will not induce the harmful effect 24


Page 25 of 29

407


on soil.

90

pH=7 pH=5.5

Removal effciency (%)

80 70 60 50 40 30 20 10 0

408

1

2

3

4

5

Dosage of CNG (mg/mL)

409

Figure 9. Removal efficiency of Cu2+ in neutral and weak acidic simulated soil

410

leachate.

411

In summary, a novel cellulose nanogel was prepared by crosslinking glyoxal

412

modified carboxymethyl cellulose (CMC) and 3,3'-dithiobis(propionohydrazide)

413

(DTP). Palmitic chloride (PCl) was used to graft hydrophobic branches on cellulose

414

chains for enhancing the loading capacity of model agrochemicals, i.e., salicylic acid

415

(SA). The acylhydrazone and disulfide bonds endow the nanogel with reversible

416

sol–gel transitions when exposed to stimulation of pH and redox changes. The

417

maximum loading capacity of PCl grafted nanogels reaches 40.6% which is 31%

418

higher than unmodified nanogels. Controlled-release experiment showed that HCl

419

and GSH solutions accelerated the release of SA significantly Aabundant carboxyl

420

and thiol groups make nanogels capable of complexing heavy metal ions; and 89%

421

of copper (II) ions can be removed from simulated soil leachate. The pH and redox

422

dual-responsive nanogel developed in this work is of great potential for the precise 25



Page 26 of 29

423

controlled-release of agrochemicals and meanwhile stabilizing heavy metal ions to

424

improve soil remediation.

425

ACKNOWLEDGEMENTS

426

This work was supported by Fundamental Research Funds for the Central

427

Universities (2018QN089), Dean Project of Guangxi Key Laboratory of

428

Petrochemical Resource Processing and Process Intensification Technology

429

(2018k001), NSF China (No. 21466005 and No. 51379077) and NSERC Canada.

430

AUTHOR INFORMATION

431

Corresponding Authors:

432

*Email: [email protected]; [email protected]

433

References

434 435 436 437 438 439 440 441 442 443 444 445 446

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Controlled Release of Agrochemicals Using pH and Redox Dual

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