Development of nanocomplexes for curcumin vehiculization using

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Development of nanocomplexes for curcumin vehiculization using ovalbumin and sodium alginate as building blocks: Improved stability, bioaccessibility, and antioxidant activity Jin Feng, Huiqing Xu, Lixia Zhang, Hua Wang, Songbai Liu, Yujiao Liu, Wanwei Hou, and Chunyang Li J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b02567 • Publication Date (Web): 19 Dec 2018 Downloaded from http://pubs.acs.org on December 19, 2018

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Journal of Agricultural and Food Chemistry

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Development of nanocomplexes for curcumin vehiculization using

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ovalbumin and sodium alginate as building blocks: Improved

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stability, bioaccessibility, and antioxidant activity

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Jin Feng†*, Huiqing Xu†, Lixia Zhang†, Hua Wang‡, Songbai Liu‡, Yujiao

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Liu§, Wanwei Hou§, and Chunyang Li†*

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†Department

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Processing, Jiangsu Academy of Agricultural Sciences, 50 Zhongling Street, Nanjing

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210014, China

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‡Department

of Functional Food and Bio-active compounds, Institute of Agro-product

of Food Science and Nutrition, Fuli Institute of Food Science, Zhejiang

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Key Laboratory for Agro-Food Processing, Zhejiang R&D Center for Food Technology

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and Equipment, Zhejiang University, 866 Yuhangtang Road, Hangzhou 310058, China

12

§

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Xining 810016, China

Academy of Agriculture and Forestry Science, Qinghai University, 251 Ningda Road,

ACS Paragon Plus Environment


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ABSTRACT: Type I (Complex I) and type II nanocomplexes (Complex II) were

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created in this work for curcumin (Cur) delivery using ovalbumin (OVA, 1.0% w/w)

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and sodium alginate (ALG, 0.5% w/w) as building blocks. OVA was heated at 90 °C

17

for 5 min at pH 7.0 and then coated with ALG at pH 4.2 to produce Complex I; OVA–

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ALG electrostatic complex was created at pH 4.0, which was treated at 90 °C for 5 min

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thereafter yielding Complex II. Complex I presented an irregular elliptical shape with

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a diameter of ~ 250 nm, whereas Complex II adopted a defined spherical structure of a

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smaller size (~ 200 nm). Complex II did not dissociate at the pH range of 5–7, which

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was different from Complex I. Cur was loaded into the non–polar matrix of

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nanocomplexes through hydrogen bonding and hydrophobic interactions, and Complex

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II displayed a higher loading capacity than Complex I. Nanocomplexes were resistant

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to pepsinolysis during simulated gastrointestinal digestion, which enhanced the stability

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and controlled release of loaded Cur, thereby improving Cur bioaccessibility from ~ 20%

27

(free form) to ~ 60%. Additionally, nanocomplexes contributed to the cellular

28

antioxidant activity (CAA) of Cur by promoting its cellular uptake. The CAA of Cur

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was also better preserved in nanocomplexes especially in Complex II after digestion

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owing to the increased stability and bioaccessibility. Results from this work highlighted

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the effect of nanocomplex encapsulation on the performance of Cur and revealed the

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critical role of preparation method in the physicochemical attributes of nanocomplexes.

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KEYWORDS: ovalbumin–sodium alginate nanocomplexes; curcumin; simulated

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gastrointestinal digestion; proteolysis; cellular antioxidant activity


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INTRODUCTION

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Curcumin (Cur) is a hydrophobic polyphenol extracted from the powdered rhizomes

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of turmeric spices. This compound is traditionally used as a spice and food-coloring

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reagent and displays various health-benefiting attributes ranging from anti-oxidant,

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anti-inflammatory, anti-carcinogenic, anti-obesity, anti-diabetes, and antitumor

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activities.1 Nevertheless, the solubility of Cur is low (~ 11 ng/mL) and it readily

41

undergoes

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bioavailability in vivo.2 Constructing nanocomplexes using natural biopolymers, such

43

as proteins and polysaccharides, as building blocks for Cur delivery has been reported

44

to be an effective way to improve its bioavailability and preserve its biological

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activities.3-5 In such vehicles, proteins usually serve as a cargo space for Cur binding,

46

whereas the polysaccharide segments are expected to improve the stability and

47

digestive resistance of nanocomplexes by adding repulsive electrostatic and/or steric

48

forces.

degradation

under

physiological

conditions,

which

restricts

its

49

Nanocomplexes with different composites, structures, and dimensions were created

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depending on the nature of biopolymers and assembly principle utilized.6 During the

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preparation of nanocomplexes, proteins are usually thermally treated to improve their

52

surface hydrophobicity, which is favorable for the binding of lipophilic molecules.7,8

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Polysaccharide can be introduced to interact with proteins through electrostatic and

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hydrophobic forces either before or after thermal treatment. For example, in previous

55

works, protein nanoparticles were fabricated by heating lactoferrin (LF) or soy protein

56

isolates (SPI) above their thermal denaturation temperature, which were then coated

57

with polysaccharides under acidic conditions to produce type I nanocomplexes.9-11



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Besides, nanocomplexes can also be fabricated via heating protein/polysaccharide

59

mixtures (e.g., β-lactoglubulin (BLG)/pectin and low-density lipoprotein (LDL)/pectin)

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above the thermal denaturation temperature of protein under pH conditions where they

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are weakly electrically attracted to each other (type II nanocomplexes).12-14 The

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formation of this type of nanocomplexes can be interpreted in terms of nucleation and

63

growth mechanism.15 In the work of Jones et al., both type I and type II nanocomplexes

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were created utilizing BLG/pectin combination as building blocks.15 They

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demonstrated that type II nanocomplex presented a higher surface charge and better

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stability against aggregation under acidic and high ionic strength circumstance

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compared with type I nanocomplex. This result suggests that, although the overall

68

biopolymer compositions were similar, the physicochemical properties of these two

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types of nanocomplexes are different, which would affect the bioavailability and

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functionality of loaded nutraceuticals. However, comparisons on the effect of

71

encapsulation by each type of nanocomplex on the performance of nutraceuticals

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especially Cur have not been reported.

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Ovalbumin (OVA) is a globular monomeric phosphoglycoprotein of 42–47 kDa

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molecular weight; it is the main component of egg white protein.8 OVA has been widely

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utilized to prepared nanoparticles or nano–emulsion for the delivery of bioactive

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compounds, such as Cur,8 polyunsaturated fatty acids,16,17 and retinol,18 to improve their

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water–solubility, stability, and antioxidant activity. In recent works, sodium alginate

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(ALG), an anionic polysaccharide extracted from the cell walls of seaweeds,19 has been

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proven to effectively improve the stability of protein or protein-stabilized vehicles20


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and retard their hydrolysis during digestion.21 We speculate that stable nanocomplexes

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with excellent biocompatibility and biodegradability can be created using OVA–ALG

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combination as building blocks because they are natural biopolymers.

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In this work, both type I (Complex I) and type II OVA–ALG nanocomplexes

84

(Complex II) were fabricated, and the effects of these two types of nanocomplexes on

85

the performance of loaded Cur were compared. The interaction between entrapped Cur

86

and Complex I or Complex II were investigated by a combination of FT-IR and steady-

87

state fluorescence study. The proteolysis of delivery systems, stability of encapsulated

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Cur, and the portion of Cur solubilized in mixed micelles that can be absorbed by small

89

intestine epithelium (i.e., bioaccessibility of Cur) were monitored during the simulated

90

digestion. Besides, the effect nanocomplex encapsulation on the antioxidant activity of

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Cur was evaluated on a HepG2 model as Cur offers many health-benefiting effects

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through antioxidant mechanisms.22 OVA nanoparticles (ONP) created by heating OVA

93

at neutral condition were also included in the present work as a control. Information

94

from this work will provide some insights into the application of biopolymer

95

nanocomplexes in the delivery of bioactive compounds.

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MATERIALS AND METHODS

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Materials. Cur (a mixture of Cur, demethoxycurcumin, and bisdemethoxylcurcumin;

98

curcuminoid content ≥ 94%; Cur content ≥ 80%), ALG (from brown algae), bile salts

99

(for microbiology, cholic acid sodium salt ≈ 50%; deoxycholic acid sodium salt ≈ 50%),

100

and 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide (MTT, 98%) were

101

purchased from Sigma–Aldrich Corp. (St. Louis, USA). OVA (from egg white,



102

purity >80%), 2,2ʹ-azobis(2-amidinopropane) dihydrochloride (ABAP, 97%), 2ʹ,7ʹ-

103

dichlorofluorescin diacetate (DCFH-DA, ≥ 97%), pepsin (activity: 3000–3500 U mg-

104

1),

105

U mg-1), and lipase (activity: 56 U mg-1) were purchased from Sangon Biotech Co., Ltd.

106

(Shanghai, China). All other chemicals were of analytical grade and used as purchased.

107

and pancreatin consisting of protease (activity: 285 U mg-1), amylase (activity: 288

Biopolymer solution preparation. Stock solutions of OVA (5.0% w/w) or ALG (4.0%

108

w/w) were prepared by dispersing powdered sample in a 10 mM phosphate buffered

109

saline (PBS, pH 7.0) for 5 h under mild magnetic stirring (RCT 5 digital, IKA-Werk

110

Co., Staufen, Germany). ALG solution was also homogenized with a T-25 blender

111

(IKA-Werk Co., Staufen, Germany) for 1–2 min because of its high viscosity. Stock

112

solutions were then kept overnight at 4 °C to ensure complete hydration. Biopolymer

113

solutions with lower concentrations were obtained by diluting the stock solution with

114

the same buffer.

115

OVA–ALG nanocomplexes fabrication.

116

ONP. A stock solution of OVA was diluted with PBS (pH 7.0, 10 mM) to reach a

117

final concentration of 2.0% (w/w). Thereafter, 40 mL of the diluted solution was

118

transferred into a plastic tube, which was then heated at 90 °C for 5 min in a MS-100

119

incubator (Allsheng Instruments Co., Hangzhou, China) and cooled to room

120

temperature in an ice bath. The Z-average diameter (DZ), polydispersity index (PDI),

121

and ζ-potential of ONP at pH 7.0 were determined to be 107.4 nm, 0.279, and -16.9

122

mV, respectively.

123

Complex I. ONP suspension mentioned above was titrated into an ALG solution


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under magnetic stirring to reach a final protein of 1.0% (w/w) and ALG concentration

125

of 0.5% (w/w). The mixture was then adjusted to pH 4.2 with HCl solution (1 M) and

126

left overnight at 4 °C to promote the adsorption of ALG onto ONP surface.

127

Complex II. Mixed OVA–ALG solution with an 1.0% OVA concentration and 0.5%

128

ALG concentration was adjusted to pH 4.0 using a 1 M HCl solution to produce

129

electrostatic complexes. Thereafter, 40 mL of the mixed solution was transferred into a

130

plastic tube, which was then heated at 90 °C for 5 min and cooled to room temperature

131

in an ice bath.

132 133

Schematic illustration for the preparation of ONP, Complex I, and Complex II are presented in Figure 1.

134

Particle size and surface charge. The DZ, PDI, count rate, and ζ-potential of three

135

delivery systems were determined using a commercial Nano-ZS 90 zeta-sizer (Malvern

136

Instrument Ltd., Malvern, United Kingdom) at 25 °C with a He/Ne laser (λ = 633 nm)

137

and 90° scattering angle. Samples were properly diluted to avoid multiple scattering

138

phenomena. Each parameter was calculated as the average of at least triplicate

139

measurements, and each measurement was obtained from the mean of at least 10

140

readings for a sample.

141

TEM observation. The morphology of three delivery systems was characterized by

142

TEM observation. One drop of dispersion was placed on a carbon Formvar-coated

143

copper grid (200 mesh) for 5 min, after which the excess solution was wiped away with

144

filter paper to form a thin liquid film layer in the copper grid. Thereafter, one drop of

145

aqueous uranyl acetate (UA) or phosphotungstic acid (PA) was placed on the copper



146

grid, and the excess liquid was also removed with filter paper. The stained samples

147

were dried in air before being observed by a JEM-1230 (HR) transmission electron

148

microscope (Jeol Ltd., Tokyo, Japan) at a working voltage of 200 kV.

149

Cur loading. Before thermal treatment, the stock solution of Cur (4.0 mg/mL in

150

ethanol) was titrated into protein solution under magnetic stirring to reach a mass ratio

151

of Cur to OVA 1:20. The mixture was stirred at room temperature for another 2 h, and

152

Cur-loaded ONP or nanocomplexes were fabricated as described above. The remaining

153

ethanol in suspension was evaporated under reduced pressure. The colloidal systems

154

were then centrifuged at 1000 g for 15 min to remove free Cur crystals. The

155

encapsulated Cur was extracted by chloroform and diluted properly to be analyzed by

156

a UV-VIS spectrophotometer at 419 nm according to our previous report.3 The

157

encapsulation efficiency (EE) and loading content (LC) of Cur were respectively

158

calculated using the following equations:

159

EE (% ) =

160

and

161

LC (% ) =

Encapsulated amount of Cur Total amount of Cur

Encapsulated amount of Cur Total amount of biopolymers in the delivery system

.

162

FT-IR spectra. The FT-IR spectra of empty delivery systems and those loaded with

163

Cur were recorded on a Tensor 27 instrument (Bruker Co., Karlsruhe, Germany) using

164

a KBr disk with 1% finely ground samples. The spectra were acquired in the range of

165

400–4000 cm-1 at a resolution of 4 cm-1 and analyzed using an OMNIC software version

166

8.0.

167

Steady-state fluorescence analysis. Fluorescence emission spectra were recorded on


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a Cary Eclipse fluorescence spectrometer (Agilent technologies Co., Santa Clara, USA)

169

at 25 °C. Intrinsic fluorescence spectra: samples were diluted with 10 mM PBS (pH

170

7.0) to reach a final protein concentration of 2.0 mg mL-1, excitation was performed at

171

295 nm, and the emission spectra were scanned over the range of 300–450 nm with an

172

excitation slit width of 2.5 nm and emission slit width of 5 nm. Fluorescence spectra

173

of free and encapsulated Cur: samples were diluted with the same buffer to reach a

174

final Cur concentration of 50 µg mL-1, excitation was conducted at 420 nm, and

175

emission spectra were scanned over the range of 450–750 nm with an excitation slit

176

width of 5 nm and emission slit width of 10 nm.

177

Simulated digestion. An in vitro gastrointestinal model was utilized to investigate

178

the digestion of delivery systems and the effect of nano-encapsulation on the stability

179

and bioaccessibility of Cur according to our previous work3 with a few modifications.

180

In brief, an aliquot (100 mL) of ONP, Complex I, or Complex II encapsulating Cur with

181

a protein concentration of 10 mg/mL was incubated at 37 °C for 10 min and then mixed

182

with preheated simulated gastric fluids (prepared by mixing 2 g NaCl, 7 mL HCl and

183

3.2 g pepsin to a flask and then adding ultrapure water to 1 L) at a 1:1 mass ratio. The

184

mixture was then adjusted to pH 3.0 and placed in a shaking incubator at 100 rpm and

185

37 °C to mimic gastric digestion. After 60 min, the chyme collected from gastric phase

186

was mixed with an equal volume of simulated intestinal fluid containing 0.30 mM

187

CaCl2, 30.72 mM NaCl, 5 mg mL-1 bile salts, and 8 mg mL-1 pancreatin. The resultant

188

mixture was adjusted to pH 7.0 and then shook continuously at 370 rpm and 37 °C for

189

2 h to mimic intestinal digestion. For free Cur, 5 mL of the Cur stock solution (4 mg



190

mL-1) was dispersed in 95 mL of ultrapure water and subjected to the same digestion

191

process as mentioned above.

192

The proteolysis of delivery systems was characterized by sodium dodecyl sulphate–

193

polyacrylamide gel electrophoresis (SDS-PAGE) and trichloroacetic acid (TCA)-

194

soluble nitrogen analysis. SDS-PAGE analysis was carried out with an 8% acrylamide

195

separating gel and a 5% stacking gel. An aliquot (10 µL) of sample was obtained from

196

digesta at a specific digestion time and immediately mixed with 5 µL of ultrapure water

197

and 10 µL of 2 × SDS sample buffer (pH 6.8, containing 100 mM Tris, 4% SDS, 0.2%

198

bromophenol blue, 20% glycerol, and 200 mM 2-mercaptoehanol) and then heated in

199

boiling water for 10 min. A total of 20 µL of each sample was loaded to designated

200

wells for electrophoresis at 100 mV with a Tris-HEPES running buffer (pH 8.0,

201

containing 100 mM Tris, 100 mM HEPES and 3 mM SDS). The gels were stained with

202

Coomassie Brilliant Blue R-250 and destained in an ethanol/acetic acid/water (3:1:6)

203

solution for 24 h. For TCA-soluble nitrogen analysis, 2 mL of sample was obtained

204

from digesta at specific digestion times and immediately mixed with 1 mL of 20% TCA

205

solutions to precipitate OVA or OVA fragments with high molecular mass.4,7 The

206

mixture was placed at room temperature for 30 min and then centrifuged at 5000 g for

207

10 min. The nitrogen content in resultant supernatants was determined by the micro-

208

Kjeldahl method, and the percentage of TCA-soluble nitrogen was calculated from the

209

following equation:

210

TCA - soluble nitrogen (%) =

211

amount of nitrogen in the supernatant × 100. total amount of nitrogen

During simulated digestion, 1 mL of the sample was obtained from the digesta at


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specific digestion times, and Cur was extracted and measured with a UV-visible

213

spectrometer. The retention rate of Cur was calculated using the following equation:

214

Retention rate (%) =

215

where Cdigesta refers to the Cur concentration in the overall digesta; Cinitial denotes the

216

Cur concentration before simulated digestion.

Cdigesta Cinitial

× 100.

217

After the simulated digestion, 30 mL of raw digesta from each sample was

218

centrifuged at 15600 g and 4 °C for 10 min. The digesta was separated into a clear

219

micelle phase at the top and a sediment phase at the bottom. The Cur content in both

220

phases was measured, and the bioaccessibility of Cur was calculated using the

221

following equation:3

222

Bioaccessibility (%) =

223

where Cdigesta and Cmicelle represent the concentrations of Cur in the overall digesta and

224

micelle fraction after simulated digestion, respectively.

Cmicelle Cdigesta

× 100.

225

Cell culture and cytotoxicity assay. The HepG2 cells was cultured in Dulbecco’s

226

Modified Eagle Medium (DMEM) (supplemented with 10% fetal bovine serum (FBS),

227

1% penicillin–streptomycin, and 1% HEPES buffer) at 37 °C and 5% CO2 atmosphere.

228

The MTT test was used to evaluate the cytotoxicity of free or encapsulated Cur on

229

HepG2 cells. After incubation in a 96-well plate (6 × 105 cells per well) for 24 h, the

230

cells were cultured with free or encapsulated Cur of different concentrations (0.5, 1.0,

231

5.0, 10.0, 20.0 and 100 µM) for 12 h. An aliquot (10 µL) of MTT solution with a

232

concentration of 5 mg mL-1 was then added to each well, and the plate was incubated

233

for an additional 4 h. Formazan crystals formed by active cells were dissolved with 100



234

µL of dimethyl sulfoxide, and the absorbance at 570 nm was measured. Cell viability

235

was calculated by the following equation:

236

cell viability (%) =

237

where Asample and Acontrol refer to the absorbance of wells treated with and without Cur,

238

respectively.

Asample Acontrol

× 100%.

239

Cell antioxidant activity (CAA). The CAA test of free and encapsulated Cur was

240

carried out according to the method of Wolfe and Liu (2007) with slight modification.23

241

The HepG2 cells were seeded on a transparent 96-well plate with a density of 6 × 105

242

cells per well. After 24 h of incubation, DMEM was removed, and the HepG2 cells

243

were rinsed with PBS. Triplicate wells were treated with 100 µL of free or encapsulated

244

Cur in FBS–free DEME at Cur concentrations of 1, 2.5, 5, 10, 20, and 50 µM. DCFH-

245

DA with a final concentration of 25 µM was then added, and the cells were cultured for

246

1 h. Afterward, HepG2 cells were rinsed thrice with PBS, and 100 µL of ABAP (600

247

µM) was added to each well. The 96-well plate was read immediately by a SpectraMax

248

M2e reader (Molecular Devices, California, USA) at 37 °C for 1 h with a time interval

249

of 5 min. Excitation and emission wavelengths were set at 485 and 538 nm, respectively.

250

Triplicate control wells treated with DCFH-DA and ABAP were included in each plate.

251

The CAA unit of each antioxidant was calculated as follows:

252

CAA unit = 100 - (∫SA/∫CA ) × 100.

253

where ∫SA denotes the integrated area under the sample fluorescence versus time

254

curve and ∫CA is the integrated area from the control curve. Log (fa/fu) (fa = CAA, fu

255

= 100 – CAA) was further plotted against log (Cur concentration), and the medium


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effective dose (EC50) of free and encapsulated Cur was determined from the plot of log

257

(fa/fu) = log 1 = 0.

258

Cellular uptake of Cur. HepG2 cells were seeded in a 6-well plate at a density of 6

259

× 105 cells per well. After reaching ~ 90% confluence, they are treated with free or

260

encapsulated Cur (20 or 50 μM) in FBS–free DMEM for 1 h. Cells were then washed

261

with 4 °C PBS three times to remove any residual Cur, and their fluorescent images

262

were taken with a confocal laser scanning microscopy (CLSM, Olympus, Japan).

263

Afterward, cells were lysed and the total protein content in each well were determined

264

using the BCA protein assay kit. Cur in HepG2 cells was extracted with 1 mL of ethyl

265

acetate under sonication. The collected organic phase was evaporated to dryness and

266

dissolved in acetonitrile for Cur analysis. Cur extracted from HepG2 cells was

267

determined by HPLC rather than UV-VIS spectrophotometer owing to its low detection

268

limit. The result was expressed as nmol Cur/mg cellular protein.

269

HPLC analysis. An Agilent 1290 Infinity LC fitted with a Zorbax eclipse analytical

270

XDB-C18 column (4.6 × 150 mm, 5 μM) was utilized in this work. Mobile phase

271

solvents were 2.0% acetic acid in water (A) and acetonitrile (B). Gradient elution was

272

applied by varying the proportion of the mobile phases. The initial composition of the

273

mobile phases, consisting of 60% A and 40% B, was held for 5 min. Phase B was then

274

increased linearly to 70% at 22 min, kept constant at 70% for 2 min, and decreased

275

linearly to 40% at 25 min. The flow rate was set at 1.0 mL/min and the detection

276

wavelength was 420 nm. A calibration curve with acceptable linearity (R2 = 0.9987)

277

was constructed by plotting the peak area versus Cur concentration (15–1000 ng/mL).



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278

Statistical analysis. All tests were performed in triplicate, and the data were

279

presented as means ± standard deviation. The results were subjected to least significant

280

difference and one-way analysis of variance using PASW Statistics 18 software to

281

analyze the differences. Differences with a P value of < 0.05 were considered

282

significant.

283

CHARACTERIZATION OF NANOCOMPLEXES

284

TEM observation. As illustrated in Figure 2a, native OVA molecules did not self-

285

assembly to produce nanoparticles at pH 7.0. They aggregated slightly owing to the

286

drying process before observation. When heated at 90 °C, the tertiary structure of OVA

287

dissociated and the interior non–polar amino acid resides shifted onto the surface of

288

protein,

289

formation/interchange between OVA molecules.3 Therefore, the denatured OVA

290

molecules interacted with each other leading to the formation of nanoparticles (ONP)

291

with a diameter of ~ 100 nm (Figure 2b), which is in consistence with our previous

292

work.3 Complex I presents an irregular elliptical shape with a larger particle size (DZ:

293

254.38 nm) and higher magnitude of ζ-potential (-31.07 mV) than ONP (Figure 2c),

294

which may be attributed to the aggregation of nanoparticles and electrostatic deposition

295

of ALG chains onto particle surface during preparation. By contrast, the shape of

296

Complex II is spherical with defined edges (Figure 2d). Besides, Complex II displays

297

a smaller particle size but higher magnitude of surface charge compared with Complex

298

I. These results suggest that, thought with the same biopolymer composition, the

299

structure and matrix biopolymer distribution between Complex I and Complex II are

which

promoted

the

hydrophobic

interactions


and

disulfide

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300

different. On the other hand, the PDI values of Complex I and II are below 0.3, and

301

their size distributions are narrow and homogenous (Figure S1), suggesting their

302

potential application in the delivery of bioactive compounds.

303

pH stability. The stability of delivery systems (ONP and Complex I and II) upon pH

304

change was further investigated. The surface charges on ONP shift from positive at pH

305

3.0 to near zero at ~ pH 4.5 (pI), and become more negative at higher pH values (Figure

306

3b). Extensive aggregation of ONP occurs between pH 3.5 and 5.5 owing to the loss of

307

electrostatic repulsion as evidenced by the dramatic increase in particle size (Figure 4a)

308

and subsequent phase separation. By contrast, Complex I and II are negatively charged

309

and show no phase separation throughout the studied pH range. The capping

310

polysaccharides contribute to the stability of nanocomplexes at the pH range around pI

311

by adding electrostatic and steric repulsion.18 The particle size of Complex I or II is

312

nearly doubled and accompanied by an appreciable decrease in the magnitude of ζ-

313

potential when pH alters from 4.0 to 3.0; this result originates from the enhanced

314

protonation of carboxyl groups on ALG and NH2 groups on OVA.

315

The diameter of Complex I in the pH range of 3.0–5.0 is ~ 330 nm, which decreases

316

remarkably to ~ 150 nm when pH increases from 5.0 to 7.0 (Figure 3a). The protein

317

portion of Complex I is unexpected to bind tightly with ALG at pH values above 5.0

318

because they are both negatively charged. The negative ζ-potential of Complex I

319

decreases gradually from -40.9 to -24.5 mV when pH changes from 4.0 to 7.0 (Figure

320

4b). The decrease in particle size and magnitude of ζ-potential of Complex I with pH

321

reveals the dissociation of its structure; this result is attributed to the enhanced repulsive



322

electrostatic forces between the protein core and exterior polysaccharide layer. The DZ

323

and ζ-potential of Complex I at pH 7.0 are very similar with those of ONP, suggesting

324

that the solution of Complex I consists mainly of individual ONP and free ALG chains.

325

Nevertheless, it has been demonstrated that the structure of nanocomplexes fabricated

326

by mixing thermally denatured LF with pectin remained intact across a wide pH range

327

of 2.0–7.0. This discrepancy may arise from the higher pI of LF (~ 8.5) in comparison

328

with that of OVA. LF and pectin were therefore tightly associated with each other

329

through electrostatic attraction at the studied pH range.10

330

By contrast, both the particle size and magnitude of ζ-potential of Complex II increase

331

gradually with pH in the range of 4.0–7.0, indicating the different biopolymer

332

arrangement within Complex I and II. Complex II was formed by heating OVA and

333

ALG together, resulting in homogenous distribution of protein molecules and

334

polysaccharide chains.12 During ripening of the nanocomplex, the electrostatic bonds

335

between OVA and ALG continuously broke down and reformed in a reversible

336

manner.24 The OVA clusters can therefore move independently along the ALG chains

337

toward the interior of particles owing to their high surface hydrophobicity. Meanwhile,

338

ALG chains preferentially located at the particle surface with an adequate segment

339

extending into solution and the remaining segment anchored in the matrix.25 Complex

340

II therefore adopted a more compact microstructure than Complex I because ALG

341

chains were partly entrapped and fixed in the inner protein core. Dai et al. suggested

342

that the structure integrity of Complex II is primarily maintained by hydrophobic

343

interactions rather than ionic bonds, although they contribute significantly to the initial


Page 16 of 48

Page 17 of 48


344

structure development during thermal treatment.26 Therefore, the structure of Complex

345

II unlikely undergoes dissociation upon pH change. Complex II swells remarkably

346

when pH increases from 4.0 to 7.0 (Figure 3a) due to the enhanced electrostatic

347

repulsion between composite biopolymers.27 Similar results have also been observed in

348

Complex II prepared with egg yolk LDL/carboxymethyl cellulose combination.28

349

Nevertheless, contrasting evidence proved that nanocomplex formed by heating β-lg

350

and pectin together dissociated to a certain extent at neutral conditions.12 This

351

discrepancy suggests that the structure of Complex II is closely correlated with the

352

physicochemical properties of biopolymers.

353

The negative ζ-potential of Complex II increases with pH (Figure 3b) owing to the

354

enhanced deprotonation of carboxyl groups on ALG and NH3+ groups on OVA. The

355

surface charge of Complex II is highly similar with that of ALG in the studied pH range

356

(data not shown), suggesting that the electrical properties of Complex II are largely

357

determined by the exterior polysaccharide layer.

358

CUR INCORPORATION

359

EE and LC. The EE and LC of Cur in three delivery systems decrease with the

360

following trend: Complex II > Complex I ≈

ONP (Figure 4a). Results have

361

demonstrated that the Cur-loading capacity of the proteins is highly dependent on their

362

surface hydrophobicity and specific surface area.8 When preparing Complex II, OVA–

363

ALG electrostatic complex is fabricated at first, which significantly inhibits the heat-

364

induced aggregation of OVA molecules by blocking their hydrophobic interactions.12

365

In this sense, more hydrophobic binding sites are accessible for Cur in Complex II. By



366

contrast, in the absence of polysaccharides, OVA molecules readily aggregate during

367

thermal treatment owing to the weaker electrostatic repulsion, which therefore produces

368

minor specific surface area for Cur binding.8 The binding capacity of the delivery

369

systems is also supposed to feature a close relationship with the tertiary conformation

370

of proteins.29 The three delivery systems experience a slight increase in particle size

371

after Cur incorporation (Figure 4b). On the other hand, the PDI and ζ-potential of ONP,

372

Complex I, and Complex II are maintained (data not shown). These results indicte that

373

Cur was loaded into the mesh space between the hydrogel networks of the delivery

374

systems without changing their structure, except causing particle expansion by about

375

20–36 nm. Similar results were reported by Zhou et al., who showed that the dimension

376

of LDL/pectin nanocomplexes increased slightly after Cur incorporation.14

377

FT-IR analysis. FT-IR analysis was performed to reveal the type of interactions that

378

occurred in assembled systems containing OVA, ALG, and Cur. As depicted in Figure

379

5, native OVA displayed typical bands centered at 3274.5 (amide A, attributed to O-H

380

stretching vibration of hydroxyl-bound water), 2959.2 (amide B, attributed to CH2

381

asymmetric stretching vibration), 1632.4 (amide I, attributed to C=O stretching

382

vibration of peptide linkage), and 1520.7 cm-1 (amide II, attributed to C-N stretching

383

vibration and N-H bending vibration).17 The intermolecular interactions during the

384

formation of ONP or nanocomplexes can be interpreted by several major changes in

385

the FT-IR spectra. First, the amide A band becomes flattened and shifts to a lower

386

wavenumber, suggesting the formation of strong hydrogen bonds due to the

387

participation of hydroxyl groups.14 CH2 asymmetric stretching almost disappears in the


Page 18 of 48

Page 19 of 48


388

delivery systems, revealing the involvement of hydrophobic interactions. Besides, the

389

intensities of amide I and II bands decrease appreciably and shift to either lower (amide

390

I) or higher (amide II) wavenumbers, confirming the heat-induced protein denaturation

391

and strong hydrophobic interactions.3 A more notable blue-shift of amide A and amide

392

I bands while an inhibited red-shift of amide II band are observed for delivery systems

393

containing Cur compared with the corresponding empty ones. For example, the amide

394

I band shifts from 1632.4 cm-1 to 1625.2 and 1624.3 cm-1 for empty ONP and Cur-ONP,

395

respectively. This result confirms that Cur binds to the protein portion of delivery

396

systems through hydrogen bonding and hydrophobic interactions. Similar phenomenon

397

has been observed by Sun et al., who reported a more remarkable blue-shift of amide I

398

band in zein-propylene glycol alginate nanoparticles containing quercetagetin than in

399

the empty ones.30

400

Steady-state fluorescence analysis. Steady-state fluorescence analyses of Cur and

401

OVA were performed at pH 3.0 and 7.0 to mimic the stomach and small intestine

402

conditions, respectively. As illustrated in Figure 6a, free Cur exhibits a weak

403

fluorescence at pH 3.0 with a flat emission peak centered at 553 nm. The fluorescence

404

intensity of Cur increases by about 10–25 folds and emission maxima blue-shifts to

405

528, 524, and 519 nm after incorporating into ONP, Complex I, and Complex II,

406

respectively. In general, the lower accessibility of water will result in stronger extent of

407

blue-shift of λmax and fluorescence intensity of Cur.31 Therefore, this result reveals the

408

improved hydrophobicity of the surrounding environment of encapsulated Cur

409

compared with that dispersed in water. A similar phenomenon has been reported



410

previously for Cur entrapped within caseinate-zein-polysaccharide complexes32 and

411

surfactant vesicles.31

412

The polysaccharide layer on nanocomplexes is expected to improve their surface

413

thickness and rigidity, which blocks the penetration of water molecules into the protein

414

core where Cur resides. Cur in nanocomplexes therefore presents higher fluorescence

415

efficiency compared with that in ONP. Cur encapsulated by Complex II displays the

416

highest fluorescence intensity, accompanied by the most notable blue-shift of emission

417

maxima; this result confirms that Complex II adopts a more compact structure than

418

ONP and Complex I. The encapsulated Cur undergoes a significant decrease in

419

fluorescence intensity when pH increases from 3.0 to 7.0 (Figure 6b), which would be

420

attributed to the enhanced deprotonation of its hydroxyl groups under neutral condition.

421

Notably, the gap of fluorescence intensity between Cur in Complex II and I improves,

422

whereas that between Cur in Complex I and ONP decreases after pH adjustment (from

423

3.0 to 7.0). This result unequivocally supports the detachment of ALG chains from the

424

protein core in Complex I at a pH range where they are both negatively charged.

425

The intrinsic fluorescence of protein exited at 295 nm has been widely applied to

426

investigate the polarity of the microenvironment around Trp residues and

427

conformational changes in protein structure.17 The intrinsic fluorescence properties of

428

native OVA will be considered as the average contribution of Trp148, Trp267, and Trp184

429

residues.8 As illustrated in Figure 6c, regardless of Cur loading, the fluorescence

430

intensity of the three delivery systems at a protein concentration of 2 mg mL-1 decreases

431

with the following trend: ONP > Complex I > Complex II. ALG coating is expected to


Page 20 of 48

Page 21 of 48


432

shield the Trp residues in protein. Therefore, the intrinsic fluorescence intensity of

433

Complex I is significantly lower than that of ONP. The λmax of Complex II blue-shifts

434

by about 2 nm compared with that of Complex I and ONP, indicating the stronger

435

hydrophobicity of the microenvironment around the Trp residues in Complex II. This

436

finding will arise from the differences in the molecular organization between Complex

437

II and Complex I (or ONP) as mentioned above. The intrinsic fluorescence intensity of

438

the three delivery systems decreases remarkably after Cur incorporation, accompanied

439

by the blue-shift of emission maxima. This phenomenon suggests that the binding of

440

Cur will quench the fluorescence of Trp residues and alter the spatial structure of

441

protein, leading to the orientation of inner fluorophores to a more lipophilic

442

environment. Similar fluorescence spectra are observed at pH 7.0 for empty delivery

443

systems or those loaded with Cur, presenting slight differences in fluorescence intensity

444

(Figure 6d).

445

SIMULATED DIGESTION

446

Proteolysis. The hydrolysis of protein will be closely related with the release and

447

degradation of entrapped Cur because it serves as the building blocks of the three

448

delivery systems. In this work, OVA proteolysis during simulated digestion was

449

monitored by SDS-PAGE and the release of TCA-soluble nitrogen. As illustrated in

450

Figure 7a, all samples present a dense OVA band at ~ 45 kDa prior to digestion (0 min).

451

ONP suffers a rapid pepsinolysis, and intact OVA molecules totally disappear within

452

15 min. In the intestinal tract, the derived fragments (25–30 kDa) appear for further

453

hydrolysis by pancreatin to produce peptides with very low molecular weight.



454

Meanwhile, the amount of TCA-soluble nitrogen increases sharply with digestion time,

455

reaching a value of 77.5% at 30 min, and nearly levels off afterward (Figure 7b). This

456

result is consistent with those of previous studies, where heating at 90 °C or 80 °C for

457

a certain time facilitates the proteolysis of OVA and its derived fragments.33,34 These

458

authors suggested that denaturation and aggregation of OVA induced by thermal

459

treatment promoted the accessibility of cleavage sites to digestive enzymes, thereby

460

accelerating its hydrolysis.

461

By contrast, Complex I and II are more resistant to pepsinolysis compared with ONP.

462

The majority of OVA band in Complex I and II is maintained, and very few peptides

463

are observed at the end of in vitro gastric digestion (Figure 7a), accompanied by the

464

generation of small amount (< 20%) of TCA-soluble nitrogen (Figure 7b). The

465

interfacial ALG chains shield the cleavage sites to pepsin, thereby inhibiting the rate

466

and extent of proteolysis. The electrostatic repulsion between pepsin and ALG chains

467

have also contributed to the restricted pepsin binding because they are both negatively

468

charged under gastric condition (pH 3.0). Sarkar et al. (2017) also observed a reduced

469

susceptibility of β-lg to pepsinolysis after its complexation with anionic

470

polysaccharide.35

471

On the other hand, Complex I and II are readily hydrolyzed in the small intestine

472

phase. Intact OVA band disappears rapidly (Figure 7a), and nearly 70% of TCA-soluble

473

nitrogen is released from the nanocomplexes at the end of small intestine digestion

474

(Figure 7b). The structure of nanocomplexes dissociates (Complex I) or swells

475

(Complex II) under neutral conditions, promoting the penetration of intestinal fluids. In


Page 22 of 48

Page 23 of 48


476

addition, the highly surface-active bile salts may displace the adsorbed polysaccharide

477

chains and destabilize the tertiary structure of protein, thereby accelerating its

478

proteolysis.36 The TCA-soluble nitrogen analysis suggests that Complex II is more

479

resistant to proteolysis than Complex I, especially during intestinal digestion. For

480

example, the release of TCA-soluble nitrogen at 75 min is 67.44% for Complex I and

481

45.55% for Complex II, respectively. This finding arises from the better stability of

482

Complex II against pH change compared with Complex I (Figure 3a).

483

Cur stability and bioaccessibility. The degradation of free and encapsulated Cur

484

during simulated digestion was investigated. The retention rate of Cur decreases

485

slightly during stomach digestion, and the degradation of Cur becomes more

486

pronounced when samples are transferred into the small intestine phase (Figure 8a).

487

Less than 50% of free Cur is remained after simulated digestion owing to its direct

488

exposure to the intestinal tract, consistent with our previous report.3 The stability of Cur

489

improves significantly after nano-encapsulation because these delivery systems will

490

segregate Cur from the exterior environment, thereby retarding its degradation. Cur

491

encapsulated in Complex II displays the highest stability, followed by that in Complex

492

I and ONP. Interestingly, the ranking of the Cur stability during digestion is consistent

493

with that of the resistance to proteolysis mentioned above (Figure 7). The exposure of

494

Cur to the gastrointestinal tract is hypothesized to be closely related with the disruption

495

of the structure of delivery systems resulting from proteolysis.3 Delivery systems with

496

better resistance to proteolysis will therefore more effectively segregate Cur from the

497

exterior harsh environment.37 Besides, results of fluorescence analyses (Figure 6b)



498

suggest that the surrounding microenvironment of Cur in Complex II is more

499

hydrophobic than that of Cur in ONP or Complex I. Therefore, Complex II is expected

500

to better inhibit the diffusion of loaded Cur across matrix into the small intestinal fluids

501

than other delivery systems.

502

Bioaccessibility refers to the proportion of hydrophobic materials solubilized in

503

mixed micelles, which are available for absorption by small intestine epithelium, after

504

sequential gastric and intestinal digestion.3 Free Cur displays a low bioaccessibility

505

(~20%) given the inefficient incorporation of Cur crystals into mixed micelles (Figure

506

8b). On the contrary, Cur loaded within delivery systems is released stepwise with the

507

progression of protein digestion and dissolved within the mixed micelles afterward,

508

which contributes to improved bioaccessibility.4 The bioaccessibility of Cur in ONP is

509

significantly lower (P < 0.05) than that in Complex I or II. One possible explanation is

510

that rapid proteolysis of ONP in gastrointestinal tract led to the burst release of Cur,

511

which exceeded its dissolution rate in mixed micelles. Therefore, a considerable amount

512

of Cur precipitated in the continuous phase. There is no significant difference (P > 0.05)

513

between the bioaccessibility of Cur in Complex I and II, which may be attributed to the

514

very similar proteolysis process of the nanocomplexes during the simulated digestion

515

(Figure 7).

516

ANTIOXIDANT ACTIVITY

517

CAA assay provides a middle ground between assays in vitro and animal or human

518

clinic trials. CAA assay is also a useful tool for assessing the bioavailability of food-

519

derived antioxidants because it considers factors such as cellular uptake and


Page 24 of 48

Page 25 of 48


520

metabolism.23,38 A free-radical generator, ABAP, is introduced to the system to create

521

peroxyl radicals. If an antioxidant can enter cells, it can quench these radicals, thus

522

preventing intracellular DCHF from being oxidized to fluorescent DCF.

523

In this work, the CAA of free and encapsulated Cur was measured with a HepG2

524

model by monitoring the intracellular fluorescence from DCF over a time course of 60

525

min. As depicted in Figures 9a–d, the peroxyl radical-induced DCHF oxidation is

526

visibly inhibited after introducing free or encapsulated Cur compared with the control

527

group (0 µM). The CAA value of each antioxidant increases in a dose-dependent

528

manner and follows a curvilinear pattern as this effect tapers off at higher

529

concentrations. For cytotoxicity assay, free and capsulated Cur were applied to cell

530

lines at concentrations ranging from 0.5 µM to 100 µM for 24 h, and the cell viability

531

was generally above 90% (Figure S2, Supporting Information). Therefore, all samples

532

showed no toxicity to cells at any of the studied concentrations, and the reduced

533

fluorescence intensity resulted from antioxidant defenses. The EC50 value is the half-

534

maximal inhibitory concentration at which log (fa/fu) = 0 (i.e., CAA unit = 50). Based

535

on the linear regression of the log (fa/fu) versus the concentration curve, EC50 values

536

are calculated to be 15.42 µM for free Cur and 9.61, 6.58, and 6.28 µM for Cur loaded

537

within ONP, Complex I, and Complex II, respectively (Figure 10). This result suggests

538

that the antioxidant activity of Cur in HepG2 model improves remarkably after nano–

539

encapsulation. Similarly, Fan et al. (2018) reported that Cur loaded within bovine serum

540

albumin–dextran nanocomplex displayed higher CAA than the free one.5

541

The cellular uptake of both free and encapsulated Cur was further carried out at



542

concentrations of 20 and 50 μM. Data in Figure 11 revealed that both free and

543

encapsulated Cur was internalized into HepG2 cells in a dose–dependent manner. The

544

absorption rate of Cur decreases in the order: Cur in Complex I ≈ Cur in Complex II

545

> Cur in ONP > free Cur, either at 20 or 50 μM. This is further confirmed by the CLSM

546

images of HepG2 cells after being incubated with free or encapsulated Cur at a

547

concentration of 50 μM. Weak green fluorescence is observed for cells treated with free

548

Cur, and the fluorescence becomes more obvious when they are treated with Cur

549

entrapped within ONP. Nanocomplex groups present the strongest fluorescence

550

intensity, indicating more Cur was internalized. The trend of Cur absorption is well

551

correlated with that of antioxidant activity by CAA test (Figure 11), which suggests that

552

the nano–delivery systems in the present work improve the antioxidant activity of Cur

553

by enhancing its absorption in HepG2 cells. Only solubilized Cur is expected to

554

permeate across the cell membrane through passive diffusion.39 Free Cur in medium is

555

in a supersaturation state and the majority of Cur crystallizes, which restricts its

556

absorption. By contrast, encapsulated Cur is well dispersed in the medium and released

557

stepwise from the matrix of nano–delivery systems in free solubilized form to be

558

absorbed by HepG2 cells. On the other hand, free Cur suffers from a rapid degradation

559

when being incubated in FBS–free medium, and only 40% of Cur is remained 60 min

560

later (Figure S3a). On the contrary, the retention rate at the end of the test are 66.34%,

561

81.23%, and 84.56% for Cur in ONP, Complex I, and Complex II, respectively. Cur in

562

delivery systems presents improved stability because it is segregated from the harsh

563

conditions in medium. Therefore, ONP and nanocomplexes also contribute to the


Page 26 of 48

Page 27 of 48


564

cellular uptake of Cur by improving its stability during CAA test. The protective effect

565

is more pronounced in nanocomplexes than in ONP owing to the capping

566

polysaccharide on the surface of nanocomplexes. This result can partially explain why

567

Cur in nanocomplexes presented higher cellular uptake (or lower IC50 values) than that

568

in ONP. According to previous reports, apart from passive diffusion, nano–delivery

569

systems promotes the cellular uptake by of Cur through pinocytotic pathways, such as

570

macropinocytosis, clathrin–mediated endocytosis, caveolae–mediated endocytosis, and

571

clathrin– and caveolae–independent endocytosis.37,40 More work is required in the

572

future to elucidate the effect of pinocytotic pathways on the cellular absorption of

573

encapsulated Cur.

574

The digested samples of free and encapsulated Cur were collected and evaluated for

575

CAA (see Supporting Information for details). Figures S4a–d summarize the DCHF

576

oxidation-inhibitory effect and CAA values of four digested samples at different

577

concentrations. In general, the EC50 values of both free and encapsulated Cur increases

578

appreciably after simulated digestion (Figure 10) owing to their degradation and

579

precipitation during the simulated digestion. The increase in EC50 value is the highest

580

for free Cur (37.54 µM), followed by that in ONP (10.76 µM), Complex I (7.81 µM),

581

and Complex II (4.76 µM), and it is negatively related with the results of retention rate

582

and bioaccessibility analyses (Figure 8). Hence, the antioxidant activity of Cur is better

583

preserved in nanocomplexes especially in Complex II because they are more effective

584

in blocking Cur degradation and promoting its micellization during the simulated

585

digestion.



586

In summary, two types of OVA–ALG nanocomplexes (Complex I and II) were

587

developed for Cur delivery in this work. Though with the same biopolymer composition,

588

Complex I and II presented different morphology and stability against pH change. The

589

stability and bioaccessibility of Cur improved remarkably after encapsulation by

590

nanocomplexes because they were resistant to pepsinolysis during the simulated

591

digestion, which enabled a controlled Cur release in the gastrointestinal tract.

592

Additionally, nanocomplexes contributed to the antioxidant activity of Cur on a HepG2

593

by enhancing its cellular uptake. The CAA of Cur was also better preserved in

594

nanocomplexes especially in Complex II after simulated digestion owing to the

595

improved stability and micellization of Cur. Therefore, the development of protein–

596

polysaccharide nanocomplexes for bioactive compounds delivery is an effective and

597

convenient way to improve their stability and functional attributes.

598

ASSOCIATED CONTENTS

599

Supporting information

600

Particle size distribution of Complex I (a) and Complex II (b) (Figure S1). Effect of

601

free and encapsulated Cur on the viability of HepG2 cells (Figure S2). Retention rate

602

of free Cur (a) and those encapsulated in ONP (b), Complex I (c), or Complex II (d)

603

during incubation in FBS–free medium under cell culture conditions (Figure S3).

604

Peroxyl radical-induced oxidation of DCFH to DCF in HepG2 cells over time and

605

inhibition of oxidation by the digested samples of free Cur (a) and those encapsulated

606

by ONP (b), Complex I (c), or Complex II (d). Insets in a–d are the dose–dependent

607

CAA values for each antioxidant (Figure S4).


Page 28 of 48

Page 29 of 48


608

AUTHOR INFORMATION

609

Corresponding authors

610

*Tel.:

611

*Tel.: 86-25-84392191, Fax: 86-25-84392191, E-mail: [email protected] (L.-

86-25-84392191, Fax: 86-25-84392191, E-mail: [email protected] (F.-J.).

612

C.Y.).

613

Funding

614

This work was supported by the National Postdoctoral Program for Innovative

615

Talents (BX201700101), the China Postdoctoral Science Foundation funded project

616

(2017M621668), the Qinghai Special projects for science and technology cooperation

617

(2017-HZ-816), the Natural Science Foundation of Jiangsu Province (BK20180298),

618

the National Natural Science Foundation of China (31801555), and the Qinghai Science

619

and Technology Program (2017-ZJ-Y06).

620

Notes

621

The authors declare no conflict of interest.

622

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623

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arabic in their coacervates. Langmuir 2004, 20, 6389-6395.

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25. Zeeb, B.; Mi-Yeon, L.; Gibis, M.; Weiss, J., Growth phenomena in biopolymer complexes composed

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of heated WPI and pectin. Food Hydrocolloid. 2018, 74, 53-61.

687

26. Dai, Q. Y.; Zhu, X. L.; Yu, J. Y.; Karangwa, E.; Xia, S. Q.; Zhang, X. M.; Jia, C. S., Mechanism of

688

Formation and Stabilization of Nanoparticles Produced by Heating Electrostatic Complexes of WPI-

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Dextran Conjugate and Chondroitin Sulfate. J. Agric. Food Chem. 2016, 64, 5539-5548.

690

27. Feng, J.; Lin, C.; Wang, H.; Liu, S., Decoration of gemini alkyl O-glucosides based vesicles by

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electrostatic deposition of sodium carboxymethyl cellullose: Mechanism, structure and improved

692

stability. Food Hydrocolloid. 2016, 58, 284-297.

693

28. Zhou, M.; Hu, Q.; Wang, T.; Xue, J.; Luo, Y., Effects of different polysaccharides on the formation

694

of egg yolk LDL complex nanogels for nutrient delivery. Carbohydr. Polym. 2016, 153, 336-344.

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29. Perez, A. A.; Andermatten, R. B.; Rubiolo, A. C.; Santiago, L. G., β-Lactoglobulin heat-induced


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aggregates as carriers of polyunsaturated fatty acids. Food Chem. 2014, 158, 66-72.

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30. Sun, C.; Wei, Y.; Li, R.; Dai, L.; Gao, Y., Quercetagetin-Loaded Zein-Propylene Glycol Alginate

698

Ternary Composite Particles Induced by Calcium Ions: Structure Characterization and Formation

699

Mechanism. J. Agric. Food Chem. 2017, 65, 3934-3945.

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31. Feng, J.; Wu, S.; Wang, H.; Liu, S., Stability of trianionic curcumin enhanced by gemini alkyl O-

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Glucosides and alkyl trimethyl ammonium halides mixed micelles. Colloid. Surface. A 2016, 504, 190-

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200.

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32. Chang, C.; Wang, T.; Hu, Q.; Luo, Y., Caseinate-zein-polysaccharide complex nanoparticles as

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potential oral delivery vehicles for curcumin: Effect of polysaccharide type and chemical cross-linking.

705

Food Hydrocolloid. 2017, 72, 254-262.

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33. Jimenez-Saiz, R.; Belloque, J.; Molina, E.; Lopez-Fandino, R., Human Immunoglobulin E (IgE)

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Binding to Heated and Glycated Ovalbumin and Ovomucoid before and after in Vitro Digestion. J. Agric.

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Food Chem. 2011, 59, 10044-10051.

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34. Nyemb, K.; Guerin-Dubiard, C.; Dupont, D.; Jardin, J.; Rutherfurd, S. M.; Nau, F., The extent of

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ovalbumin in vitro digestion and the nature of generated peptides are modulated by the morphology of

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protein aggregates. Food Chem. 2014, 157, 429-438.

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35. Sarkar, A.; Zhang, S. N.; Murray, B.; Russell, J. A.; Boxal, S., Modulating in vitro gastric digestion

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of emulsions using composite whey protein-cellulose nanocrystal interfaces. Colloid. Surface. B 2017,

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158, 137-146.

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36. Martos, G.; Contreras, P.; Molina, E.; Lope-Fandino, R., Egg White Ovalbumin Digestion Mimicking

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Physiological Conditions. J. Agric. Food Chem. 2010, 58, 5640-5648.

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37. Liu, C.; Cheng, F. F.; Yang, X. Q., Fabrication of a Soybean Bowman-Birk Inhibitor (BBI)



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Nanodelivery Carrier To Improve Bioavailability of Curcumin. J. Agric. Food Chem. 2017, 65, 2426-

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2434.

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721

assay to study phenolic antioxidants in a Caco-2 cell line. Food Chem. 2018, 244, 359-363.

722

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2 Cell Monolayers. J. Agric. Food Chem. 2011, 59, 9120-9126.

724

40. Wang, J.; Ma, W.; Tu, P., The mechanism of self-assembled mixed micelles in improving curcumin

725

oral absorption: In vitro and in vivo. Colloid. Surface. B 2015, 133, 108-119.

726 727

FIGURE CAPTION

728

Figure 1. Schematic illustration for the fabrication process of ONP, Complex I, and

729

Complex II.

730

Figure 2. TEM images of native OVA (a), ONP (b), Complex I (c), and Complex II (d)

731

under different pH conditions. The DZ, ζ-potential, and PDI of sample are summarized

732

in the inset of each subfigure. UA: stained by uranyl acetate; PA: stained by

733

phosphotungstic acid. Bar: 500 nm.

734

Figure 3. Effect of pH on the particle size (a) and surface charge (b) of ONP, Complex

735

I, and Complex II.

736

Figure 4. (a) EE and LC of Cur in ONP, Complex I, and Complex II; (b) influence of

737

Cur loading on the particle size of three delivery systems. Different lowercase and

738

uppercase letters in (a) represent significant differences in EE and LC, respectively (P

739

< 0.05).


Page 34 of 48

Page 35 of 48


740

Figure 5. FT-IR spectra of native OVA, empty delivery systems (ONP, Complex I, and

741

Complex II), and those loaded with Cur (Cur-ONP, Cur-Complex I, and Cur-Complex

742

II).

743

Figure 6. Steady-state fluorescence spectra of free and encapsulated Cur at pH 3.0 (a)

744

and 7.0 (b); intrinsic fluorescence spectra of empty delivery systems and those loaded

745

with Cur at pH 3.0 (c) and 7.0 (d).

746

Figure 7. SDS-PAGE patterns (a) and the release kinetics of TCA-soluble nitrogen (b)

747

of three delivery systems encapsulating Cur during the simulated digestion.

748

Figure 8. Degradation kinetics of free and encapsulated Cur during the simulated

749

digestion (a) and their bioaccessibility after the whole digestion process (b). Different

750

lowercase letters in (b) represent significant differences (P < 0.05).

751

Figure 9. Peroxyl radical-induced oxidation of DCFH to DCF in HepG2 cells over time

752

and inhibition of oxidation by free Cur (a) and those encapsulated by ONP (b), Complex

753

I (c), or Complex II (d). The insets in a–d are dose-dependent CAA values for each

754

antioxidant.

755

Figure 10. EC50 values for inhibition of peroxyl radical-induced DCFH oxidation by

756

free and encapsulated Cur. Different lowercase letters represent significant differences

757

(P < 0.05) between antioxidants before or after simulated digestion. Different uppercase

758

letters represent significant differences (P < 0.05) between the digested and undigested

759

samples of the same antioxidant.

760

Figure 11. Cellular uptake of free and encapsulated Cur by HepG2 cells. (a) Qualitative

761

Cur uptake monitored by CSLM at a Cur concentration of 50 μM. (b) Quantitative Cur



762

uptake at Cur concentrations of 20 and 50 μM. Different lowercase letters represent

763

significant differences (P < 0.05) between samples of the same concentration. Different

764

uppercase letters represent significant differences (P < 0.05) between different

765

concentrations of the same sample.


Page 36 of 48

Page 37 of 48


766 767 768

Figure 1. Schematic illustration for the fabrication process of ONP, Complex I, and

769

Complex II.



770 771 772

Figure 2. TEM images of native OVA (a), ONP (b), Complex I (c), and Complex II (d)

773

under different pH conditions. The DZ, ζ-potential, and PDI of sample are summarized

774

in the inset of each subfigure. UA: stained by uranyl acetate; PA: stained by

775

phosphotungstic acid. Bar: 500 nm.


Page 38 of 48

Page 39 of 48


776 777 778

Figure 3. Effect of pH on the particle size (a) and surface charge (b) of ONP, Complex

779

I,

and

Complex


II.


781 782 783

Figure 4. (a) EE and LC of Cur in ONP, Complex I, and Complex II; (b) influence of

784

Cur loading on the particle size of three delivery systems. Different lowercase and

785

uppercase letters in (a) represent significant differences in EE and LC, respectively (P

786

< 0.05).


Page 40 of 48

Page 41 of 48


787 788 789

Figure 5. FT-IR spectra of native OVA, empty delivery systems (ONP, Complex I, and

790

Complex II), and those loaded with Cur (Cur-ONP, Cur-Complex I, and Cur-Complex

791

II).



792

793 794 795

Figure 6. Steady-state fluorescence spectra of free and encapsulated Cur at pH 3.0 (a)

796

and 7.0 (b); intrinsic fluorescence spectra of empty delivery systems and those loaded

797

with Cur at pH 3.0 (c) and 7.0 (d).


Page 42 of 48

Page 43 of 48


798

799 800 801

Figure 7. SDS-PAGE patterns (a) and the release kinetics of TCA-soluble nitrogen (b)

802

of three delivery systems encapsulating Cur during the simulated digestion.



803

804 805 806

Figure 8. Degradation kinetics of free and encapsulated Cur during the simulated

807

digestion (a) and their bioaccessibility after the whole digestion process (b). Different

808

lowercase letters in (b) represent significant differences (P < 0.05).


Page 44 of 48

Page 45 of 48


809

810 811 812

Figure 9. Peroxyl radical-induced oxidation of DCFH to DCF in HepG2 cells over time

813

and inhibition of oxidation by free Cur (a) and those encapsulated by ONP (b), Complex

814

I (c), or Complex II (d). The insets in a–d are dose-dependent CAA values for each

815

antioxidant.



816 817 818

Figure 10. EC50 values for inhibition of peroxyl radical-induced DCFH oxidation by

819

free and encapsulated Cur. Different lowercase letters represent significant differences

820

(P < 0.05) between antioxidants before or after simulated digestion. Different uppercase

821

letters represent significant differences (P < 0.05) between the digested and undigested

822

samples of the same antioxidant.


Page 46 of 48

Page 47 of 48


823 824

Figure 11. Cellular uptake of free and encapsulated Cur by HepG2 cells. (a) Qualitative

825

Cur uptake monitored by CSLM at a Cur concentration of 50 μM. (b) Quantitative Cur

826

uptake at Cur concentrations of 20 and 50 μM. Different lowercase letters represent

827

significant differences (P < 0.05) between samples of the same concentration. Different

828

uppercase letters represent significant differences (P < 0.05) between different

829

concentrations of the same sample.



830

TOC Graphic

831


Page 48 of 48

Development of nanocomplexes for curcumin vehiculization using

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