Quercetagetin-Loaded Composite Nanoparticles Based on Zein and

Subscriber access provided by McMaster University Library

Food and Beverage Chemistry/Biochemistry

Quercetagetin-loaded composite nanoparticles based on zein and hyaluronic acid: formation, characterization and physicochemical stability Shuai Chen, Cuixia Sun, Yingqi Wang, Yahong Han, Lei Dai, Arzigül Abliz, and Yanxiang Gao J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b01046 • Publication Date (Web): 13 Jun 2018 Downloaded from http://pubs.acs.org on June 13, 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 39

Journal of Agricultural and Food Chemistry

1

Quercetagetin-loaded composite nanoparticles based on zein and

2

hyaluronic acid: formation, characterization and physicochemical

3

stability

4

Shuai Chen, Cuixia Sun, Yingqi Wang, Yahong Han, Lei Dai, Arzigül Abliz,

5

Yanxiang Gao*

6 7

Beijing Advanced Innovation Center for Food Nutrition and Human Health,

8

Beijing Laboratory for Food Quality and Safety, Beijing Key Laboratory of

9

Functional Food from Plant Resources, College of Food Science & Nutritional

10

Engineering, China Agricultural University, 100083, China

11 12 13 14 15 16

*Corresponding author.

17

Tel.: + 86-10-62737034

18

Fax: + 86-10-62737986

19

Address: Box 112, No.17 Qinghua East Road, Haidian District, Beijing 100083,

20

China

21

E-mail: [email protected]

ACS Paragon Plus Environment


22

Abstract

23

Zein and hyaluronic acid (HA) composite nanoparticles were self-assembly

24

fabricated using anti-solvent co-precipitation (ASCP) method to deliver quercetagetin

25

(Que). FTIR, CD and FS results revealed that electrostatic attraction, hydrogen

26

bonding, and hydrophobic effect were the dominant driving forces among zein, Que,

27

and HA. With the increasing of HA level, the morphological structure of

28

zein-Que-HA complex was changed from nanoparticle (from 100: 5: 5 to 100: 5: 20)

29

to microgel (from 100: 5: 25 to 100: 5: 30). The encapsulation efficiency of Que has

30

significantly increased from 55.66% (zein-Que, 100:5) to 93.22% (zein-Que-HA, 100:

31

5: 20), and Que in the zein-Que-HA composite nanoparticles exhibited obviously

32

enhanced photochemical, thermal and physical stability. After 8 months of storage

33

(4°C), the retention rate of Que also up to 77.93%. These findings interpreted that

34

zein-HA composite nanoparticle would be an efficient delivery system for

35

encapsulating and protecting bioactive compounds.

36 37

Keywords: Quercetagetin; Zein; Hyaluronic acid; Composite nanoparticles;

38

Interaction.

39 40 41 42


Page 2 of 39

Page 3 of 39


43 44

INTRODUCTION Quercetagetin (Que) is a kind of alcohol soluble bioactive substance, with many hydroxyl

groups.1

45

phenolic

46

anti-inflammatory capacities,2-3 and a potential in the prevention of tumor,

47

cardiovascular disease and other chronic diseases. 4-5 Thus, Que can be applied to

48

health food, however, the application of Que was limited owing to its weak chemical

49

stability, poor water solubility, and low oral bioavailability.

It

has

excellent

antioxidant,

antimicrobial,

50

To overcome the aforementioned disadvantages, many delivery systems have

51

been designed, e.g. hydrogel, liposome, micelle, and particle.6 Compared to other

52

delivery systems, nanoparticles have unique advantages, e.g. small mean size

53

(10-1000 nm), high encapsulation efficiency, slow degradation rate, effective

54

targetability and penetration ability.6 Food-grade nanoparticles are usually prepared

55

from protein and polysaccharide, which are non-toxic, biodegradable, and

56

biocompatible,7 such as soy protein,8 lactoferrin,9 gelatin,10 chitosan,11 and alginate.12

57

Zein is a vegetable protein with much hydrophobic amino acids (over 50%).12 It

58

is easy to form nanoparticles by the anti-solvent precipitation (ASP) method for

59

delivering polyphenols.7 However, zein nanoparticle is unstable when confronted with

60

certain pH, high salt concentration, and thermal processing. In order to enhance the

61

stability of zein nanoparticle, some food biopolymers have been used to coat its

62

sufurce.13-14 Moreover, the composite nanoparticles can provide a better protecting

63

function and higher encapsulation efficiency (EE) for bioactive components.14-15 Such



Page 4 of 39

64

as curcumin,16 resveratrol17, and epigallocatechin gallate.18 Recently, the investigation

65

into delivery system based on the complexation between protein and polysaccharide

66

has been becoming a research hotspot.

67

Hyaluronic acid (HA) is a linear polysaccharide,19 which is naturally found in

68

human bodies with important biological and physicochemical functions,20 such as

69

anti-aging,

70

angiogenesis, and embryonic development.19-20 Due to the rapid turnover (about 1/3

71

of HA is degraded and regenerated daily) in human body,21 oral intake of HA can

72

replenish the raw material of HA needed for metabolism. Based on the particular

73

physicochemical properties and functional attributes, HA has been widely used as a

74

raw material for the production of soft-tissue filler, anti-aging matrices, and drug

75

delivery vehicles. 21 Previous researchers mainly concentrated on the application of

76

HA in medicine through applying ointment or injection.22 Some drug or gene delivery

77

systems were fabricated based on HA, such as HA-chitosan nanoparticles,23-24

78

HA-silk hydrogels,21 HA-curcumin nanogels.25 However, the investigations into HA

79

as a matrix to design food-grade delivery systems for oral intake are very limited.

80

Little information focused on interaction among HA, protein, and polyphenols was

81

found in previous literatures.

cell-matrix

interactions,

anti-inflammation,

immunoregulation,

82

In the present work, the zein-Que-HA composite nanoparticles were fabricated

83

using a new and simple (anti-solvent co-precipitation, ASCP) method, which was ease

84

of operation and required a small amount of HA. The influence of the proportion of


Page 5 of 39


85

zein, Que, and HA on the physicochemical property and microstructure of

86

zein-Que-HA was investigated. The physical, photochemical, thermal stability, as

87

well as a long-term storage stability of Que in zein-HA composite nanoparticles were

88

evaluated. The findings from present work would have a potential application in the

89

field of nutraceutical delivery system.

90

MATERIALS AND METHODS

91

Materials. Zein (95%) was obtained from Gaoyou Co. Ltd. (Jiangsu, China).

92

Hyaluronic acid (99%, molecular weight 1.0×105) was purchased from Xi’an

93

Baichuan Co. Ltd. (Xi’an, China). Ethanol (99.9%) was purchased from Beijing

94

chemical Co., Ltd (Beijing, China). Quercetagetin (95%, w/w) was prepared from

95

marigold (Tagetes erectaL.) using the procedure described in our previous report.26

96

All other chemicals were analytical grade unless stated otherwise.

97

Preparation of zein-Que-HA composite nanoparticles. Zein-Que-HA composite

98

nanoparticles were prepared using the ASCP method. Briefly, Different quantities (50,

99

100, 150, 200, 250, and 300 mg) of HA were dissolved in distilled water (30 mL), and

100

then the solutions were diluted with 70 mL of ethanol. Zein (1.0 g) and Que (50 mg)

101

were added to 100 mL HA aqueous ethanol solution (0.5, 1.0, 1.5, 2.0, 2.5, and 3.0

102

mg/mL). After ultrasonic treatment for 20min, the final mixed solution (40 mL) was

103

injected into the 120 mL distilled water in 2 min, and stirred for 20 min at 600 rpm.

104

To obtain zein-Que-HA aqueous dispersions, ethanol was removed using rotary

105

evaporation (45°C, -0.1 MPa). Zein, zein-Que, and Que-HA aqueous dispersions were



Page 6 of 39

106

prepared by the aforementioned method as controls. Part of the colloidal dispersions

107

were stored at 4°C, and others were freeze-dried (-50°C, 48 h) for solid-state analysis.

108

In the study, the different mass ratios of zein-Que-HA complexes at 100:5:5,

109

100:5:10, 100:5:15, 100:5:20, 100:5:25 and 100:5:30 were termed as zein:Que:HA

110

(100:5:5),

111

(100:5:20) , zein:Que:HA (100:5:25) and zein:Que:HA (100:5:30), respectively.

zein:Que:HA

(100:5:10),

zein:Que:HA

(100:5:15),

zein:Que:HA

112

Characterization of zein-Que-HA composite nanoparticles

113

Particle size, zeta-potential, entrapment efficiency (EE) and loading capacity

114

(LC). The particle size (nm) and zeta-potential (mV) of zein-Que-HA composite

115

nanoparticles were analyzed according to our previous report.27 Three parallel samples

116

were performed, and result was calculated as cumulative mean value. The EE and LC

117

of zein-Que-HA composite nanoparticles were analyzed using our reported method.28

118

They were centrifuged at 15000 g for 30 min (25 °C). The supernatant liquor was

119

sucked out and diluted using ethanol aqueous solution (70%, v/v), and determined

120

using a UV-1800 spectrophotometer at 360 nm. The EE and LC were calculated by

121

the following equations:

122

EE =

Total Que - Free Que × 100% ………………..………(1) Total Que

123

LC =

Total Que − Free Que × 100% ………..… (2) Total mass of composite particle

124

Field

emission

scanning

electron

microscopy


(FE-SEM)

and

Page 7 of 39


125

Fourier-transform infrared (FTIR) spectroscopy. The microstructure of the

126

freeze-dried zein-Que-HA composite nanoparticles was observed by the field

127

emission scanning electron microscope (FE-SEM, SU8010, Hitachi). The surfaces of

128

samples were sputter-coated with a gold layer, and the accelerating voltage was 5.0

129

kV. The Spectrum 100 Fourier transform spectrophotometer (PerkinElmer, U.K.) and

130

the potassium bromide (KBr) pellet method were used in the work. 29 11 scans were

131

taken in the range of 400-4000 cm-1, and the resolution was 4 cm-1. The infrared

132

spectra of zein, zein-Que, HA-Que, and zein-Que-HA composite nanoparticles were

133

analyzed using Omnic v8.0 (Thermo Nicolet, USA).

134

Fluorescence spectroscopy (FS), circular dichroism (CD) and differential

135

scanning calorimetry (DSC) analysis. The intrinsic fluorescence of zein-Que-HA

136

composite nanoparticles was measured at 0.25 mg/mL of zein with Que (12.5 µg/mL)

137

and HA at different levels. The spectral region was 290-450 nm, the scanning speed

138

was 100 nm/min, excitation wavelength at 280 nm, both excitation and emission slit

139

widths were set at 10 nm. CD measurement conditions: far-UV region (190-260 nm),

140

path length was 0.1 cm. recorded speed was 100 nm/min, 2.0 nm band width, 20

141

accumulations, and 0.2 nm resolution. The contents of secondary structure of

142

zein-Que-HA

143

http://dichroweb.cryst.bbk.ac.uk, which was an online Circular Dichroism Website.31

144

The thermal characteristics of zein-Que-HA composite nanoparticles were analyzed

145

using DSC. Briefly, the freeze-dried sample (3.0 mg) was placed into an aluminium

composite

nanoparticles

were

analyzed


using

Dichroweb:


146

pan and hermetically sealed. DSC measurement conditions: temperature range was

147

30-150 oC, temperature rate was 10 oC/min. The thermal curve were collected by the

148

DSC-60 workstation.

149

Stability evaluation

150

Physical stability. The physical stability of the zein-Que-HA colloidal dispersions

151

were measured using the LUMiSizer (LUM, Germany) according to our previous

152

reported.32 The measurement parameters were as follows: sample amount, 1.8 mL;

153

rotational speed, 4000 rpm; time interval, 30 s; temperature, 25°C.

154

Photochemical stability. The free Que, zein-Que, HA-Que, and zein-Que-HA

155

colloidal dispersions were placed into a light cabinet. The light condition was 0.68

156

W/m2 and 45°C. 32 The sampling was carried out at time points of 0, 2, 4, 6, 8 and 10

157

h. In general, the degradation of flavonoids followed the first order kinetics.33 The

158

half-life (t1/2) and degradation rate constant (k) were calculated using the following

159

equations:

160

Ln (C / C 0 ) = − kt ……………..(3)

161

t1/ 2 =

Ln2 ……………………..(4) k

162

Where C0 represents the concentration of Que at the initial and C represents the

163

concentration of Que at time t (h).

164

Thermal stability. According to the method of Liu et al., 34 50 mL samples of free

165

Que, zein-Que, HA-Que, and zein-Que-HA colloidal dispersions were placed into test

166

tubes and incubated in a water-bath at 60, 70 and 80 °C for 10 h. After that, the


Page 8 of 39

Page 9 of 39


167

samples were cooled in ice water. The Que in the samples were quantified according

168

to the aforementioned method.

169

Storage stability. The storage stability of zein-Que-HA colloidal dispersions was

170

performed on a refrigerated condition (4 °C) for 8 months. Particle size of composite

171

colloidal nanoparticles was detected by the method as aforementioned above, and the

172

retention rate of Que after storage was estimated by the following equation:

173

Retention rate (%) =

the amount of Que after storge the initial amount of Que

×100%............（5）

174

Statistical analysis. The parallel experiments were performed at least in three times.

175

Results were expressed as mean ± SD. Data were analyzed by ANOVA, and p < 0.05

176

was regarded as significant.

177

RESULTS AND DISCUSSION

178

Particle size, zeta-potential, entrapment efficiency (EE), and loading capacity

179

(LC). The photographs of samples were shown in Fig. 1a. It's clearly observed that

180

hyaluronic acid (HA) aqueous solution, Que in 70% ethanol aqueous solution and

181

HA-Que aqueous solution were clear and transparent. However, zein-Que and

182

zein-Que-HA were homogeneous colloidal dispersions. The mean size and

183

zeta-potential were given in Fig. 1b and c. The zein-Que nanoparticles had a smaller

184

particle size than that of zein nanoparticles. It could be explained by the fact that

185

polyphenols tend to intimate connection with proline-rich zein, and form a small and

186

compact structure.35 When HA was added, the particle size was significantly (p < 0.05)

187

increased from 89.54 nm (zein-Que) to 315.93 nm (zein-Que-HA), and the



188

zeta-potential was changed from 29.43 mV (zein-Que) to -16.47 mV (zein-Que-HA).

189

The surface charge of native zein nanoparticle was positive (31.97 mV), while native

190

HA was negative (-49.30 mV). In general, the charge attribute of composite

191

nanoparticle was dominated by the charge characteristics of the outer layer

192

biopolymer.36 It indicated that the negatively charged HA coated on the surface of

193

zein nanoparticles. Similarly, Patel et al.13 had reported that positively charged zein

194

nanoparticles was covered by the negatively charged sodium caseinate. On the one

195

hand, the addition of HA resulted in the increased volume of zein-Que-HA spherical

196

nanoparticle; On the other hand, the decrease of zeta-potential resulted in the reduced

197

electrostatic repulsion, the aggregation of zein-Que-HA composite nanoparticles

198

might be formed. Therefore, zein-Que-HA composite nanoparticles had a larger

199

particle size than zein-Que nanoparticles.

200

Continuously increasing the concentration of HA from 100:5:5 to 100:5:15

201

(zein:Que:HA), both zeta-potential and the particle size were decreased. It could be

202

explained by the fact that the increasing negative charge led to the increased

203

electrostatic repulsion, which could prevent the aggregation of zein-Que-HA

204

composite nanoparticles. However, it was interesting to find that with the mass ratio

205

(zein: Que: HA) increasing from 100:5:20 to 100:5:25, the particle size was obvious

206

increased from 233.73 nm to 398.9 nm. It might be caused by the crosslinking of

207

excessive HA.37 A similar observation was reported that high level of shellac could

208

increase the size of zein-shellac composite nanoparticles through intermolecular


Page 10 of 39

Page 11 of 39


209

cross-linking.29

210

The effects of different formulations on EE and LC were presented in Table 1. All

211

the zein-Que-HA composite nanoparticles exhibited significantly (P < 0.05) higher

212

EE values than that of zein-Que nanoparticles. With the mass ratio of zein: Que: HA

213

increased from 100:5:5 to 100:5:20, there was a gradual increase in the EE and LC.

214

Particularly, the highest EE and LC values of Que in zein-Que-HA (100:5:20)

215

composite nanoparticles reached up to 93.22% and 3.85%, respectively. The result

216

may be due to the long molecular chain and high molecular weight of HA (1.0×105

217

Da), which was 4.5 times than zein (2.2×104 Da). Liang et al.15 reported that chitosan

218

derivative could entrap more curcumin and lead to a high EE ascribing to its longer

219

chain of molecule structure. They were consistent with our previous study focused on

220

the Que-loaded zein-propylene glycol alginate complexes.28

221

Overall, the negatively charged HA could be combined with positively charged

222

zein, and formed a compact-structure nanoparticle (zein:Que:HA=100:5:20) with

223

strong ability of encapsulation. These results revealed that HA and zein had a

224

synergistic effect on improving the EE and LC of Que, and zein-HA binary composite

225

nanoparticles could be an ideal carrier for functional component.

226

Field emission scanning electron microscopy (FE-SEM) analysis. The

227

morphological images of samples were shown in Fig. 2. Zein nanoparticles (Fig. 2a)

228

were typically spherical, which was the same as reported in previous study.30 HA (Fig.

229

2b) exhibited a fine dendritic network structure. When zein and HA were combined



230

together to encapsulate Que, the morphology of zein-Que-HA composite

231

nanoparticles were also spherical, but their surface was coarse (Fig. 2c, d, e, f). The

232

result could be explained by that HA covered and convolved on the zein nanoparticles

233

by electrostatic attraction, and Que was embedded in zein nanoparticles by

234

hydrophobic interaction.38

235

As shown in Fig. 2g and h, it was interesting to find that the crosslinking occurred

236

among excessive HA. Zein-Que-HA complex presented a 3D network-like structure,

237

and seemed to be clusters of grapes. The result demonstrated that the increasing

238

concentration of HA led to the zein-Que-HA complex change from spherical particle

239

to network-like microgel. It was similar to that propylene glycol alginate-human

240

serum albumin complex presented a vesicular structure or a lacework-like structure

241

depending on the polymer concentrations. 39

242

Fourier-transform infrared (FTIR) spectroscopy. Zein nanoparticles showed a

243

major band at 3313.40 cm-1 (Fig. 3a), which was ascribed to the stretching vibration of

244

hydroxy groups. The band at 1659.81 cm-1 represented the C-O stretching (amide I)

245

and 1533.51 cm-1 was related to the C-N stretching coupled with N-H bending modes

246

(amide II).30 Compared with individual zein, the peaks of amide I and amide II of

247

zein-Que were changed to 1658.88 cm-1 and 1534.63 cm-1, respectively, and their

248

peak intensity was increased. The findings demonstrated that hydrophobic interaction

249

might exist between zein and Que, which was ascribed to the high percentage of

250

hydrophobic amino acids (over 50%) of zein and aromatic rings of Que.40 In addition,


Page 12 of 39

Page 13 of 39


251

the presence of Que induced significant shift of –OH groups to 3310.12 cm-1, which

252

revealed that the hydrogen bonding occurred between zein and Que. When HA was

253

incorporated, the peak of hydrogen bond (–OH) was shifted to 3304.11 cm-1 (zein:

254

Que: HA=100:5:5). These findings suggested that the intermolecular forces between

255

zein and HA included not only electrostatic attraction due to their opposite charges,

256

but also hydrogen bonding. Similarly, Luo et al.14 had reported that hydrogen bonding

257

was one of the dominant driving forces in the formation of zein-carboxymethyl

258

chitosan-vitamin D3 nanoparticle. Compared the low level (zein: Que: HA=100:5:5)

259

with high level (zein: Que: HA=100: 5: 25) of HA, the peaks of amide II was changed

260

from 1536.12 cm-1 to 1541.10 cm-1, the peaks of C–O stretching of –COO− was

261

changed from 1451.17 cm-1 to 1450.76 cm-1, and the peaks of –OH groups was

262

changed from 3304.11 cm-1 to 3311.49 cm-1. When HA was at a high level, the

263

hydrogen bonding formed among the –COOH, –NHCOCH3 and –OH groups on the

264

backbone of HA molecules.37 The hydrogen bonding resulted in the formation of a 3D

265

network-like structure microgel, whose microtopography was confirmed by FE-SEM

266

(Fig. 2g and h).

267

Fluorescence spectra analysis. Fig. 3b showed that the fluorescence emission

268

peak of zein nanoparticles was at 304 nm,27 and the presence of Que caused the

269

fluorescence quenching. It might be attributed to multiple intermolecular interactions

270

such as energy transfer, rearrangement, collisional quenching, and ground state

271

complex formation.41 The incorporation of HA into zein-Que (zein: Que: HA, 100:5:5



272

and 100: 5: 10) resulted in the decreased intensity of fluorescence. The result might be

273

explained by the fact that a slight precipitation phenomenon occurred when zein was

274

combined with the low concentration of HA, and total quantity of zein-Que-HA

275

colloidal nanoparticles decreased, which led to the decrease in the fluorescence

276

intensity. However, when the concentration of HA was increased (zein:Que:HA,

277

100:5:15 and 100:5:20), the zein-Que-HA colloidal dispersions were stable, and the

278

fluorescence intensity of zein-Que-HA colloidal nanoparticles were increased. It

279

could be explained by that HA was combined with hydrophilic groups of zein, and

280

induced the tryptophan residues exposed, which were originally inside the zein.36

281

With the increase of HA concentration, excessive HA might wrap around zein-Que

282

nanoparticles, therefore, the fluorescence intensity of zein-Que-HA (100:5:25 and

283

100:5:30) was slightly less than that of zein-Que-HA (100:5:15 and 100:5:20).

284

Circular dichroism (CD) analysis. Fig. 3c showed that zein had a positive peak

285

(195 nm), 2 negative peaks (208 and 224 nm), and a zero-crossing (203 nm). It was

286

attributed to the high content of α-helix.42 When zein was combined with HA, the

287

secondary structure was significant changed, which could be reflected by the changes

288

in the ellipticities of spectral curve at both 208 and 224 nm. The interaction between

289

the protein and polymer made the alteration in secondary structure of the protein was

290

a common phenomenon.43-44

291

The DICHROWEB procedure (SELCON3) was performed to calculate the

292

percentages of α-helix, β-sheet, β-turn and unordered coil, 31 and the data were shown


Page 14 of 39

Page 15 of 39


293

in supporting information Table 1. The secondary structure of zein contained 49.7%

294

α-helix, 8.8% β-sheet, 16.8% β-turn and 25.6% unordered coil, respectively. However,

295

when Que was incorporated, the percentage of α-helix was significantly (p < 0.05)

296

reduced from 49.7% (zein) to 35.7% (zein-Que). At the same time, the percentages of

297

β-sheet and unordered coil were significantly (p < 0.05) increased from 8.8% and 25.6%

298

to 10.5% and 32.1%, respectively. The finding might be attributed to the hydrophobic

299

interaction between Que and zein, which caused the change of zein structure.40 When

300

HA was added (zein: Que: HA=100:5:5), the α-helix of zein was obviously decreased

301

from 35.7% to 9.0%, but β-sheet increased from 10.5% to 30.2%. These results could

302

be explained by that a low concentration of HA led to the slight aggregation of zein

303

nanoparticles. Lefevre and Subirade

304

in the aggregation of proteins. Continuously increasing the concentration of HA, the

305

percentage of α-helix in zein was gradually increased, however, the other secondary

306

structure decreased. The result might be ascribed to the conformational relaxation of

307

zein-Que-HA composite nanoparticles,

308

fluorescence analysis.

45

pointed that β-sheet was frequently increased

38

which was also confirmed by the

309

Differential scanning calorimetry (DSC) analysis. Thermograms of zein,

310

zein-Que, HA-Que, and zein-Que-HA composite nanoparticles were showed in Fig.

311

3d. There was a broad endothermic peak at 95.9°C in the curve of zein, it was related

312

to the denaturation of biopolymers.46 The endothermic peak temperature of zein-Que

313

(100:5) complex was lower (92.8°C) than that of zein. The decrease might be



314

attributed to hydrophobic effects and hydrogen bonding between Que and zein, which

315

were confirmed by the FTIR result. It was analogous to that curcumin could decrease

316

the melting temperature of zein though the intermolecular interactions.40 The

317

endotherms peak of zein-Que-HA (100:5:5) complex was 78.6°C, the low melting

318

temperature may be due to that it had the large particle size and loose network

319

structure, resulting in the weak stability against the thermal treatment. With the

320

increasing of HA concentration, the endothermic peak of zein-Que-HA complex

321

tended to the higher temperature. This result revealed that the incorporated HA

322

enhanced the ability to resist degeneration of zein-Que-HA composite nanoparticles.

323

In particular, zein-Que-HA composite nanoparticles (zein-Que-HA, 100:5:20) and

324

microgel (zein-Que-HA, 100:5:25) exhibited the better thermal stability than that of

325

zein-Que-HA (100:5:5, 100:5:10, and 100:5:15). It was futher cinformed that

326

polysaccharides could improve the stability of the protein by disturbing its original

327

denaturation behavior.47 Kittur et al. 48 has found that the alteration of endothermic

328

peak temperature of chitosan was related to the polymer-water interaction, namely,

329

the water holding capacity. The HA with lots of hydrophilic groups could bind some

330

water molecules into the zein-Que-HA microgel, leading to the increased endothermic

331

peak temperature.

332

Physical stability. As shown in Fig. 4, it was obvious that zein-Que-HA colloidal

333

dispersions at the mass ratios of 100:5:5 and 100:5:10 were less stable than those of

334

100:5:15, 100:5:20, 100:5:25, and 100:5:30. It was attributed to the aggregation of


Page 16 of 39

Page 17 of 39


335

zein-Que-HA nanoparticles, which was confirmed by particle size, zeta-potential and

336

FE-SEM analyses in this work. For better understanding, the curves of the integrated

337

transmission against the measuring time were plotted.32 Lower value of the slope

338

represent more stable (Fig. 4g). It was apparent that the slopes of zein-Que-HA

339

colloidal dispersions with a low level of HA (100:5:5 and 100:5:10) were significantly

340

(p < 0.05) lower than the slopes of other samples. At high levels of HA, the colloidal

341

dispersions became more stable. One of the reasons was that the negative charge of

342

zein-Que-HA composite nanoparticles was increased; electrostatic repulsion could

343

prevent the particles from being together. With the addition of HA (100:5:25 and

344

100:5:30), the viscosity of the colloidal dispersions was increased, and zein-Que-HA

345

microgel with 3D network-like structure was formed, thus induced more stable

346

dispersions.38

347

Photochemical and thermal stability. On the basis of particle size, zeta-potential,

348

EE, LC, and micro-morphology analysis results, zein-Que-HA (100:5:20) composite

349

nanoparticles were regarded as an ideal delivery carrier for Que. The photochemical

350

and thermal stability of free Que, HA-Que (20:5), zein-Que (100:5), and

351

zein-Que-HA (100:5:20) were examined in the work.

352

Fig. 5a showed the relationship between the retained Que (Ln(C/C0)) and the

353

time (h) at the level of 150 µg/mL Que in free or in composite nanoparticles. The

354

degradation of Que followed the first order kinetics. The coefficient (R2 > 0.95),

355

half-life (t1/2), and overall rate constant (k) for Que were given in supporting



356

information Table 2. The protective capability of zein, HA, and zein-HA composite

357

nanoparticles on Que according to t1/2 value was ranked as follows: Que < HA-Que

Quercetagetin-Loaded Composite Nanoparticles Based on Zein and

Recommend Documents