Stable Nanoparticles Prepared by Heating Electrostatic Complexes of

Subscriber access provided by University Libraries, University of Memphis

Article

Stable Nanoparticles Prepared by Heating Electrostatic Complexes of Whey Protein Isolate-Dextran Conjugate and Chondroitin Sulfate Qingyuan Dai, Xiuling Zhu, Shabbar Abbas, Eric Karangwa, Xiaoming Zhang, Shuqin Xia, Biao Feng, and Chengsheng Jia J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b00794 • Publication Date (Web): 06 Apr 2015 Downloaded from http://pubs.acs.org on April 14, 2015

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 free 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 accessible to all readers and 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.

Journal of Agricultural and Food Chemistry 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 44

Journal of Agricultural and Food Chemistry

1

Stable Nanoparticles Prepared by Heating Electrostatic Complexes of Whey

2

Protein Isolate-Dextran Conjugate and Chondroitin Sulfate

3

Qingyuan Dai,†,‡ Xiuling Zhu,‡ Shabbar Abbas,† Eric Karangwa,† Xiaoming Zhang,∗,†

4

Shuqin Xia,† Biao Feng,† and Chengsheng Jia†

5

†

6

Technology, Jiangnan University, Lihu Road 1800, Wuxi, Jiangsu 214122, People’s

7

Republic of China

8

‡

9

Beijing Middle Road, Wuhu, Anhui 241000, People’s Republic of China

State Key Laboratory of Food Science and Technology, School of Food Science and

College of Biological and Chemical Engineering, Anhui Polytechnic University,

∗

To whom correspondence should be addressed. Tel.: +86 510 85197217; Fax: +86 510 85884496. E-mail: [email protected] (X. Zhang).

1

ACS Paragon Plus Environment


10

ABSTRACT: A simple and green method was developed for preparing the stable

11

biopolymer nanoparticles with pH and salt resistance. The method involved the

12

macromolecular crowding Maillard process and heat-induced gelation process. The

13

conjugates of whey protein isolate (WPI) and dextran were produced by Maillard

14

reaction. The nanoparticles were fabricated by heating electrostatic complexes of

15

WPI-dextran conjugate and chondroitin sulfate (ChS) above the denaturation

16

temperature and near the isoelectric point of WPI. And then, the nanoparticles were

17

characterized by spectrophotometry, dynamic laser scattering, zeta potential,

18

transmission electron microscopy, atomic force microscopy and scanning electron

19

microscopy. Results showed that the nanoparticles were stable in the pH range from

20

1.0 to 8.0 and in the presence of high salt concentration of 200 mM NaCl.

21

WPI-dextran conjugate, WPI and ChS were assembled into the nanoparticles with

22

dextran conjugated to WPI/ChS shell and WPI/ChS core. The repulsive steric

23

interactions, from both dextran covalently conjugated to WPI and ChS

24

electrostatically interacted with WPI, were the major formation mechanism of the

25

stable nanoparticles. As a nutrient model, lutein could be effectively encapsulated into

26

the nanoparticles. Additionally, the nanoparticles exhibited a spherical shape and

27

homogeneous size distribution regardless of lutein loading. The results suggested that

28

the stable nanoparticles from proteins and strong polyelectrolyte polysaccharides

29

would be used as a promising target delivery system for hydrophobic nutrients and

30

drugs at the physiological pH and salt conditions.

31

KEYWORDS: nanoparticle, whey protein isolate, dextran, conjugate, chondroitin

2


Page 2 of 44

Page 3 of 44


32

sulfate, electrostatic complex

33

INTRODUCTION

34

Biopolymer nanoparticles have received great attention for their exceptional

35

physical characteristics, including biocompatible, biodegradability, non-antigenicity,

36

abundant renewable sources and extraordinary binding capacity of hydrophobic

37

bioactive compounds 1-3. And it has been reported that biopolymer nanoparticles were

38

able to sustain drug release for prolonged duration, enhance the stability of sensitive

39

nutraceuticals and drugs, and increase the solubility and absorption of poorly soluble

40

nutraceuticals and drugs

41

be used as the delivery systems for nutraceuticals and drugs 1-3.

4-8

. Therefore, the biopolymer nanoparticles are potential to

42

Proteins and ionic polysaccharides have been conventionally used for the formation

43

of biopolymer particles by the complex coacervation 9. The electrostatic attractions

44

between protein and ionic polysaccharide molecules drive the complex formation at a

45

specific pH where they have opposite charges. The net charge on a protein is zero at

46

the isoelectric point (pI), positive at pH values below its pI, and negative at pH values

47

above its pI

48

electrostatic complexes with proteins near their pIs. However, the electrostatic

49

complexes formed at ambient temperature are extremely unstable when they are

50

followed by adjustment of pH and/or an increase in ionic strength, e.g., the complex

51

coacervates tend to dissociate or precipitate due to the alteration of electrostatic

52

interaction between protein and polysaccharide molecules. Nowadays, an increasing

53

number of researchers are paying attention to the formation of stable nanoparticles

10

. Therefore, the cationic and anionic polysaccharides can form the

3



Page 4 of 44

54

from proteins and polysaccharides over a wide range of environmental conditions

55

(e.g., pH, ionic strength and temperature) 11-13. It has been found that the electrostatic

56

repulsion is the weakest and the hydrophobic interaction is the strongest at the pI of

57

protein. In addition, high temperature contributes to the hydrophobic interaction,

58

formation and interchange of disulfide bond, which lead to a decrease in solubility

59

and aggregation or gel formation

60

could be fabricated by heating globular protein and ionic polysaccharide above the

61

denaturation temperature at a pH close to the pI of protein

62

protein gelation is attributed mainly to ionic bonds, hydrogen bonds, hydrophobic

63

interactions and disulfide bonds 21, so that the stability of the biopolymer nanoparticle

64

could be influenced by pH and salt. The nanoparticles prepared by heat-induced

65

gelation could remain relatively stable at a certain pH range where the complex

66

coacervates were expected to dissociate or precipitate. Nevertheless, the secondary

67

aggregates of these biopolymer nanoparticles would be possible to occur at a

68

particular pH range such as physiological pH conditions. Stability problem of

69

biopolymer nanoparticles formed by heating protein and polysaccharide electrostatic

70

complexes has not yet been completely solved.

14, 15

. For example, the biopolymer nanoparticles

16-20

. The formation of

71

Whey protein isolate (WPI) is widely used as a food ingredient not only based on

72

its high nutritional values but also its excellent functional properties, including

73

emulsifying, foaming and gelation. WPI consists mainly of several globular proteins,

74

α-lactalbumin (α-la), β-lactoglobulin (β-lg), and bovine serum albumin (BSA). The

75

functional properties of WPI are mainly dominated by β-lg, the major component of

4


Page 5 of 44


76

WPI. β-lg is a suitable candidate for the preparation of drug delivery systems for

77

lipophilic compounds because of its ability to bind hydrophobic constituents

78

the best of our knowledge, WPI and β-lg can form biopolymer nanoparticles with or

79

without ionic polysaccharide through thermal treatment. However, these biopolymer

80

nanoparticles are unstable at a pH range below their pIs with and without high salt

81

concentrations, leading to the secondary aggregation. The stability problem of

82

WPI-based nanoparticles under acidic pH conditions and high salt concentrations is

83

greatly challenging.

1, 4

. To

84

Maillard reaction is a complex series of reactions between reducing carbonyl

85

groups of polysaccharide and free amino groups of protein, and it has become more

86

and more popular to prepare glycosylated proteins compared with chemical

87

glycosylation reagents. The covalent grafting of glycosyl residues to protein can

88

significantly improve the solubility, thermal stability, and emulsifying properties of

89

proteins

90

and no gelation. It has been widely used in the glycosylation of proteins to avoid the

91

complication due to the formation of electrostatic complexes. Many studies have

92

focused on the optimization process of Maillard reaction and functional properties of

93

protein-polysaccharide conjugates as well as their applications

94

less work has been reported on the influence of glycosyl residues introduction on the

95

stability of biopolymer nanoparticles against pH and salt, which were formed by

96

heating protein-polysaccharide conjugate and ionic polysaccharide electrostatic

97

complexes.

22-26

. Dextran is a neutral polysaccharide with high solubility, low viscosity

5


22-30

. However, much


Page 6 of 44

98

Chondroitin sulfate (ChS) is a mucopolysaccharide extracted from animal cartilage

99

as an important food grade material. ChS contains weakly acidic carbonyl groups and

100

strongly acidic sulfate groups. And thus, ChS has a higher negative charge density and

101

can form ionic complexes with positively charged substance. In addition, ChS has

102

been investigated as a potential carrier for drug delivery due to its numerous

103

appealing properties, including safety, biocompatibility, and biodegradability 31, 32.

104

Lutein, a non-provitamin A carotenoid, is widely distributed in fruits and vegetables

105

and is one of the two carotenoids found in the macula and lens of the human eyes,

106

which is responsible for central vision and visual acuity. In addition, lutein is a

107

powerful antioxidant which plays an important role in the prevention of excessive

108

ultraviolet light exposure, stroke, cardiovascular disease, and lung cancer

109

Unfortunately, lutein is not synthesized in the human body. Therefore, dietary

110

ingestion is the only source for supplementation of lutein. However, the

111

bioavailability of lutein is limited by its inherent poor water solubility

112

reports have demonstrated that nanoencapsulation is a promising approach to improve

113

the bioavailability of poorly water soluble compounds 34, 35.

33

.

33

. Recent

114

Objective of the present study was to prepare the stable nanoparticles against pH

115

and salt by heating WPI-dextran conjugate/ChS electrostatic complexes, and to

116

elucidate the stability mechanism of the biopolymer nanoparticles in a wide pH range

117

and high salt concentration. The biopolymer nanoparticles were characterized by

118

spectrophotometry, dynamic light scattering, zeta potential, transmission electron

119

microscopy (TEM), atomic force microscopy (AFM) and scanning electron

6


Page 7 of 44


120

microscopy (SEM). As a nutrient model, lutein was used to evaluate the encapsulation

121

efficacy of the biopolymer nanoparticles for hydrophobic compounds.

122

MATERIALS AND METHODS

123

Materials and Reagents. Whey protein isolate (WPI) was purchased from Hilmar

124

Ingredients (Hilmar, California). WPI contained about 95.6% total solid, 88.7%

125

protein, and 2.7% ash. Dextran with an average molecular weight of 40 kDa was

126

obtained from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). Chondroitin

127

sulfate (ChS) was supplied by Shandong Yibao Biologics Co., Ltd (Yanzhou, China).

128

ChS was comprised of 95.4% sodium ChS and 4.6% protein. Lutein (98% pure) was

129

obtained from Zhejiang Medicine Co., Ltd. (Shaoxing, China). Hydrochloric acid,

130

sodium hydroxide, anhydrous ethanol, phenol, sulfuric acid (98%), Coomassie

131

brilliant blue G-250 and o-phthaldialdehyde (OPA) were purchased from Sinopharm

132

Chemical Reagent Co., Ltd (Shanghai, China). All materials were used without

133

further purification. All aqueous solutions were prepared with deionized water.

134

Preparation of WPI-Dextran Conjugates. WPI-dextran conjugates were prepared

135

under macromolecular crowding conditions according to the method described

136

previously with minor modifications 23, 36. Briefly, the conditions of pH 6.5 and 60 °C

137

were used to maximize the extent of formation of Schiff base, which is the initial

138

product of the Maillard reaction. The mixtures of WPI and dextran were dissolved in

139

10 mM sodium phosphate buffer solution (PBS) (pH 6.5) containing 0.02% (w/v)

140

sodium azide to prevent bacterial growth. The degree of glycosylation (DG) increased

141

with increasing protein concentration and mass ratio of dextran to WPI. However, the

7



Page 8 of 44

142

protein concentration above 10% (w/w) would lead to the formation of WPI gelation

143

during thermal treatment

144

significantly increase the DG of WPI

145

WPI and dextran were 7.5, 22.5% (w/w), respectively. The solutions were carefully

146

adjusted to pH 6.5 using 0.1 M HCl or 0.1 M NaOH and gently stirred for 6 h at room

147

temperature to dissolve completely the mixtures. The mixed solutions were stored at

148

4 °C overnight for the complete hydration. The following morning, 6 mL aliquots of

149

the solutions were dispensed into 10 mL screw-top, glass vials sealed with teflon tape

150

to prevent evaporation, and then incubated in a water bath at 60 °C for 24, 48 and 72

151

h (denoted as WPI-dextran conjugate 1, conjugate 2, and conjugate 3, respectively).

152

These reacted solutions were then removed from the water bath and immediately

153

cooled in an ice-water bath. The reacted mixtures were diluted 4-fold with deionized

154

water (pH 6.5, containing 0.02% NaN3) and centrifuged at 10,000g for 30 min. There

155

were few white precipitates after centrifugation. The white precipitates were dissolved

156

when resuspended in deionized water and heated at 100°C for 30 min. After the

157

redissolved solution was assayed by Coomassie brilliant blue method and

158

phenol-sulfuric acid method, the white precipitates were identified as dextran

159

self-associates due to its high concentration during thermal treatment

160

supernatant solutions were kept at -20 °C until use. WPI and dextran were separately

161

incubated and centrifuged using the same treatment to serve as controls. All

162

experiments were performed in triplicate.

163

Preparation of Biopolymer Nanoparticles from Mixtures of WPI/ Polysaccharide.

15, 29, 36

, and dextran concentration above 30% could not 36

. In this work, the optimal concentrations of

8


36

. The

Page 9 of 44


164

Biopolymer nanoparticles were prepared according to the method previously

165

described

166

ChS in deionized water and stirring for 2 h at room temperature. ChS stock solution

167

was stored at 4°C overnight for complete hydration. The stability of biopolymer

168

nanoparticles prepared by the mixtures of protein and polysaccharide was investigated

169

by the following experiments. (i) The WPI supernatant (WPI incubated at 60 °C for

170

48 h) and ChS stock solution were mixed and diluted with deionized water

171

(WPI/ChS). (ii) The WPI and dextran supernatants (WPI and dextran separately

172

incubated at 60 °C for 48 h) were mixed and diluted with deionized water

173

(WPI/dextran). (iii) The WPI and dextran supernatants (WPI and dextran separately

174

incubated at 60 °C for 48 h) and ChS stock solution were mixed and diluted with

175

deionized water (WPI/dextran/ChS). WPI, dextran, and ChS concentrations were

176

adjusted to fixed concentrations at 0.2, 0.55, and 0.008% (w/w), respectively (as

177

discussed below). The concentrations of WPI and dextran in the supernatants were

178

determined by Coomassie brilliant blue method and phenol-sulfuric acid method.

179

After stirring for 2 h, the mixed solutions were adjusted to pH 5.2 (near the pI of WPI)

180

with 0.1 M HCl, and then heated at 85 °C for 15 min. The mixtures were finally

181

cooled for 10 min in an ice-water bath. The nanoparticle suspensions obtained by the

182

above process were kept at 4 °C before analysis.

183

Preparation of Biopolymer Nanoparticles from WPI-Dextran Conjugate and

184

ChS. The WPI-dextran conjugate (n) supernatant and ChS stock solution (denoted as

185

WPI-dextran conjugate n/ChS, n = 1, 2, 3) were mixed and adjusted to the fixed final

12, 13, 37

. Briefly, 1% (w/w) ChS stock solution was prepared by dissolving

9



Page 10 of 44

186

concentrations as described above. When the WPI-dextran conjugate 2 (WPI and

187

dextran mixtures incubated at 60 °C for 48 h) supernatant was adjusted to 0.2% (w/w)

188

WPI, the dextran concentration was 0.55% (w/w). To keep experimental conditions as

189

consistent as possible, the concentrations of WPI and dextran and ChS were fixed. All

190

other procedures were the same as described above.

191

pH and Salt Stability. The stability of biopolymer nanoparticles against pH and salt

192

was determined using spectrophotometry and dynamic light scattering. The

193

nanoparticle suspensions were divided into two parts: one was adjusted to the desired

194

pH values (1.0 to 8.0) using hydrochloric acid (2.0, 1.0, 0.1 and 0.01 M) or sodium

195

hydroxide solution (0.1 and 0.01 M). The other portion was firstly added with NaCl

196

stock solution (3 M NaCl) to reach a final concentration of 200 mM NaCl, and then

197

adjusted to the desired pH values as described above. In this study, the final NaCl

198

concentration was 200 mM slightly higher than physiological salt concentration,

199

which was to verify salt stability of biopolymer nanoparticles in physiological

200

concentration.

201

Lutein Loading. Lutein-loaded WPI-dextran conjugate/ChS nanoparticles were

202

prepared according to the ethanol injection-ultrasonic method

203

solution (0.2%, w/w) was obtained by dissolving lutein in anhydrous ethanol by

204

sonication treatment (SK5210HP, Shanghai Kudos Ultrasonic Instrument Co., Ltd,

205

China) for 1 min. Ten milliliters of lutein ethanol solution was rapidly injected into

206

100 mL of WPI-dextran conjugate/ChS nanoparticle suspension under agitation. After

207

stirring for 1 h, the ethanol in the coarse suspension was removed by a rotary

10


35

. Lutein ethanol

Page 11 of 44


208

evaporator under reduced pressure at 40 °C. Then the lutein-loaded suspension was

209

subjected to ultrasonic treatment in an ice bath for 4 min at 400 W with a pulse mode

210

of 1 s of sonication and 1 s of rest using a probe sonicator (JY98-IIIDN, Ningbo

211

Scientz Biotechnology Co., Ltd, China). The final suspension was kept at 4 °C in the

212

dark before analysis.

213

Lutein was absorbed on the surface and entrapped in the lutein-loaded

214

nanoparticles. To determine the amount and efficiency of lutein loading into the

215

nanoparticles, the amount of lutein unentrapped on the surface of nanoparticles was

216

measured as follows: 0.5 mL of nanoparticle suspension was added to 2.5 mL of ethyl

217

acetate and violently vortexed for 1 min, then centrifuged at 1000g for 5 min. The

218

resultant upper ethyl acetate layer was collected. The above treatment was repeated

219

twice. The collected extracts were combined and concentrated to dry under nitrogen

220

conditions and redissolved in 10 mL of ethanol for spectral analysis. Lutein

221

concentration was determined using a spectrophotometer (UV-1600; Mapada

222

Instruments Co., Ltd., China) at 446 nm and calculated by the following equation: C =

223

0.0322A - 0.0065 (R2 = 0.9992), where C and A were the concentration and

224

absorbance of lutein in anhydrous ethanol, respectively. R2 was the correlation

225

coefficient of linear regression equation. The lutein loading content (LC, % w/w) and

226

encapsulation efficiency (EE, %) were respectively calculated using the following

227

equations:

228

LC (% w/w) =

entrapped amount of lutein amount of WPI

× 100

11



229

EE (%) =

total amount of lutein - unentrapped amount of lutein total amount of lutein

Page 12 of 44

× 100

230

LC was defined as the content of lutein in the biopolymer nanoparticles measured by

231

determining the ratio of entrapped amount of lutein to the total amount of WPI. The

232

amount of lutein entrapped inside the biopolymer nanoparticles was calculated as the

233

difference between the total amount of lutein added to the nanoparticle suspensions

234

and that of lutein recovered by extraction. The amount of WPI in the biopolymer

235

nanoparticles was obtained from the initial WPI concentration. EE was defined as the

236

amount of lutein entrapped inside the biopolymer nanoparticles measured by

237

determining the ratio of entrapped amount of lutein to the total amount of lutein.

238

Turbidity Measurements. Turbidity analysis of the biopolymer nanoparticle

239

suspensions was carried out using an ultraviolet-visible spectrophotometer (UV-1600

240

spectrophotometer, Mapada Instruments Co., Ltd., China) at 633 nm with glass

241

cuvette (1 cm path length) 37. Deionized water was used as blank for all solutions. All

242

experiments were performed in triplicate.

243

Dynamic Laser Scattering (DLS) Measurements. The apparent Z-average

244

hydrodynamic diameter (Dh) and polydispersity index (PDI) of biopolymer

245

nanoparticles were measured by dynamic light scattering using a Malvern Zetasizer

246

Nano ZS analyzer (Malvern Instruments Ltd., Malvern, UK) fitted with 633 nm

247

He-Ne laser beam. Measurements were taken at 25 °C and 173° scattering angle. Dh

248

and PDI were obtained by cumulant analysis (software of Zetasizer Nano ZS,

249

Malvern Instruments Ltd., Malvern, UK). The nanoparticle samples were measured

250

directly without filtering or removing dust. 12


Page 13 of 44


251

Zeta Potential Measurements. The Zeta potential (ζ-potential) of nanoparticles was

252

determined by laser Doppler electrophoresis at 25 °C using a Malvern Zetasizer Nano

253

ZS analyzer (Malvern Instruments Ltd., Malvern, UK). ζ-potential was calculated by

254

the Zetasizer Software (Malvern Instruments Ltd., Malvern, UK) according to the

255

Henry equation

256

was analyzed in triplicate.

257

Transmission Electron Microscopy (TEM) Measurements. A drop of a 1000-fold

258

dilution of the sample suspensions (approximately 7 µL) was deposited onto a

259

carbon-coated copper grid. The samples were then allowed to dry for 72 h at ambient

260

temperature. TEM images of biopolymer nanoparticles were recorded with a

261

commercial TEM (JEM-2100, JEOL Ltd., Japan) operating at an accelerating voltage

262

of 200 kV.

263

Atomic Force Microscopy (AFM) Measurements. The nanoparticle suspensions for

264

AFM measurement (Dimension icon, Bruker Co., USA) were diluted 400-fold with

265

deionized water. AFM samples were prepared by dropping an aliquot (1 µL) of the

266

diluted suspensions on a freshly cleaved mica surface and air drying at room

267

temperature for 72 h. The ScanAsyst mode was carried out using silicon tip (TESP,

268

Bruker, nom. frep. 320 kHz, nom. Spring constant of 42 N/m) with a scan resolution

269

of 512 × 512 pixels in the dimension of 5 µm × 5 µm. Images were generated and

270

processed using NanoScopeTM software (Digital Instruments, version V614r1).

271

Scanning Electron Microscope (SEM) Measurements. A drop of a 1000-fold

272

dilution of the sample suspensions (approximately 7 µL) was deposited onto a

38

. The nanoparticle samples were measured directly. Each sample

13



273

carbon-coated copper grid. The samples were allowed to dry for 72 h at ambient

274

temperature, and then coated with gold palladium using a sputter coater. The

275

morphology of biopolymer nanoparticles was observed at 40000× magnification with

276

a field emission scanning electron microscope (S-4800, Hitachi Science Systems, Ltd.,

277

Japan) operating at an accelerating voltage of 5 kV.

278

Statistical Analysis. All experiments were performed in triplicate. Data were

279

presented as a mean value with its standard deviation. The means were compared with

280

one-way analysis of variance (ANOVA) followed by Duncan’s multiple range tests,

281

and p < 0.05 was regarded as significant. Statistical analyses were conducted using

282

SPSS 17.0 for Windows (SPSS Inc., Chicago, USA).

283

RESULTS AND DISCUSSION

284

pH and Salt Stability of WPI/Polysaccharide Nanoparticles. pH and Salt Effect on

285

Turbidity of WPI/Polysaccharide Suspensions. The effect of pH on the turbidity of

286

protein/polysaccharide suspensions was shown in Figure 1A. All the suspensions had

287

a similar trend when pH changed from pH 1.0 to 8.0. WPI solution (0.2%, w/w)

288

rapidly coagulated and the white precipitates were found at the bottom of the bottle

289

during thermal treatment near its pI. Whereas dextran did not form aggregates under

290

the same experimental conditions (data not shown). Similar phenomena have

291

previously been observed by other researchers. Jones and co-authors reported that

292

solutions of β-lg alone at pH 4.0-5.5 became very turbid after heating at 83 °C for 15

293

min

294

through hydrophobic interactions and disulfide bonds, which leaded to the formation

12

. The reason maybe related to the unfolding and self-association of protein

14


Page 14 of 44

Page 15 of 44


295

of irreversible particulate aggregates, in the absence of inter-protein charge repulsion

296

and above its thermal denaturation temperature. Additionally, Jones and co-authors

297

also found that when 0.5% (w/w) β-lg solution was heated at pH 5 in the absence of

298

pectin, large protein aggregates (d > 6000 nm) were formed, and remained large (d >

299

3000 nm) across the entire pH range

300

occurred near the pI of the globular protein was pH-irreversible.

20

. The results suggested that the aggregation

301

Although WPI/dextran solution did not rapidly form much white precipitates during

302

thermal treatment, the turbidity of WPI/dextran suspensions remained relatively high

303

and constant at high pH values (pH 6.0-8.0), and had a maximum turbidity at pH 5.0.

304

In addition, the turbidity of WPI/dextran suspensions gradually decreased with pH

305

values decreasing from 5.0 to 1.0. In most case for biopolymer mixtures, WPI and

306

dextran are co-soluble in very dilute solutions. Above WPI denaturation temperature

307

and near its pI, phase separation (aggregation) would take place, resulting in WPI-rich

308

and dextran-rich domains. Due to the very low turbidity resulting from dextran-rich

309

domain, the turbidity of WPI/dextran suspensions was dominated by WPI

310

self-association. It suggested that WPI was the core component of the nanoparticles.

311

The results also indicated that dextran could only partly suppress the formation of

312

large sediments during thermal treatment. It was possible that dextran could decrease

313

the frequency of encounters between protein molecules, and thereby reduce the

314

magnitude and range of protein-protein interaction during thermal treatment. However,

315

due to the lack of electrostatic repulsion and steric hindrance between dextran and

316

WPI molecules, dextran could not completely prevent WPI self-association. Similar

15



39

317

results were reported by Chakrabortee and co-authors

. They demonstrated that

318

dextran behaved as a molecular shield polysaccharide and used physical interference

319

to reduce protein aggregation caused by desiccation. These studies have shown that

320

the neutral dextran could indeed partly impact the aggregation or gelation process of

321

protein during thermal treatment.

322

As shown in Figure 1A, the turbidity of WPI/ChS suspensions significantly

323

decreased at pH 5.0. It was due to the fact that ChS molecules could increase the

324

electrostatic and steric forces between particles, but these forces were not sufficient to

325

prevent the particle aggregation at pH 4.0. Similar results were reported by previous

326

studies 11, 40. Pectin stabilized the β-lg/pectin particles against aggregation around the

327

pI of β-lg, which was presumably due to an increase in the electrostatic and steric

328

repulsion between the biopolymer nanoparticles. Whereas the extensive aggregation

329

was still observed at lower pH values

330

suspensions was relatively low in the pH ranges of 1.0-3.0 and 5.0-8.0, which was

331

possible due to the lack of disulfide bond formation (pH 1.0-3.0) and the increase of

332

repulsive electrostatic interaction (pH 1.0-3.0 and pH 5.0-8.0) between biopolymer

333

nanoparticles.

11

. In addition, the turbidity of WPI/ChS

334

The maximum turbidity of WPI/ChS and WPI/dextran/ChS suspensions was

335

obtained at pH 4.0 and pH 5.0, respectively (Figure 1A). It was attributed to the fact

336

that dextran could also decrease the collision frequency between WPI and ChS

337

molecules as between WPI molecules. Therefore, dextran significantly weakened the

338

suppressing effect of anionic ChS on WPI self-association. The similar results were

16


Page 16 of 44

Page 17 of 44


339

reported in previous studies, demonstrating that the incompatibility between gelatin

340

and pectin was attributed to the competitive interactions between dextran and the

341

reactive groups of gelatin

342

concentration can destabilize the polyelectrolyte complexes due to the electrostatic

343

screening effect. All suspensions in the presence of 200 mM NaCl had higher

344

turbidity over a wider pH range compared with those in the absence of salt (Figure

345

S1A and Figure 1A, respectively).

346

pH and Salt Effect on Particle Diameter of WPI/Polysaccharide Suspensions. The

347

turbidity of the biopolymer suspensions was further supported by DLS measurements

348

(Figure 1B-C and Figure S1B-C). As observed in Figure 1B, the Z-average particle

349

diameter (160-250 nm) of WPI/ChS nanoparticles remained relatively stable in the pH

350

ranges of 1.0-3.0 and 6.0-8.0. And Figure 1C showed that the biopolymer

351

nanoparticles had a small polydispersity index (PDI < 0.15) in the same pH ranges.

352

However, the particle diameter significantly (p < 0.05) increased from 550 nm at pH

353

5.0 (PDI = 0.41) to 5600 nm at pH 4.0 (PDI = 0.46) (Figure 1B and 1C). The change

354

trends of particle diameter of WPI/dextran and WPI/dextran/ChS suspensions were

355

the same as that of WPI/ChS suspensions. In the presence of salt, the large aggregates

356

in all suspensions occurred over a wider pH range compared with those in the absence

357

of salt (Figure S1B and Figure 1B, respectively).

358

pH Effect on ζ-potential of WPI and/or Polysaccharide Suspensions. ζ-potential is

359

directly related to the net charges on the surface of macromolecules and particles in

360

solutions

41

. Additionally, it is well known that the high salt

42

. Figure 1D showed that the ζ-potentials of native WPI changed from

17



361

negative at high pH values to positive at low pH values, with a zero charge point

362

between pH 4.0 and 5.0. pH value at the zero charge point was close to the pI of

363

native WPI (4.8-5.2) reported in the literature 14. ChS was negatively charged across

364

the entire pH range of 3.0-8.0. Although dextran has no charges, its solution had a

365

relatively low negative ζ-potential, which was attributed to the increased ionic

366

strength when the solution was adjusted to various pH values by adding hydrochloric

367

acid or sodium hydroxide. These results are in agreement with previous findings,

368

which reported that the ζ-potential of dextran solution (phosphate buffer 0.01 M, pH

369

7.4) was -9.9±0.5 mV 43.

370

The absolute ζ-potential values of WPI/dextran suspensions, formed by heating at

371

pH 5.2, were smaller than those of individual WPI solutions. The results confirmed

372

that the principal component of the nanoparticles was comprised of WPI, and dextran

373

could impact the interactions between WPI molecules as discussed above. The

374

ζ-potentials of WPI/ChS suspensions were between those of the individual WPI and

375

ChS solutions below pH 6.0, suggesting that there was the electrostatic attraction

376

between cationic patches on the protein surface and anionic groups on the

377

polysaccharide backbone 44. Therefore, ChS would influence the net surface charge of

378

the outer periphery of WPI/ChS particles. It was important that the ζ-potentials could

379

provide

380

WPI/polysaccharide suspensions in different pH values. For example, the ζ-potentials

381

of WPI/ChS suspensions went from negative (-36.8 mV) to positive (+15.8 mV) as

382

pH values decreased from 8.0 to 3.0 (Figure 1D). The electrostatic repulsions between

a good explanation for the turbidity and particle diameter of

18


Page 18 of 44

Page 19 of 44


383

nanoparticles decreased with decreasing the absolute ζ-potential values. Therefore, the

384

turbidity and particle diameter of WPI/ChS suspensions increased.

385

Preparation of WPI-Dextran Conjugates. Maillard reaction is a natural nontoxic

386

process, which produces conjugates from the graft reaction between reducing end

387

carbonyl groups of polysaccharide and free amino groups of protein. WPI-dextran

388

conjugates prepared by the Maillard reaction have been reported by previous studies

389

22, 23, 26, 29, 36

390

than those of the native WPI around its pI. In this study, WPI-dextran conjugates were

391

prepared with the weight ratios of WPI to dextran 1:3 under macromolecular

392

crowding conditions, which were previously reported by other researchers

393

formation of WPI-dextran conjugates was validated by o-phthalaldehyde (OPA)

394

method and Fourier transform infrared spectra (FT-IR) (Supporting Information,

395

Figure S2 and Figure S3, respectively). Meanwhile, the degree of glycosylation (DG)

396

of the WPI-dextran conjugates was determined by the OPA assay from the loss of free

397

amino groups of WPI 29, 45. The analysis revealed that DGs of WPI-dextran conjugate

398

1 (incubated for 24 h), conjugate 2 (incubated for 48 h), and conjugate 3 (incubated

399

for 72 h) were 5.2, 9.7, and 12.2%, respectively (Supporting Information, Figure S2).

400

Stability of WPI-Dextran Conjugate/ChS Nanoparticles against pH and Salt.

401

Stability of WPI-Dextran Conjugate 1/ChS Nanoparticles against pH and Salt. The

402

stability of WPI-dextran conjugate/ChS nanoparticles against pH in the absence or

403

presence of salt was shown in Figure 2 and Figure 3, respectively. Compared with

404

WPI/polysaccharide suspensions (Figure 1 and Figure S1), the turbidity and particle

. WPI-dextran conjugates exhibit higher solubility and thermal stability

19


23, 36

. The


405

diameter of WPI-dextran conjugate or WPI-dextran conjugate/ChS suspensions

406

significantly decreased (p < 0.05) at the same pH values in the absence or presence of

407

salt, which illustrated that the dextran conjugated to WPI could significantly decrease

408

the turbidity and particle diameter of the suspension systems. The results also

409

suggested that the dextran conjugated to WPI could be extended to the periphery of

410

the inner core of the nanoparticles, and prevent their aggregation. The WPI-dextran

411

conjugate 1/ChS suspensions had a relatively constant turbidity and smaller particle

412

size diameter (about 150 nm, PDI < 0.15) at low pH range of 1.0-3.0 and high pH

413

range of 5.0-8.0 (Figure 2A-C) and in the presence of 200 mM NaCl (Figure 3A-C),

414

indicating that the nanoparticles were fairly stable to association or dissociation. But

415

the turbidity and particle diameter of WPI-dextran conjugate 1/ChS were higher at the

416

pH range of 3.0-5.0 regardless of salt. The results indicated that the steric hindrance

417

provided by the dextran conjugated to WPI with only 5.2% DG could not effectively

418

inhibit the aggregation of biopolymer nanoparticles in this pH range.

419

Stability of WPI-Dextran Conjugate 2/ChS Nanoparticles against pH and Salt. It has

420

been demonstrated that nanoparticles with 50-200 nm size acquire the best properties

421

for cellular uptake

422

particle diameter about 150 nm and PDI 0.08) could remain stable in the entire pH

423

range of 1.0-8.0 and in the absence or presence of salt (Figure 2 and Figure 3,

424

respectively), which was ideal for the encapsulation and transportation of

425

nutraceuticals and drugs. Although the WPI/dextran/ChS nanoparticles had a smaller

426

particle diameter (about 170-270 nm and PDI 0.08-0.23) at the pH ranges of 1.0-4.0

46, 47

. The WPI-dextran conjugate 2/ChS nanoparticles (Z-average

20


Page 20 of 44

Page 21 of 44


427

and 6.0-8.0, the secondary aggregates of the nanoparticles occurred in the pH range of

428

4.0-6.0 and in the presence of salt, and largest aggregates (about 6400 nm and PDI

429

0.52) appeared at pH 5.0 (Figure 1B). Obviously, dextran conjugated to WPI could

430

significantly improve the stability of WPI-dextran conjugate 2/ChS nanoparticles

431

against pH and salt. However, the turbidity and particle diameter of WPI-dextran

432

conjugate 2 suspensions were fairly similar to those of WPI-dextran conjugate 1/ChS

433

suspensions (Figure 2 and Figure 3, respectively). Only the introduction of dextran,

434

from the WPI-dextran conjugate 2 with 9.7% DG, was not strong enough to stabilize

435

the nanoparticles in the pH range from 3.0 to 5.0. It suggested that the combined

436

action of the dextran conjugated to WPI with 9.7% DG and ChS could stabilize the

437

nanoparticles against pH and salt.

438

Stability of WPI-Dextran Conjugate 3/ChS Nanoparticles against pH and Salt. There

439

was no significant difference in the turbidity, particle diameter and PDI between

440

WPI-dextran conjugate 2/ChS and WPI-dextran conjugate 3/ChS suspensions over a

441

wide pH range of 1.0-8.0 and in the absence or presence of salt (Figure 2A-C and

442

Figure 3A-C, respectively). The results illustrated that both dextran covalently

443

conjugated to WPI with 9.7% DG and ChS electrostatically interacted with WPI were

444

the minimum requirement to stabilize the nanoparticles. The WPI-dextran conjugate 2

445

was more economical than WPI-dextran conjugate 3 because the latter required more

446

preparation time. Therefore, the WPI-dextran conjugate 2/ChS complexes were used

447

to further determine the characteristics of the stable nanoparticles.

448

ζ-Potentials of WPI-Dextran Conjugate 2 and WPI-Dextran Conjugate/ChS

21



Page 22 of 44

449

Suspensions. The ζ-potentials of biopolymer nanoparticle suspensions at different pH

450

values were shown in Figure 2D. The ζ-potential values of all WPI-dextran conjugate

451

or WPI-dextran conjugate/ChS suspensions were slightly lower than those of

452

WPI/dextran or WPI/dextran/ChS suspensions (Figure 2D and Figure 1D,

453

respectively). For example, the ζ-potentials of WPI-dextran conjugate 2, conjugate

454

1/ChS, conjugate 2/ChS, and conjugate 3/ChS suspensions at pH 8.0 were -31.5, -33.0,

455

-35.3, and -34.2 mV, respectively (Figure 2D). While the ζ-potentials of WPI/dextran

456

and WPI/dextran/ChS suspensions at pH 8.0 were -38.1 and -37.7 mV, respectively

457

(Figure 1D). The decrease of absolute ζ-potential values after protein glycosylation

458

was probably related to the highly hydratable dextran lowering the electrophoretic

459

mobility of WPI-dextran conjugate and WPI-dextran conjugate/ChS suspensions.

460

Similar results have been reported in previous studies 25, 27.

461

WPI-Dextran Conjugate 2/ChS Nanoparticle Formation Mechanism. As

462

evidenced above, the stability of the nanoparticles was closely related to the degrees

463

of glycosylation of WPI. It indicated that the steric repulsions would stabilize the

464

protein nanoparticles against aggregation, which were contributed by both dextran

465

covalently conjugated to WPI and ChS electrostatically interacted with WPI.

466

Therefore, the results suggested that the repulsive steric interactions were the major

467

mechanism for the formation of the stable nanoparticles against pH and salt. It has

468

been demonstrated that the steric hindrance of dextran conjugated to protein almost

469

completely suppressed the protein aggregation during thermal treatment

470

Combining with the present findings, it was further confirmed that the appropriate

22


22-25, 28

.

Page 23 of 44


471

glycosylation of protein molecules was a crucial step to prepare the size-controlled

472

nanoparticles. In addition, the ζ-potentials of WPI-dextran conjugate suspensions

473

were slightly lower than those of WPI or WPI/dextran (Figure 2D and Figure 1D,

474

respectively), which indicated that dextran chains conjugated to WPI extended in the

475

shell of the nanoparticle. Previous studies have also illuminated that steric hindrance

476

was a function of polysaccharide chains on colloidal particle surface to improve the

477

heat stability of protein, which was supported by ζ-potentials of protein and

478

glycosylated protein 24, 25, 27, 30.

479

Although the formation of the nanoparticles prepared by heating protein and ionic

480

polysaccharide complexes undergo a variety of interactions and processes, the

481

electrostatic attraction and hydrophobic interaction primarily contribute to the initial

482

structure development during gelation. Proteins might only electrostatically couple

483

with a certain amount of ionic polysaccharides during thermal treatment. At the high

484

mass ratio of WPI to ChS, most of ChS chains could be partly or completely

485

entrapped and fixed in the inner core of the nanoparticles during the process of

486

denaturation and aggregation of WPI. It has been demonstrated that the β-lg/pectin

487

nanoparticles prepared by thermal treatment were primarily composed of β-lg, but

488

some pectin was still present 12. Chakrabortee and co-authors have also demonstrated

489

that there was only a few formation of complexes between proteins and molecular

490

shields against protein aggregation 39.

491

The turbidity and particle diameter of WPI-dextran conjugate 2/ChS suspensions

492

were stable in the pH range of 1.0-8.0 regardless of salt, but the secondary aggregates

23



493

occurred in the WPI-dextran conjugate 2 suspensions at pH 4.0 in the absence and

494

presence of salt (Figure 2A-B and Figure 3A-B, respectively). The results confirmed

495

that not all ChS chains loosely distributed at the periphery of WPI-dextran conjugate

496

2/ChS nanoparticles after thermal treatment, but most of ChS chains were

497

incorporated into the nanoparticles to improve their stability, through the electrostatic

498

interaction between WPI-dextran conjugate or WPI and ChS molecules during

499

thermal treatment. Although there was a slight difference in the ζ-potentials between

500

WPI-dextran conjugate 2/ChS and WPI-dextran conjugate 2 suspensions in the pH

501

range of 3.0-8.0, the ζ-potentials of the former and latter were from -24.8 to -35.3 mV

502

and from -24.2 to -31.5 mV at the pH range of 6.0-8.0, respectively (Figure 2D),

503

where the electrostatic repulsion between WPI-dextran conjugate and ChS was

504

predominant. It suggested that some of ChS chains extended in the shell of the

505

nanoparticles. Therefore, the shell of the nanoparticles was comprised of both ChS

506

and dextran conjugated to WPI. Multiple interactions finally contributed to the

507

stability of the nanoparticles against pH and salt. As discussed above, ChS, WPI and

508

WPI-dextran conjugate with an appropriate degree of glycosylation could be

509

assembled into the stable nanoparticles with dextran conjugated to WPI/ChS shell and

510

WPI/ChS core in a wide range of pH values and high salt concentration.

511

Lutein Loading of WPI-Dextran Conjugate 2/ChS Nanoparticles. There were no

512

significant differences in the turbidity, particle diameter and ζ-potential between

513

lutein-unloaded and lutein-loaded nanoparticles in the absence or presence of salt

514

(Figure 2, Figure 3 and Figure 4). The results illustrated that lutein loading did not

24


Page 24 of 44

Page 25 of 44


515

impair the stability of the nanoparticles. The WPI-dextran conjugate 2/ChS

516

suspension with light blue opalescence was obtained as shown in Figure 5A-1 (as

517

insert), and the lutein-loaded nanoparticle suspension was uniformly stable as shown

518

in Figure 5B-1 (as insert). Previous studies showed that the Z-average particle

519

diameter of nano-emulsions decreased with sonication treatment 48. This discrepancy

520

was attributed to the good stability of the conjugate/polysaccharide nanoparticles

521

formed by thermal treatment. On the contrary, the ultrasonic cavitation could promote

522

lutein molecules to migrate into the interior of the nanoparticles. Lutein molecules

523

were trapped in the hydrophobic regions of the nanoparticle core, by hydrophobic

524

interactions. In this study, the nanoparticles could effectively encapsulate lutein with

525

the loading content and encapsulation efficiency of 8.03, 94.07%, respectively. The

526

ζ-potentials had no significant difference between lutein-unloaded and lutein-loaded

527

nanoparticles (Figure 4B). Apparently, the neutral lutein could not influence the

528

surface charge of the nanoparticles. The Z-average particle diameter of lutein-loaded

529

nanoparticles remained almost unchanged at the entire pH range (1.0-8.0) regardless

530

of salt (Figure 4A and 4C), indicating that lutein-loaded nanoparticles had good

531

stability against pH and salt.

532

Morphology of WPI-Dextran Conjugate 2/ChS Nanoparticles. TEM. The

533

morphology of lutein-unloaded and lutein-loaded nanoparticles was characterized

534

using TEM, AFM and SEM as shown in Figure 5. TEM images of the nanoparticles

535

revealed a homogeneous size distribution and a spherical shape with smooth surface,

536

as shown in Figure 5A-2 and 5B-2. The particle diameters of lutein-unloaded and

25



537

lutein-loaded nanoparticles in TEM images were approximately 70 nm and 90 nm,

538

respectively, much smaller than the hydrodynamic diameter obtained from DLS

539

(about 150 nm) (Figure 5A-1 and 5B-1). It is well known that DLS determines the

540

data for the particle swollen in solution, whereas the particle diameters in TEM

541

images are obtained by spreading and drying the nanoparticles on a carbon-coated

542

copper grid

543

nanoparticles had more density in the inner core of the nanoparticles compared with

544

the lutein-unloaded nanoparticles because of the higher gray level in the former.

545

Therefore, TEM images further confirmed that the lutein molecules were located in

546

the core of the nanoparticles.

547

AFM. AFM and SEM are both powerful instruments to observe the three dimensional

548

morphology of the nanoparticles. AFM images of the nanoparticles were shown in

549

Figure 5A-3 and 5B-3. The images of all nanoparticles appeared to be fairly uniform

550

and spheroid shape, indicating that the encapsulation of lutein did not greatly change

551

the morphology of nanoparticles. Additionally, the dimensions of the lutein-loaded

552

nanoparticles were slightly bigger than those of the unloaded nanoparticles, consistent

553

with the results of TEM. The height of the nanoparticles in the AFM images was

554

much smaller than their dimension, suggesting that the nanoparticles were very soft

555

and collapsed on the mica surface during air drying 12, 18, 27.

556

SEM. SEM images showed that the nanoparticles had a fairly uniform spherical shape

557

(Figure 5A-4 and 5B-4) as observed by AFM. The diameters in SEM images

558

exhibited a narrow size distribution from 50 nm to 100 nm, which was in good

7, 19

. Meanwhile, Figure 5B-2 also showed that the lutein-loaded

26


Page 26 of 44

Page 27 of 44


559

agreement with the dimensions obtained from TEM images. Our findings are

560

consistent with previous studies

561

nanoparticles observed by AFM and TEM. It has been demonstrated that large

562

amounts of water or biological fluids were imbibed into the three-dimensional

563

networks of the hydrogel nanoparticles, and drugs could be entrapped into the mesh

564

space between the hydrogel nanoparticle networks

565

illuminated that lysozyme-dextran nanogels had a low-density structure and could

566

contain a large amount of water by the swelling ratio of the nanogels estimated from

567

the ratio of average volumes of DLS to AFM

568

observed by TEM, AFM and SEM were smaller than those obtained by DLS was due

569

to the syneresis of the nanoparticles during air drying. These results also suggested

570

that there were adequate mesh spaces between the nanoparticle networks, which could

571

provide an opportunity to encapsulate other components within themselves.

572

Potential Application of WPI-Dextran Conjugate 2/ChS Nanoparticles. There has

573

been a great interest in applications of nanoparticles as biomaterials for delivering

574

hydrophobic bioactive compounds such as nutraceuticals and drugs. Moreover,

575

biopolymer nanoparticles can be easily prepared and scaled up during manufacture 1, 2.

576

Several nanoparticles have been launched in the area of cancer treatment. For

577

example, Abraxane® (paclitaxel-albumin nanoparticle) with an approximate diameter

578

of 130 nm is the first nanotechnology based chemotherapeutic approved by FDA, and

579

has shown significant benefit in treatment of metastatic breast cancer

580

commercial success of albumin-based nanoparticles has created a great interest in

34, 46

. The results confirmed the microstructure of the

49

. Li and co-authors have

27

. Therefore, the fact that the sizes

27


1, 4

. The


Page 28 of 44

1, 4, 5

581

other proteins

. Whey protein has been used to prepare biopolymer-based

582

nanoparticles using the bottom-up approach

583

known to be stable at low pH and highly resistant to digestion by gastric proteases.

584

β-lg has been investigated for drug delivery applications

585

nanoparticles were successfully fabricated by pH-cycling treatment and thermal

586

processing 50. Jones and co-authors discussed the effect of polysaccharide charge on

587

formation and properties of biopolymer nanoparticles, which were created by heat

588

treatment of β-lg/pectin complexes

589

prepared with the aim of developing a biocompatible carrier for the oral

590

administration of nutraceuticals 7.

5, 8

. β-lg, the major whey protein, is

1, 4

. Whey protein

12

. Chitosan/β-lg core-shell nanoparticles were

591

However, the biopolymer nanoparticles were unstable in a wide pH range

592

(especially entire physiological pH range) and high salt concentration. Glycosylation

593

of whey protein with dextran increased protein solubility and thermal stability 22. Liu

594

and co-authors illustrated that the glycosylation of whey protein with maltodextrins

595

prevented protein aggregation before and after heating, and steric hindrance was

596

concluded to be the primary mechanism responsible for transparent dispersions with

597

protein structures smaller than 12 nm after heating

598

glycosylation of protein would offer an excellent opportunity to improve the stability

599

of biopolymer nanoparticles. Therefore, the stable biopolymer nanoparticles against

600

pH and salt, having spherical shapes with smooth surface, would be used as a

601

promising delivery system for hydrophobic nutrients or drugs in physiological

602

conditions. Additionally, it has been demonstrated that complexes of proteins and

24

. The results suggested that

28


Page 29 of 44


603

strong polyelectrolytes like sulfated or phosphate polysaccharides tend to form

604

precipitates or solid-like structures, and there is a narrow range of physicochemical

605

conditions where electrostatic complexes are formed and stay in solution 9. Sulfated

606

polysaccharide like ChS may be suitable for delivering active compounds to target

607

cells 32. This method would provide an approach to prepare target nanoparticles from

608

protein and sulfated polysaccharide.

609

ASSOCIATED CONTENT

610

Supporting Information

611

Additional experimental details. This material is available free of charge via the

612

Internet at http://pubs.acs.org.

613

AUTHOR INFORMATION

614

Corresponding Author

615

Postal address: State Key Laboratory of Food Science and Technology, School of

616

Food Science and Technology, Jiangnan University, Lihu Road 1800, Wuxi, Jiangsu

617

214122, People’s Republic of China. E-mail: [email protected] (X. Zhang).

618

Tel.: +86 510 85197217. Fax: +86 510 85884496.

619

Funding

620

This research was financially supported by projects of the National 125 Program of

621

China (2011BAD23B04, 2012BAD33B05, and 2013AA102204), projects of the

622

National Natural Science Foundation of China (31471624), and open projects of the

623

Key Laboratory of Carbohydrate Chemistry Biotechnology & Ministry of Education

624

(KLCCB-KF201202)

29



625

Notes

626

The authors declare no competing financial interest

627

REFERENCES

628

(1) Elzoghby, A. O.; Samy, W. M.; Elgindy, N. A. Protein-based nanocarriers as

629

promising drug and gene delivery systems. J. Controlled Release 2012, 161, 38-49.

630

(2) Nitta, S. K.; Numata, K. Biopolymer-based nanoparticles for drug/gene delivery

631

and tissue engineering. Int. J. Mol. Sci. 2013, 14, 1629-1654.

632

(3) Liu, Z.; Jiao, Y.; Wang, Y.; Zhou, C.; Zhang, Z. Polysaccharides-based

633

nanoparticles as drug delivery systems. Adv. Drug Del. Rev. 2008, 60, 1650-1662.

634

(4) Lohcharoenkal, W.; Wang, L.; Chen, Y. C.; Rojanasakul, Y. Protein nanoparticles

635

as drug delivery carriers for cancer therapy. Biomed Res Int. 2014, DOI:

636

10.1155/2014/180549.

637

(5) Chen, L.; Remondetto, G. E.; Subirade, M. Food protein-based materials as

638

nutraceutical delivery systems. Trends Food Sci. Technol. 2006, 17, 272-283.

639

(6) Paillard, A.; Passirani, C.; Saulnier, P.; Kroubi, M.; Garcion, E.; Benoit, J. P.;

640

Betbeder, D. Positively-charged, porous, polysaccharide nanoparticles loaded with

641

anionic molecules behave as 'stealth' cationic nanocarriers. Pharm. Res. 2010, 27,

642

126-133.

643

(7) Chen, L.; Subirade, M. Chitosan/β-lactoglobulin core-shell nanoparticles as

644

nutraceutical carriers. Biomaterials 2005, 26, 6041-6053.

645

(8) Gunasekaran, S.; Ko, S.; Xiao, L. Use of whey proteins for encapsulation and

646

controlled delivery applications. J. Food Eng. 2007, 83, 31-40.

30


Page 30 of 44

Page 31 of 44


647

(9) de Kruif, C. G.; Weinbreck, F.; de Vries, R. Complex coacervation of proteins and

648

anionic polysaccharides. Curr. Opin. Colloid Interface Sci. 2004, 9, 340-349.

649

(10) Shaw, K. L.; Grimsley, G. R.; Yakovlev, G. I.; Makarov, A. A.; Pace, C. N. The

650

effect of net charge on the solubility, activity, and stability of ribonuclease Sa. Protein

651

Sci. 2001, 10, 1206-1215.

652

(11) Jones,

653

protein-polysaccharide nanoparticle fabrication methods: impact of biopolymer

654

complexation before or after particle formation. J. Colloid Interface Sci. 2010, 344,

655

21-29.

656

(12) Jones, O. G.; Lesmes, U.; Dubin, P.; McClements, D. J. Effect of polysaccharide

657

charge on formation and properties of biopolymer nanoparticles created by heat

658

treatment of β-lactoglobulin–pectin complexes. Food Hydrocolloids 2010, 24,

659

374-383.

660

(13) Jones, O. G.; McClements, D. J. Biopolymer nanoparticles from heat-treated

661

electrostatic

662

characteristics. J. Food Sci. 2010, 75, N36-N43.

663

(14) Damodaran, S. Food proteins: an overview. In Food Proteins and Their

664

Applications, Damodaran, S.; Paraf, A., Eds.; Marcel Dekker Inc.: New York, 1997;

665

pp 1-24.

666

(15) Zhu, H.; Damodaran, S. Heat-induced conformational changes in whey protein

667

isolate and its relation to foaming properties. J. Agric. Food Chem. 1994, 42, 846-855.

668

(16) Jones, O. G.; McClements, D. J. Stability of biopolymer particles formed by heat

O.

G.;

Decker,

E.

protein-polysaccharide

A.;

McClements,

complexes:

D.

factors

31


J.

Comparison

affecting

of

particle


669

treatment of β-lactoglobulin/beet pectin electrostatic complexes. Food Biophys. 2008,

670

3, 191-197.

671

(17) Jones, O. G.; McClements, D. J. Recent progress in biopolymer nanoparticle and

672

microparticle

673

complexes. Adv. Colloid Interface Sci. 2011, 167, 49-62.

674

(18) Peinado, I.; Lesmes, U.; Andres, A.; McClements, J. D. Fabrication and

675

morphological characterization of biopolymer particles formed by electrostatic

676

complexation of heat treated lactoferrin and anionic polysaccharides. Langmuir 2010,

677

26, 9827-9834.

678

(19) Yu, S.; Hu, J.; Pan, X.; Yao, P.; Jiang, M. Stable and pH-sensitive nanogels

679

prepared by self-assembly of chitosan and ovalbumin. Langmuir 2006, 22,

680

2754-2759.

681

(20) Jones, O. G.; Decker, E. A.; McClements, D. J. Formation of biopolymer particles

682

by thermal treatment of β-lactoglobulin–pectin complexes. Food Hydrocolloids 2009,

683

23, 1312-1321.

684

(21) Sun, X. D.; Arntfield, S. D. Molecular forces involved in heat-induced pea

685

protein gelation: Effects of various reagents on the rheological properties of

686

salt-extracted pea protein gels. Food Hydrocolloids 2012, 28, 325-332.

687

(22) Jiménez-Castaño, L.; Villamiel, M.; López-Fandiño, R. Glycosylation of

688

individual whey proteins by Maillard reaction using dextran of different molecular

689

mass. Food Hydrocolloids 2007, 21, 433-443.

690

(23) Zhu, D.; Damodaran, S.; Lucey, J. A. Physicochemical and emulsifying properties

formation

by

heat-treating

electrostatic

32


protein-polysaccharide

Page 32 of 44

Page 33 of 44


691

of whey protein isolate (WPI)-dextran conjugates produced in aqueous solution. J.

692

Agric. Food Chem. 2010, 58, 2988-2994.

693

(24) Liu, G.; Zhong, Q. Glycation of whey protein to provide steric hindrance against

694

thermal aggregation. J. Agric. Food Chem. 2012, 60, 9754-9762.

695

(25) Liu, G.; Zhong, Q. Thermal aggregation properties of whey protein glycated with

696

various saccharides. Food Hydrocolloids 2013, 32, 87-96.

697

(26) Sun, W.; Yu, S.; Zeng, X.; Yang, X.; Jia, X. Properties of whey protein

698

isolate–dextran conjugate prepared using pulsed electric field. Food Res. Int. 2011, 44,

699

1052-1058.

700

(27) Li, J.; Yu, S.; Yao, P.; Jiang, M. Lysozyme-dextran core-shell nanogels prepared

701

via a green process. Langmuir 2008, 24, 3486-3492.

702

(28) Qi, J.; Yao, P.; He, F.; Yu, C.; Huang, C. Nanoparticles with dextran/chitosan shell

703

and BSA/chitosan core-doxorubicin loading and delivery. Int. J. Pharm. 2010, 393,

704

176-184.

705

(29) Sun, W.; Yu, S.; Yang, X.; Wang, J.; Zhang, J.; Zhang, Y.; Zheng, E. Study on the

706

rheological properties of heat-induced whey protein isolate–dextran conjugate gel.

707

Food Res. Int. 2011, 44, 3259-3263.

708

(30) Pan, X.; Yao, P.; Jiang, M. Simultaneous nanoparticle formation and

709

encapsulation driven by hydrophobic interaction of casein-graft-dextran and

710

β-carotene. J. Colloid Interface Sci. 2007, 315, 456-463.

711

(31) Dawlee, S.; Sugandhi, A.; Balakrishnan, B.; Labarre, D.; Jayakrishnan, A.

712

Oxidized chondroitin sulfate-cross-linked gelatin matrixes: a new class of hydrogels.

33



713

Biomacromolecules 2005, 6, 2040-2048.

714

(32) Lee, C. M.; Tanaka, T.; Murai, T.; Kondo, M.; Kimura, J.; Su, W.; Kitagawa, T.;

715

Ito, T.; Matsuda, H.; Miyasaka, M. Novel chondroitin sulfate-binding cationic

716

liposomes loaded with cisplatin efficiently suppress the local growth and liver

717

metastasis of tumor cells in vivo. Cancer Res. 2002, 62, 4282-4288.

718

(33) Johnson, E. J. A Biological Role of Lutein. Food Rev. Int. 2004, 20, 1-16.

719

(34) Hu, D.; Lin, C.; Liu, L.; Li, S.; Zhao, Y. Preparation, characterization, and in vitro

720

release investigation of lutein/zein nanoparticles via solution enhanced dispersion by

721

supercritical fluids. J. Food Eng. 2012, 109, 545-552.

722

(35) Tan, C.; Xia, S.; Xue, J.; Xie, J.; Feng, B.; Zhang, X. Liposomes as vehicles for

723

lutein: preparation, stability, liposomal membrane dynamics, and structure. J. Agric.

724

Food Chem. 2013, 61, 8175-8184.

725

(36) Zhu, D.; Damodaran, S.; Lucey, J. A. Formation of whey protein isolate

726

(WPI)-dextran conjugates in aqueous solutions. J. Agric. Food Chem. 2008, 56,

727

7113-7118.

728

(37) Zhang, S.; Zhang, Z.; Lin, M.; Vardhanabhuti, B. Raman spectroscopic

729

characterization of structural changes in heated whey protein isolate upon soluble

730

complex formation with pectin at near neutral pH. J. Agric. Food Chem. 2012, 60,

731

12029-12035.

732

(38) Deshiikan, S. R.; Papadopoulos, K. D. Modified Booth equation for the

733

calculation of zeta potential. Colloid Polym. Sci. 1998, 276, 117-124.

734

(39) Chakrabortee, S.; Tripathi, R.; Watson, M.; Schierle, G. S.; Kurniawan, D. P.;

34


Page 34 of 44

Page 35 of 44


735

Kaminski, C. F.; Wise, M. J.; Tunnacliffe, A. Intrinsically disordered proteins as

736

molecular shields. Mol. BioSyst. 2012, 8, 210-219.

737

(40) Santipanichwong, R.; Suphantharika, M.; Weiss, J.; McClements, D. J. Core-shell

738

biopolymer nanoparticles produced by electrostatic deposition of beet pectin onto

739

heat-denatured β-lactoglobulin aggregates. J. Food Sci. 2008, 73, N23-N30.

740

(41) Antonov, Y. A.; Zubova, O. M. Phase state of aqueous gelatin-polysaccharide

741

(1)-polysaccharide (2) systems. Int. J. Biol. Macromol. 2001, 29, 67-71.

742

(42) Murray, M. J.; Snowden, M. J. The preparation, characterization and applications

743

of colloidal Microgels. Adv. Colloid Interface Sci. 1995, 54, 73-91.

744

(43) Morais, M.; Subramanian, S.; Pandey, U.; Samuel, G.; Venkatesh, M.; Martins,

745

M.; Pereira, S.; Correia, J. D. G.; Santos, I. Mannosylated dextran derivatives labeled

746

with fac-[M(CO)3]+ (M =

747

Mol. Pharm. 2011, 8, 609-620.

748

(44) Chanasattru, W.; Jones, O. G.; Decker, E. A.; McClements, D. J. Impact of

749

cosolvents on formation and properties of biopolymer nanoparticles formed by heat

750

treatment of β-lactoglobulin–pectin complexes. Food Hydrocolloids 2009, 23,

751

2450-2457.

752

(45) Markman, G.; Livney, Y. D. Maillard-conjugate based core-shell co-assemblies

753

for nanoencapsulation of hydrophobic nutraceuticals in clear beverages. Food Funct.

754

2012, 3, 262-270.

755

(46) Teng, Z.; Li, Y.; Luo, Y.; Zhang, B.; Wang, Q. Cationic beta-lactoglobulin

756

nanoparticles as a bioavailability enhancer: protein characterization and particle

99m

Tc, Re) for specific targeting of sentinel lymph node.

35



757

formation. Biomacromolecules 2013, 14, 2848-2856.

758

(47) Lerch, S.; Dass, M.; Musyanovych, A.; Landfester, K.; Mailander, V. Polymeric

759

nanoparticles of different sizes overcome the cell membrane barrier. Eur. J. Pharm.

760

Biopharm. 2013, 84, 265-274.

761

(48) Mishra, V.; Mahor, S.; Rawat, A.; Gupta, P. N.; Dubey, P.; Khatri, K.; Vyas, S. P.

762

Targeted brain delivery of AZT via transferrin anchored pegylated albumin

763

nanoparticles. J. Drug Target. 2006, 14, 45-53.

764

(49) Hamidi, M.; Azadi, A.; Rafiei, P. Hydrogel nanoparticles in drug delivery. Adv.

765

Drug Del. Rev. 2008, 60, 1638-1649.

766

(50) Giroux, H. J.; Houde, J.; Britten, M. Preparation of nanoparticles from denatured

767

whey protein by pH-cycling treatment. Food Hydrocolloids 2010, 24, 341-346.

36


Page 36 of 44

Page 37 of 44


768

Figure Captions

769

Figure 1. Effect of pH on turbidity (A), hydrodynamic diameter (Dh) (B),

770

polydispersity index (PDI) (C) of WPI/polysaccharide suspensions, and effect of pH

771

on ζ-potential (D) of WPI/polysaccharide suspensions and WPI or polysaccharide

772

solutions.

773

WPI/polysaccharide mixtures at pH 5.2 and 85 °C for 15 min. WPI or polysaccharide

774

solutions were prepared by dissolving WPI or polysaccharide in deionized water.

775

Figure 2. Effect of pH on turbidity (A), hydrodynamic diameter (Dh) (B),

776

polydispersity index (PDI) (C), and ζ-potential (D) of WPI-dextran conjugate 2 (9.7%

777

degree of glycosylation) and WPI-dextran conjugate/ChS suspensions. Suspensions

778

were prepared by heating WPI-dextran conjugate 2 solutions or WPI-dextran

779

conjugate/ChS electrostatic complexes at pH 5.2 and 85 °C for 15 min. The legends

780

are as follows: Conjugate 1, WPI-dextran conjugate with 5.2% degree of

781

glycosylation; Conjugate 2, WPI-dextran conjugate with 9.7% degree of glycosylation;

782

Conjugate 3, WPI-dextran conjugate with 12.2% degree of glycosylation.

783

Figure 3. Effect of pH and 200 mM NaCl on turbidity (A), hydrodynamic diameter

784

(Dh) (B), and polydispersity index (PDI) (C) of WPI-dextran conjugate 2 (9.7%

785

degree of glycosylation) and WPI-dextran conjugate/ChS suspensions. Suspensions

786

were prepared by heating WPI-dextran conjugate 2 solutions or WPI-dextran

787

conjugate/ChS electrostatic complexes at pH 5.2 and 85 °C for 15 min. The legends

788

are as follows: Conjugate 1, WPI-dextran conjugate with 5.2% degree of

789

glycosylation; Conjugate 2, WPI-dextran conjugate with 9.7% degree of glycosylation;

WPI/polysaccharide

suspensions

were

37


prepared

by

heating


Page 38 of 44

790

Conjugate 3, WPI-dextran conjugate with 12.2% degree of glycosylation.

791

Figure 4. Effect of pH on turbidity and hydrodynamic diameter (Dh) of

792

lutein-unloaded and lutein-loaded nanoparticle suspensions (A), effect of pH on

793

ζ-potential of lutein-unloaded and lutein-loaded nanoparticle suspensions (B), and

794

effect of pH and 200 mM NaCl on turbidity and hydrodynamic diameter (Dh) of

795

lutein-unloaded and lutein-loaded nanoparticle suspensions (C). Suspensions were

796

prepared by heating WPI-dextran conjugate 2 (9.7% DG)/ChS electrostatic complexes

797

at pH 5.2 and 85 °C for 15 min, and then lutein-unloaded and lutein-loaded

798

nanoparticle suspensions were evaporated by a rotary evaporator and subjected to

799

ultrasonic treatment.

800

Figure 5. Particle size distribution of lutein-unloaded (A-1) and lutein-loaded (B-1)

801

WPI-dextran

802

lutein-unloaded (A-2) and lutein-loaded (B-2) WPI-dextran conjugate 2 (9.7%

803

DG)/ChS nanoparticles, AFM images of lutein-unloaded (A-3) and lutein-loaded (B-3)

804

WPI-dextran conjugate 2 (9.7% DG)/ChS nanoparticles, SEM images of

805

lutein-unloaded (A-4) and lutein-loaded (B-4) WPI-dextran conjugate 2 (9.7%

806

DG)/ChS nanoparticles. The inserts are direct images of lutein-unloaded and

807

lutein-loaded WPI-dextran conjugate 2 (9.7% DG)/ChS suspensions.

conjugate

2

(9.7%

DG)/ChS

suspensions,

38


TEM

images

of

Page 39 of 44


Figure 1

39



Figure 2

40


Page 40 of 44

Page 41 of 44


Figure 3

41



Figure 4

42


Page 42 of 44

Page 43 of 44


Figure 5

43



Table of Contents (TOC)

44


Page 44 of 44

Stable Nanoparticles Prepared by Heating Electrostatic Complexes of

Recommend Documents