Enhanced chemical stability, intestinal absorption, and intracellular

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Food and Beverage Chemistry/Biochemistry

Enhanced chemical stability, intestinal absorption, and intracellular antioxidant activity of cyanidin-3O-glucoside by composite nanogel encapsulation Jin Feng, Yinghui Wu, Lixia Zhang, Ying Li, Songbai Liu, Hua Wang, and Chunyang Li J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b04778 • Publication Date (Web): 30 Aug 2019 Downloaded from pubs.acs.org on August 30, 2019

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Journal of Agricultural and Food Chemistry

Enhanced chemical stability, intestinal absorption, and intracellular antioxidant activity of cyanidin-3-O-glucoside by composite nanogel encapsulation

Jin Feng,*, † Yinghui Wu,† Lixia Zhang,† Ying Li,† Songbai Liu,‡ Hua Wang,§ and Chunyang Li*,†

†

Institute of Agro-product Processing, Jiangsu Academy of Agricultural Sciences, 50

Zhongling Street, Nanjing 210014, China ‡

Department of Food Science and Nutrition, Zhejiang University, 866 Yuhangtang

Road, Hangzhou 310058, China §

Center of Analysis and Measurement, Zhejiang University, 866 Yuhangtang Road,

Hangzhou 310058, China

*Corresponding author, (Tel: 86-25-84392191; Fax: 86-25-84392191; E-mail: [email protected]); *Corresponding author, (Tel: 86-25-84392191; Fax: 86-25-84392191; E-mail: [email protected]).

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ACS Paragon Plus Environment


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ABSTRACT: A composite nanogel was developed for cyanidin-3-O-glucoside (C3G)

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delivery by combining Maillard reaction and heat-gelation. The starting materials

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utilized were ovalbumin, dextran, and pectin. C3G-loaded nanogel was spherical with

4

a diameter of ~185 nm, which was maintained over a wide range of pH and NaCl

5

concentration. The composite nanogel enhanced the chemical stability of C3G under

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accelerated degradation models and simulated gastrointestinal tract. Clathrin-,

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caveolae-mediated and macropinocytosis-related endocytosis contributed to the higher

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cellular uptake of nano-C3G than that of free-C3G. The apparent permeability

9

coefficients of C3G increased 2.16 times after nanoencapsulation. The transcytosis of

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C3G-bearing nanogel occurred primarily through clathrin-related pathway and

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macropinocytosis and followed the “common recycling endosomes–endoplasmic

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reticulum–Golgi complex–basolateral plasma membrane” route. Moreover, nano-C3G

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was more efficient in restoring the viability of cells and activities of endogenous

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antioxidant enzymes than free-C3G in oxidative models, which may be attributed to the

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former’s high cellular absorption.

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KEYWORDS: Composite nanogel, cyanidin-3-O-glucoside, cellular uptake,

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monolayer permeability, intracellular antioxidant activity

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INTRODUCTION

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Cyanidin-3-O-glucoside (C3G) is the most common naturally occurring monomeric

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anthocyanin (ATC) present in edible berries, dark grapes, and other pigmented foods.

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C3G holds many potential health-promoting attributes, including antioxidant,

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anticancer, antidiabetic, anti-inflammatory, antiaging, and eye- and brain-benefitting

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properties.1 These properties demonstrate their potential utilization as functional

24

ingredients. Nevertheless, C3G is chemically unstable. It degrades rapidly during food

25

processing when exposed to neutral or basic pH values, elevated temperatures, oxygen,

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enzymes, and other reactive substances such as ascorbic acid.2-5 Dietary C3G undergoes

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extensive metabolisms in the gastrointestinal (GI) tract,6 such as de-glycosylation,

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glucuronidation, and sulfatation; furthermore, it is poorly absorbed by intestinal

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epithelia owing to its high polarity (log P ≈ 0.39),7-10 limiting its bioavailability and

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functionalities in vivo.

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Constructing nanocarriers is an important strategy that enhances the stability,

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controlled release, intestinal absorption, and bioavailability of nutraceuticals.11-13 Food-

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derived proteins, polysaccharides, and phospholipids are preferred starting materials

34

because they are non-toxic, biodegradable, biocompatible, and “generally recognized

35

as safe”. Specific nanocarriers regarding C3G delivery have included whey protein

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isolate-glucose (WPI-Glu) nanoparticle,4 ferritin nanocage,9 chitosan nanoparticle,14

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chitosan hydrochloride-carboxymethyl chitosan nanocomplex,3 and liposome.15

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Encapsulated C3G is provided with a physiochemical barrier against exterior

39

environment resulting in enhanced chemical stability and shelf life. The functional 3



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attributes of C3G in vitro and in vivo may also be enhanced by nanoencapsulation. For

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example, C3G in chitosan nanoparticles can better reduce UVB-induced epidermal

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damage through the p53-mediated apoptosis signaling pathway compared with free

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C3G.14 This finding may be due to the ability of nanocarriers to improve C3G

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bioavailability. This hypothesis has been confirmed by Zhang et al. (2014), who

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revealed that the level of C3G transported across Caco-2 monolayers increases from

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2.51 μg to 2.74 μg after being loaded into ferritin nanocage. Nevertheless, information

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on the detailed transport mechanisms of C3G-bearing nanocarriers in the epithelium

48

cells is currently limited. The transcytosis of nanocarriers is very complicated,

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involving the internalization of nanocarriers from the apical side (AP) through

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endocytosis pathways, intracellular transportation to different organelles and structures,

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and fusion with the plasma membrane leading to exocytosis.16,17 An improved

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understanding of the transcytosis mechanisms will contribute to the fabrication of

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optimized C3G nanocarriers with high bioavailability.

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Stable nanocarriers from proteins and polysaccharide over a wide range of

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environmental stresses such as pH, temperature, and ionic strength, have been studied.

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However, at present, the stability of the C3G nanocarriers is primarily maintained by

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electrostatic forces.3,4,9,14 C3G nanocarriers readily aggregate when the environmental

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pH is around the pI of proteins or below the pKa of charged groups on polysaccharides

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(i.e., sulfate group, carboxyl group, etc.) and in the presence of high NaCl concentration,

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which results in unfavorable precipitation and sedimentation.18,19 Glycation with

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dextran through Maillard reaction shows potential in improving the stability of proteins 4


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and protein-stabilized delivery systems by adding steric hindrance.20,21 Furthermore,

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composite nanogels with small size can be fabricated by heating these Maillard

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products near the pI of protein. Composite nanogels loaded with hydrophobic

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molecules, such as curcumin22 and lutein,20 have been reported in previous works, but

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information about their utilization on the delivery of hydrophilic ATCs is currently not

67

available. In preliminary experiments, we found that the encapsulation efficiency of

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C3G in ovalbumin–dextran nanogel22 is only approximately 30% owing its hydrophilic

69

nature, which is nearly doubled when pectin is included as a starting material. We

70

speculate that the electrostatic attraction between flavylium cation and the carboxyl

71

groups on pectin will enhance the encapsulation of C3G.

72

Therefore, this work aims to investigate the feasibility of developing composite

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nanogels for C3G delivery utilizing ovalbumin, dextran, and pectin as starting materials.

74

The physiochemical properties and the stability under various environmental stresses

75

and simulated digestion of C3G-bearing nanogel were studied. The endocytosis and

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transcytosis mechanisms of C3G-bearing nanogel were elucidated on Caco-2 cell

77

models by using various inhibitors. Finally, the protective effect of the C3G entrapped

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within nanogel (nano-C3G) against H2O2-induced cell damage was evaluated and

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compared with that of free-C3G. The findings of this work indicate a new strategy of

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preparing stable nanocarrier for C3G delivery by an ecofriendly process.

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MATERIALS AND METHODS

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Materials. C3G (chloride salt, ≥ 95%), ovalbumin (albumin from egg white, ≥ 95%), 5



83

pectin (from citrus peel, galacturonic acid ≥ 74.0%, degree of esterification 63.50%,

84

determined

85

diphenyltetrazolium bromide (MTT, 98%), sodium azide (≥ 99.5%), chlorpromazine

86

(analytical standard), methyl-β-cyclodextrin (MβCD, ≥ 95%), 5-(N-ethyl-N-isopropyl)

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amiloride (EIPA, ≥ 98%), brefeldin A (≥ 95%), monensin (≥ 90%), nocodazole (≥ 99%),

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and 2ʹ,7ʹ-dichlorofluorescin diacetate (DCFH-DA, ≥ 97%) were purchased from

89

Sigma–Aldrich Corp. (St. Louis, USA). Dextran (60 kDa), H2O2 (30 wt. % in water),

90

and bafilomycin A1 (≥ 95%) were purchased from aladdine Co., Ltd. (Shanghai, China).

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Caco-2 cell lines were purchased from the Institute of Biochemistry and Cell Biology

92

(Shanghai, China). Dulbecco’s modified Eagle’s medium (DMEM, high-glucose) and

93

fetal bovine serum (FBS) were purchased from Gibco BRL (USA). All other chemicals

94

were of analytical grade and used as purchased.

by

the

titrimetric

method),

3-[4,5-dimethylthiazole-2-yl]-2,5-

95

Fabrication of C3G-bearing composite nanogel. Ovalbumin–dextran conjugate

96

was first prepared.21 In a typical procedure, ovalbumin (1.00%, w/w) and dextran

97

(1.00%, w/w) with a molecular weight of 60 kDa was individually solubilized in 10

98

mM phosphate buffered saline (PBS, pH 7.0) and stored overnight at 4 °C to ensure

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complete hydration. Thereafter, two samples were mixed at a 1:1 mass ratio under

100

constant stirring at ambient temperature for 30 min, which led to a final protein

101

concentration of 0.50% (w/v). The mixture was then lyophilized and the powder was

102

heated at 60 °C for 72 h in a desiccator containing saturated KBr solution, which

103

afforded 79% relative humidity. After reaction, the samples were dried, grounded, and

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stored at -80 °C before use. 6


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Stock solutions of pectin or ovalbumin–dextran conjugate were prepared by

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dissolving powdered samples in 10 mM PBS (pH 7.0) to reach a final concentration of

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1.00% (w/w), which was then left overnight at 4 °C to ensure complete hydration.

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Afterward, these two solutions were mixed under constant stirring and diluted

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appropriately reaching a final conjugate concentration of 0.40% and pectin

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concentration of 0.10% (w/w). A small volume of C3G stock solution (5.00 mg/mL in

111

10 mM acetic acid–sodium acetic buffer, pH 4.0) was then slowly titrated into the

112

mixed solution to achieve a C3G-to-conjugate mass ratio of 1: 20. After stirring 2 h,

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the mixed system was adjusted to pH 4.5 and heated at 80 °C for 10 min to promote

114

biopolymer assembly. The mixture was finally cooled to room temperature in an ice-

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water bath.

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Thereafter, C3G-loaded nanogel was carefully transferred into an Amicon® Ultra-15

117

centrifugal device equipped with a filter of 3 kDa cutoff. The solution was then

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centrifuged at 10 000g for 15 min. The filtrate was collected and subjected to C3G

119

analysis by HPLC. The entrapment efficiency (EE) and loading capacity (LC) were

120

calculated as follows:

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EE (%) =

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LC (%) =

Total C3G - free C3G Total C3G

× 100%

Total C3G - free C3G Total mass of the C3G - bearing nanogel

× 100%

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Herein, total C3G is the initial content of C3G added in the formulation, where free

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C3G is the content of C3G recovered in the filtrate phase.

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HPLC analysis of C3G. An Agilent 1290 Infinity LC coupled with a Zorbax eclipse

126

analytical XDB-C18 column (4.6 × 150 mm, 5 μM) was used in this work for C3G 7



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analysis. Mobile phase A was 6.00% acetic acid in water while mobile phase B was

128

6.00% acetic acid in acetonitrile. The injection volume was 2.50 μL and the column

129

temperature was maintained at 30 °C with an Agilent column oven. The separation was

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carried out according to the following program: 0-25 min, 5-30% B; 25-30 min, 30-80%

131

B; 30-35 min, 80%-5% B. The flow rate was set at 1.0 mL/min and the detection

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wavelength was 520 nm. A calibration curve was constructed by plotting the peak area

133

versus C3G concentration.

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Dynamic light scattering (DLS) analysis. The DZ and ζ-potential of C3G-bearing

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

136

Instrument Ltd., United Kingdom). Samples were equilibrated at 25 °C for 120 s before

137

analysis. Each parameter was calculated as the average of at least triplicate, and each

138

measurement was obtained from the mean of at least 10 readings for a sample.

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Transmission electron microscopy (TEM) observation. One drop of the

140

suspension of C3G-bearing nanogel was placed on carbon formvar-coated cooper grid

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(200 mesh) for 5 min, after which of the excess solution was removed to form a thin

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liquid film layer in the cooper grid. The sample was further stained with aqueous uranyl

143

acetate and dried in the air before observed with a JEM-1230 (HR) transmission

144

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

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Scanning electron microscopy (SEM) observation. The lyophilized powder of

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C3G-bearing nanogel was mounted on a holder with double-sided adhesive tape.

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Samples were coated with a thin layer of gold by a sputter coater (JEOL JFC-1200 fine

148

coater, Japan) before observed under SEM (JSM-633OF, JEOL Ltd., Tokyo, Japan). 8


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Fourier-Transform Infrared (FTIR) Spectroscopy. The FTIR spectra of the

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freeze-dried powders of empty and C3G-bearing nanogels were recorded on a Tensor

151

27 instrument (Bruker Co., Karlsruhe, Germany) using a KBr disk with 1% finely

152

grounded samples. The spectra were acquired in the range of 400-4000 cm-1 at a

153

resolution of 4 cm-1.

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Chemical stability analysis. To investigate the thermal and pH stability of C3G, we

155

adjusted aliquots (50 mL) of free and nano-C3G solutions to pH 4.0 or 7.0 utilizing 1

156

M HCl and NaOH, which were then transferred into sealed glass tubes and incubated

157

at 60 °C for 24 h. To investigate the effect of ascorbic acid on C3G degradation, we

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titrated a small volume of ascorbic acid stock solution into free- and nano-C3G

159

solutions (50 mL) under constant stirring to reach final ascorbic acid concentrations of

160

0.05% and 0.10%, respectively. The solutions were then incubated at 40 °C and pH 4.0

161

for 24 h. For these tests, the initial concentration of free- and nano-C3G was 50.00

162

μg/mL, which was obtained by diluting the stock solutions with ultrapure water. Five

163

hundred microliters of sample was withdrawn from the reaction solution at a time

164

interval of 4 h and thoroughly mixed with 2 mL of acetonitrile supplemented with 6.00%

165

acetic acid. The mixture was then centrifuged at 10 000g for 10 min. The supernatant

166

was collected and filtered through a 220 nm membrane for C3G analysis. The retention

167

rate of C3G was calculated according to the equation:

168

Retention rate (%) = Ct/C0 × 100%,

169

where Ct and C0 are the C3G content (μg/mL) at time zero and t.

170

The degradation of both free- and nano-C3G in this work followed the first-order 9



171

kinetics. Therefore, the half-life (t1/2) of C3G was calculated using the following

172

equations:

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ln Ct/C0 = - kt

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t1/2 = ln (2)/k.

175

Herein, k represents the rate constant for C3G degradation (h-1).

176

Simulated digestion. An in vitro gastrointestinal GI model was utilized herein to

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investigate the digestibility of C3G-bearing nanogel. The simulated gastric fluids

178

contained 120 mM NaCl, 5 mM KCl, 6 mM CaCl2, and 3.2 mg mL-1 pepsin. The

179

simulated small intestinal fluids contained 0.3 mM CaCl2, 30 mM NaCl, 5 mg mL-1 bile

180

salts, and 8 mg mL-1 pancreatin.23 C3G stock solution and C3G-bearing nanogel

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solution were diluted properly with ultrapure water to reach a C3G concentration of

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100.00 μg/mL. After being incubated at 37 °C for 10 min, the solutions were mixed

183

with preheated simulated gastric fluids at a 1:1 mass ratio. The mixtures were then

184

adjusted to pH 2.0 and shaken continuously at 100 rpm and 37 °C for 60 min. Thereafter,

185

the chyme collected from the gastric phase was mixed with an equal volume of

186

simulated small intestinal fluids. The mixtures were then adjusted to pH 7.0 and shaken

187

continuously at 37 °C for 120 min.

188

During the simulated digestion, aliquots of digesta were removed from the mixture

189

at a time interval of 30 min to test the C3G retention rate, content of trichloroacetic acid

190

(TAC)-soluble nitrogen, and DZ and ζ-potential of nanogel. For TAC-soluble nitrogen

191

analysis,13,23 2.00 mL of the digesta was thoroughly mixed with 1.00 mL of 20% TCA

192

solution to precipitate proteins with large molecular size. The mixtures were placed at 10


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ambient temperature for 30 min and centrifuged at 5000g for 10 min. The nitrogen

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content in the supernatant was determined by the micro-Kjeldahl method, and the

195

percentage of TCA-soluble nitrogen was calculated according to the following equation:

196

TCA - soluble nitrogen (%) =

amount of nitrogen in the supernatant total amount of nitrogen

× 100

197

Cell culture and cytotoxicity analysis. Caco-2 Cells were cultured in DEME

198

containing 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin at 37 °C and

199

5% CO2. Cells between passages 40 and 60 were selected for experiments to maintain

200

relatively constant cellular phenotypes. MTT test was utilized to investigate the

201

cytotoxicity of both free- and nano-C3G. The Caco-2 cells were pipetted into 96-well

202

plates at a density of 5 × 104 cells per well. After 24 h, the cells were exposed to different

203

concentrations of free- or nano-C3G at 37 °C for 48 h. An aliquot (10 μL) of 5 mg/mL

204

MTT solution was then added to each well, and the plates were incubated for 4 h.

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Formazan crystals formed by the active cells were dissolved with 100 μL of DMSO,

206

and absorbance at 570 nm was measured. The cell viability was calculated using the

207

following equation:

208

Cell viability =

209

Herein, Asample and Acontrol are the absorbance of wells treated with and without C3G,

210

respectively.

Asample Acontrol

× 100%

211

Cellular uptake analysis. Caco-2 cells were cultured at a density of 5 × 105 cells

212

per well in 6-well plates. When the cells reached confluence, the culture medium was

213

replaced with Hanks’ Balanced Salt Solution (HBSS), and the cells were pre-incubated

214

at 37 °C for 30 min. After equilibration, the cells were treated with different 11



215

concentrations (5.00, 10.00, 20.00, and 30.00 μg/mL) of free- or nano-C3G for 2 h. To

216

investigate the effect of incubation time on cellular uptake, we pretreated Caco-2 cells

217

with 20.00 μg/mL of free- or nano-C3G for 0.5, 1, 2, and 4 h, respectively. The medium

218

was then aspirated, and Caco-2 cells were washed three times with PBS at 4 °C to

219

remove unabsorbed C3G. Afterward, Caco-2 cells were lysed, and the absorbed C3G

220

was extracted and analyzed by HPLC. Total protein content in the cell lysates was

221

determined using the BCA protein assay kit. The cellular uptake of free- and nano-C3G

222

was expressed as μg C3G/mg protein.

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Determination of the endocytosis pathways of C3G-bearing nanogel. To study

224

the pathways involved in the internalization of C3G-bearing nanogel, we pre-incubated

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Caco-2 cells with 20 mM sodium azide (inhibitor of energy-dependent procedure), 30

226

μM chlorpromazine (inhibitor of clathrin-mediated endocytosis), 2 mM MβCD

227

(inhibitor of caveolae-mediated endocytosis), 100 μM EIPA (inhibitor of

228

macropinocytosis), or 100 μM quercetin (inhibitor of caveolae- and clathrin-

229

independent endocytosis) at 37 °C for 1 h.17,24 Subsequently, Caco-2 cells were treated

230

with 20.00 μg/mL nano-G3G in the presence of corresponding inhibitors for 4 h. To

231

investigate the effect of temperature on endocytosis, we pre-incubated Caco-2 cells at

232

4 °C for 1 h and then treated them with 20.00 μg/mL nano-G3G under the same

233

temperature for 4 h. After co-incubation, Caco-2 cells were washed with 4 °C PBS three

234

times and analyzed for C3G and total cellular protein content as mentioned above.

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Monolayer permeability assay. The monolayer permeation rate of free- and nano-

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C3G was determined in accordance with a previous protocol with modifications.24 12


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Caco-2 cells were seeded at a density of 1 × 105/cm2 on insets with polycarbonate

238

membranes (0.4 μM pore size, 12 mm diameter, Corning, Inc.) with 0.50 mL of medium

239

in the AP side and 1.50 mL of medium in the basolateral (BL) side. The medium was

240

changed every 2 days, and Caco-2 monolayers were obtained at least 21 days after

241

seeding. The transepithelial electrical resistance (TEER) of the monolayers was

242

measured using a Millicell-ERS voltmeter (Millipore Co., Billerica, MA). Only

243

monolayers with a TEER value above 400 Ω cm2 were selected for experiments.

244

After the cell monolayers were equilibrated at 37 °C for 30 min, 0.40 mL of HBSS

245

containing 20.00 μg/mL of free- or nano-C3G was added to the AP side, and 1.20 mL

246

of HBSS was added to the BL side. Aliquots (0.50 mL) of sample were collected from

247

the BL at 0.5, 1, 1.5, 2, 2.5, and 3 h, and fresh media of the same volume was

248

replenished. The samples were then diluted with acetonitrile containing 6.00% acetic

249

acid and subjected to HPLC analysis. The apparent permeability coefficients (Papp) of

250

C3G were calculated using the following equation:

251

Papp (cm/s) =

252

where dQ/dt is the permeation rate of C3G (μg/s) of the receiver side, A is the membrane

253

surface area of the inserts (1.12 cm2), and C0 is the initial concentration of free- or nano-

254

C3G (20.00 μg/mL).

( )( ) dQ 1 dt AC0

255

Determination of the transcytosis pathways of C3G-bearing nanogel. The

256

monolayers were pre-incubated for 1 h with different endocytosis inhibitors as

257

mentioned above or with intracellular trafficking inhibitors, namely, brefeldin A (90

258

μM) which inhibits the delivery between endoplasmic reticulum (ER) and Golgi 13



259

complex; monensin (48 μM), which inhibits the delivery between Golgi complex and

260

cell membrane; nocodazole (20 μM), which disrupts the structure of microtubules; and

261

bafilomycin A1 (0.15 μM), which inhibits the maturation process of lysosomes.

262

Afterwards, the Caco-2 monolayers were incubated with 20.00 μg/mL nano-C3G in the

263

presence of corresponding inhibitors for 3 h. The Papp values were then calculated as

264

mentioned above to confirm the transcytosis pathways of nano-C3G.

265

Cellular antioxidant activity assay. Caco-2 cells were seeded on 96-well plates at

266

a density of 5 × 104 cells per well and cultivated for 24 h to allow attachment. The

267

supernatant was then aspirated, and the cells were treated with 100 μL of free- or nano-

268

C3G (2.50 and 5.00 μg/mL) for 12 h. The cells were then washed twice with fresh

269

DMEM medium to remove the unabsorbed C3G and treated with H2O2 (700 μM) for 4

270

h. After simulation, the viability of Caco-2 cells was measured using the MTT assay as

271

mentioned above. Caco-2 cells treated only with DEME medium (control) or H2O2

272

(oxidative group) were also included for comparison.

273

Intracellular reactive oxygen species (ROS) levels were monitored using DCFH-DA

274

as a fluorescence precursor in accordance with the ROS assay kit. After treatment with

275

free- or nano-C3G and H2O2, the Caco-2 cells were washed twice with DMEM medium.

276

One hundred μL of DCFH-DA solution (10 μM) was then introduced to each well, and

277

the cells were incubated at 37 °C for 30 min to produce the strong green fluorescent

278

material dichlorofluorescein (DCF). The fluorescence in each well (λex: 483 nm; λem:

279

535 nm) was recorded with a SpectraMax M5 reader (Molecular Devices, California,

280

USA) and photographed by an inverted fluorescence microscope (ECLIPSE TE200014


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S, Nikon, Tokyo, Japan). Fluorescence intensity relative to cell viability was calculated

282

to reflect the ROS level in cells.

283

The malondialdehyde (MDA), superoxide-dismutase (SOD), catalase (CAT), and

284

glutathione-peroxidase (GSH-px) levels were analyzed using assay kits according to

285

the manufactures’ protocols. Caco-2 cells were cultured in 6-well plates at a density of

286

5 × 105 cells per well. After 24 h of incubation, cells in each well were treated with

287

free- or nano-C3G and H2O2 as mentioned above. Five hundred μL of lysis buffer was

288

then added to each well to dissociate the cells, and the mixture was then centrifuged at

289

5000g and 4 °C for 10 min. The supernatant was collected and subjected to MDA, SOD,

290

CAT, and GSH-px analysis. The protein concentrations in the cell lysates were

291

determined by BCA method to normalize the levels. The results were expressed as nmol

292

per mg of protein (nmol/mg protein) or units of enzymatic activity per mg of protein

293

(U/mg protein).

294

Statistical analysis. All data were expressed as mean values ± standard deviations

295

(SD) of three independent experiments. One-way analyses of variance (ANOVA) with

296

Dunnett’s post-test or t tests with two-tailed P values were utilized to analyze the

297

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

298

RESULTS AND DISCUSSION

299

Characterization of C3G-bearing nanogel. For the preparation of composite

300

nanogel, ovalbumin-dextran conjugate was first synthesized in this work through

301

Maillard reaction. Thereafter, conjugate, anionic polysaccharide pectin, and C3G were 15



302

heated together at the pI of ovalbumin to promote protein denaturation and nanogel

303

formation. Pectin chains were still expected to bind to the positive patches on

304

ovalbumin-dextran conjugate via electrostatic interaction owing to the “charge patch

305

concept”, even though the net charge on conjugate at pI was neutral.25 C3G would

306

interact favorably with the ovalbumin through hydrophobic forces and the carboxyl

307

groups on pectin via electrostatic interaction. During the thermal treatment, the pectin

308

chains and C3G were simultaneously incorporated into the gel networks, whereas the

309

dextran portion of the conjugate tended to be oriented at the aqueous–particle interface,

310

forming a polysaccharide corona due to its hydrophilic nature.22

311

The DZ of the C3G-bearing nanogel in this work was ~ 185 nm, and its size

312

distribution was homogenous and unimodal (PDI < 0.3) (Figure 1A). Though fabricated

313

at the pI of ovalbumin (pH 4.5), the composite nanogel presented a moderate negative

314

surface charge (ζ-potential: -23.45 mV) owing to deprotonation of the carboxyl groups

315

on pectin. By contrast, heating egg yolk low density lipoprotein (LDL)/polysaccharide

316

combinations at pH 7.0 afforded small nanogels (DZ < 85 nm) with high magnitude of

317

ζ-potential (approximately -30 mV to -60 mV).26 One possible explanation is that the

318

anionic polysaccharides carried many negative charges at pH 7.0 to prevent protein

319

aggregation during the thermal treatment. The diameter of the nanogel fabricated by

320

heating biopolymer mixture at pH 5.2 was approximately 150 nm, which is more closer

321

to that of the present work.20

322

TEM and SEM observations indicated that the C3G-bearing nanogel adopted a

323

compact spherical shape with distinct boundaries (Figure 1B), which may be attributed 16


Page 16 of 62

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324

to the effective interactions between the component biopolymers induced by thermal

325

treatment. Spherical structures have been also reported in other nanocarriers utilizing

326

nature macromolecules as building blocks.14,22,23,26 The diameter of nanogel

327

(approximately 100 nm) by TEM and SEM analyses appeared to be smaller than the DZ

328

obtained by DLS analysis; this result was due to the samples that underwent drying

329

before TEM or SEM observation.22

330

The EE and LC of C3G in the composite nanogel were calculated as 65.43% and

331

2.55%, respectively. Similarly, Ge et al. (2019)27 reported that the EE of ATCs was

332

69.33% in nanocomplexes using β-lg and chitosan derivatives as wall materials. Even

333

lower EE values for ATCs/C3G were observed in other delivery systems such as

334

liposome (approximately 32–50%)5 and chitosan nanoparticle (44.90%).14 Therefore,

335

C3G seems to be less efficiently encapsulated compared with hydrophobic molecules,

336

such as curcumin.26 This phenomenon would be attributed to the hydrophilic nature of

337

C3G that promotes its partition from the matrix exterior into the aqueous phase during

338

the preparation process. Therefore, more molecules were recovered in the filtrate after

339

ultrafiltration. The LC value of C3G in the aforementioned chitosan nanoparticle was

340

4.30%,14 which was 1.68 times that in the composite nanogel in the present work. One

341

possible explanation is that nanogel preparation involves a two-step procedure, which

342

requires additional starting materials.

343

As depicted in Figure 1C, the amide I band at 1624 cm-1 (corresponding to the C=O

344

stretching vibration of peptide bond) and amide II band at 1529 cm-1 (corresponding to

345

the C–N stretching vibration and N–H bending vibration of amino groups)28 in the 17



346

composite nanogel suffered a red-shift after C3G incorporation, accompanied by a

347

remarkable decrease in intensity. This result seemed to arise from the interactions

348

between the composite nanogel and core agent via hydrophobic forces and hydrogen

349

bonding. A similar result has been observed in previous reports regarding Cur-loaded

350

biopolymer nanocomplexes.23,26 Notably, a new band was detected in the FTIR spectra

351

of C3G-bearing nanogel, that is, 1395 cm-1 associated with the C–O angular

352

deformations of polyphenols.29 This result suggests that C3G was successfully

353

entrapped into the matrix of composite nanogel. Similarly, the characterized bands of

354

C3G representing the vibration of pyranyl ring were distinguished in the FTIR spectra

355

after being incorporated into chitosan nanoparticles.14

356

The pH and salt stability of C3G-bearing nanogel was further investigated in this

357

work. As presented in Figure 1D, C3G-bearing nanogel was highly anionic (ζ-potential

358

-33.23 mV) at pH 8.0. The negative surface charge decreased stepwise with decreased

359

pH values and experienced a charge reversal to adopt a positive ζ-potential at ~ pH 2.2.

360

The ζ-potential increased thereafter with decreased pH values. The surface charge on

361

C3G-bearing nanogel was highly pH-dependent owing to the protonation/deprotonation

362

of the carboxyl groups on pectin chains and amino groups and carboxyl groups on

363

protein.23 NaCl with a final concentration of 10–500 mM was introduced to screen the

364

surface charge. As expected, the magnitude of ζ-potential of C3G-bearing nanogel

365

decreased consistently with increased NaCl concentration (Figure 1E). However, the

366

DZ of C3G-bearing nanogel was similar throughout the studied pH range or NaCl

367

concentration. In this work, the conjugated dextran chains contributed to the stability 18


Page 18 of 62

Page 19 of 62


368

of the C3G-bearing nanogel by providing steric hindrance, which was insensitive to

369

changes in pH or ionic strength. Similar results have also been observed in previous

370

reports regarding whey protein isolate–dextran–chondroitin sulfate nanoparticles21 and

371

zein nanoparticles stabilized by casein–dextran conjugates.20

372

Chemical stability. Accelerated models were utilized herein to investigate the effect

373

of nano-encapsulation on the stability of C3G. As presented in Figure 2A, the retention

374

rate of free-C3G decreased to 58.54% after 24 h when heated at 60 °C and pH 4.0, and

375

the corresponding t1/2 value was 29.62 h (Figure 2C). Sadilova et al. (2006)2 reported

376

that during thermal treatment, C3G first underwent deglycosylation, leading to the

377

formation of cyanidin aglycones, which further decomposed into phloroglucinaldehyde,

378

protocatechuic acid, and residues of A- and B-rings. C3G solution was previously

379

heated at 80 °C and pH 3.0 for 2 h.4 The t1/2 value of C3G estimated by the first-order

380

kinetic model was 109.2 min (6.44 h), which is approximately one-fifth of that in the

381

present work. Moreover, Sadilova et al. reported that the t1/2 value of C3G in strawberry

382

extract was only 1.95 h when incubated at 95 °C.2 Therefore, C3G appeared to degrade

383

more rapidly at elevated temperatures. Similar results have been observed by Ge et al.,

384

who studied C3G in blueberry extract.3

385

The C3G degradation at pH 7.0 was more obvious compared with that at pH 4.0

386

(Figure 2B). Only 4.33% of free-C3G remained after 16 h, after which the level was

387

below the detection limit. The t1/2 value of free-C3G at pH 7.0 was 3.03 h (Figure 2C),

388

10% of that at pH 4.0. Similarly, Qin et al. suggested that the C3G degradation was pH-

389

dependent, being 3.5 times faster at pH 6.0 than at pH 3.0.4 Furthermore, ATCs are 19



390

stable at low pH values (2–3) but unstable at high pH conditions (4–6), and the

391

degradation rate of ATCs increases with increased pH value.27

392

In this work, the retention rate of nano-C3G was remarkably higher than of free-C3G

393

at all time points (Figures 2A and B). The t1/2 values of nano-C3G were 144.41 h at pH

394

4.0 and 23.26 h at 7.0 (Figure 2C), which are 4.87 and 7.68 times that of the free-C3G,

395

respectively. Improved thermal stability of C3G via nano-encapsulation has been well

396

documented. For example, the t1/2 value at 37 °C is 2.45 h for free C3G molecules but

397

4.25 h for those loaded within ferritin nanocage.9 The degradation rate of C3G at pH

398

3.0 and 6.0 is decreased by approximately 70% and 84%, respectively, after binding

399

with WPI-Glu nanoparticle.4 These nanocarriers interact with the loaded C3G through

400

non-covalent forces, such as hydrophobic interaction, hydrogen bonding, and

401

electrostatic attraction, which blocks the hydration of stable flavylium cation or

402

quinoidal base forms into carbinol or chalcone structures.30Additional energy is

403

required to disrupt the structure of delivery systems, thereby improving the thermal

404

stability of C3G.4

405

Ascorbic acid is a common additive in food products owing to its preservation and

406

nutritious effects. Ascorbic acid-catalyzed ATC degradation can be attributed to two

407

mechanisms. The first one involves a condensation reaction of ascorbic acid on position

408

4 of the flavylium salt, whereas the second one is a free radical reaction leading to the

409

decomposition of ATCs into phenolic acids.5 As depicted in Figure 3A, free-C3G was

410

degraded stepwise with incubation time in the presence of 0.05% ascorbic acid, and

411

approximately one fifth of C3G was detected at the end of the test. The degradation of 20


Page 20 of 62

Page 21 of 62


412

free-C3G became increasingly pronounced with increased ascorbic acid concentration.

413

The retention rate of free-C3G was below 5% after incubation with 0.10% ascorbic acid

414

for 24 h. This observation was in accordance with previous works, where the chemical

415

stability of ATCs constantly decreased with increased ascorbic acid content from 0.01%

416

to 0.10%.3,5 Nevertheless, contrasting evidence shows that the addition of 0.04% or

417

0.012% ascorbic acid clearly enhances the chemical stability of ATCs, and the adverse

418

effects of ascorbic acid are only observed when its content increased to 0.036%.31 This

419

discrepancy arises because in this report, the chemical stability of ascorbic acid was

420

investigated in soft drink models rather than in aqueous solutions. Therefore, low

421

ascorbic acid concentrations may protect ATCs against degradation by scavenging

422

peroxyl radicals in soft drink.

423

Nano-C3G displayed a remarkably higher retention rate than free-C3G after

424

incubation with 0.05% ascorbic acid for 12 h, whereas remarkable differences in

425

retention rate between free- and nano-C3G are observed at 8 h or later in the presence

426

of 0.10% ascorbic acid. The protective effect of nanogel was also reflected by the

427

improved t1/2 values (Figure 3C). The t1/2 values of nano-C3G with 0.50% and 0.10%

428

ascorbic acid were 37.27 and 29.00 h, respectively, which were 4.40- and 5.72-fold that

429

of free-C3G. Improved ATC stability against ascorbic acid has been realized by

430

liposome encapsulation, and the protective effect is positively correlated with lecithin

431

concentration.5 Xu et al. showed that the t1/2 of purple sweet potato ATCs incubated

432

with 0.60% ascorbic acid is almost doubled after binding with gellan gum.32 The

433

composite nanogel in this work and other delivery systems in references are expected 21



434

to contribute to the chemical stability of C3G by serving as a physical barrier against

435

the exterior harsh environment, inhibiting the penetration of ascorbic acid and other

436

induced free radicals to react with loaded C3G.

437

Simulated digestion. The retention rates of free- and nano-C3G presented a similar

438

evolution pattern during the simulated GI digestion (Figure 4A). Both free- and nano-

439

C3G are chemically stable in the stomach phase, retaining above 90% of their initial

440

level after 1 h of digestion. Flavylium cation is the dominant conformation of

441

monomeric ATCs in the stomach phase, which is favorable for their chemical

442

stability.33 Slight C3G loss is attributed to the release of sugar moieties from its aglycon

443

under extremely acid conditions.34 However, contrasting evidence shows that the C3G

444

content in red wine33 and blueberry35 is increased remarkably after passing through the

445

simulated gastric phase. This discrepancy may arise from the fact that the red wine and

446

blueberry contains high amount of polymeric and bound ATCs, which can be

447

hydrolyzed yielding monomeric C3G. Evident C3G loss occurred when transferred to

448

the small intestinal phase, which was consistent with these previous reports.

449

Furthermore, 35.34% of free-C3G and 78.54% of nano-C3G were recovered from the

450

intestinal fluids, respectively, at the end of the digestion test (Figure 4A). The pH,

451

C3G’s releasability from the carrier matrix, and actions of pancreatic enzymes are

452

responsible for the metabolisms of C3G. Notably, remarkable differences in retention

453

rate between free- and nano-C3G were observed at 90 min (30 min for intestinal

454

digestion) and thereafter. Nanogel contributes to the chemical stability of C3G by

455

segregating it from the digestive fluids in the GI tract. Furthermore, the binding with 22


Page 22 of 62

Page 23 of 62


456

nanogel blocks the transition of C3G flavylium cation conformation into other unstable

457

structures.30

458

The protective effects of nanogel on the C3G are hypothesized to have a close

459

relationship with its proteolysis in the GI tract. Herein, the release of TCA-soluble

460

nitrogen was utilized to investigate the digestion of nanogel (Figure 4B). The nanogel

461

were resistant to the pepsinolysis because only 3.66% of TCA-soluble nitrogen was

462

released from the matrix at the end of the stomach digestion. The covalently attached

463

dextran through Maillard reaction and non-covalently attached pectin by electrostatic

464

forces are hypothesized to shield the cleavage sites of OVA to pepsin, thereby retarding

465

its digestion. Improved pepsinolysis stability of protein by polysaccharide coating has

466

also been confirmed by Sarker et al.36 and Feng et al.23 By contrast, the proteolysis of

467

nanogel became pronounced when they moved from the stomach phase into the small

468

intestinal phase. The amount of released TCA-soluble nitrogen was 27.31% after the

469

whole digestion process. We speculate that the structure of nanogel was disrupted in

470

the small intestinal tract, which facilitated its proteolysis.

471

In the stomach phase, the diameter of nanogel increased appreciably with digestion

472

time, and the DZ was nearly doubled after 60 min (Figure 4C). The surface charge on

473

the C3G-bearing nanogel was slightly positive because the pH condition in stomach

474

was below the pI of OVA, and the carboxyl groups on pectin were almost fully

475

protonated. The digestion of soy proteins promotes the exposure of their hydrophobic

476

clusters onto surface, thereby causing protein aggregation through hydrophobic

477

interactions.13 However, in this work, the aggregation of nanogel were not expected to 23



478

arise from pepsinolysis because the release of TCA-soluble nitrogen in the stomach

479

phase was limited (Figure 4B). One possible explanation is that anionic pepsin

480

molecules in the stomach readily bind to the positively charged spots on different

481

nanogel vehicles. They may build bridges to neighboring vehicles, thereby resulting in

482

aggregation (bridging flocculation).37 Similar results have been reported by Toro-Uribe

483

et al. regarding polyphenol liposomes, where aggregation occurs without obvious

484

phospholipid lysis and membrane disruption.38

485

The diameter of nanogel decreases remarkably when they are transferred to the small

486

intestinal phase (90 min), suggesting that the aggregates formed in the stomach phase

487

has been partially dissociated. This result can be attributed to the dilution effect, which

488

reduces the pepsin concentration and therefore weakens the bridging flocculation.

489

Simultaneously, the nanogel experienced a charge reversal from slightly positive to

490

highly negative (< -30 mV) because of the enhanced deprotonation of both OVA and

491

pectin under neutral conditions (pH 7.0). Both the particle size and magnitude of surface

492

charge increased with prolonged small-intestinal digestion from 90 min to 120 min. The

493

surface-active compounds in the digesta, such as bile salts, displaced the polysaccharide

494

chains absorbed at the surface of nanogel and disrupted the tertiary conformation of

495

protein.23 The structure of nanogel swells as a result of the penetration of the digestive

496

fluids. Consequently, rapid nanogel digestion occurred to nanogel as evidenced by the

497

burst release of TCA-soluble nitrogen at the time range of 120–150 min (Figure 4B),

498

accompanied by a reduction in particle size owing to proteolysis. Thereafter, the

499

particle size and surface charge of nanogels remained almost constant, and the released 24


Page 24 of 62

Page 25 of 62


500

amount of TCA-soluble nitrogen levelled off. These results suggest that nanogel in this

501

work are more resistant to proteolysis than many nanocarriers reported previously,13,22

502

which contributes to the controlled release and chemical stability of encapsulated C3G

503

in the GI tract.

504

Cellular uptake and endocytosis pathways. The cytotoxicity of free- and nano-

505

C3G toward Caco-2 cells was evaluated at first. As depicted in Figure S1, the viability

506

of Caco-2 cells remained above 90% after incubation with free-C3G for 48 h throughout

507

the studied concentrations. By contrast, the cell viability of Caco-2 cells in the nano-

508

C3G group was above 90% only at concentrations below 50.00 μg/mL. The reduced

509

cell viability in the presence of free- or nano-C3G may be associated with the their anti-

510

cancer effects.1 The cell viability in nano-C3G group was obviously lower than that in

511

free-C3G group at 50.00 μg/mg and above. This result may be attributed to the

512

encapsulation by nanogel causing promotion of C3G cellular absorption and therefore

513

its anti-cancer activity. Alternatively, the wall materials of nano-C3G may exert a

514

synergistic effect with C3G in preventing cell proliferation. Consequently, free- and

515

nano-C3G at concentration of 30.00 μg/mg or below were subjected to the subsequent

516

experiments to ensure safety and better comparison.

517

To investigate the effect of nano-encapsulation on the cellular uptake of C3G, we

518

incubated Caco-2 cells with free- and nano-C3G at predetermined concentrations and

519

times, after which the C3G contents in cells were quantitatively analyzed and compared.

520

The major route for ATC absorption by enterocytes is passive diffusion, and many

521

active transporters are involved depending on their structure feature.7,39 As depicted in 25



522

Figure 5A, the cellular uptake of free-C3G was only 0.38 μg/mg after 2 h of incubation

523

at 5.00 μg/mL and increased in a concentration-dependent manner, reaching a plateau

524

of approximately 1 μg/mg protein at 20.00 μg/mL. As depicted in Figure 5B, the C3G

525

accumulation in Caco-2 cells was also time-dependent, yielding 0.92 μg/mg protein

526

after 4 h of incubation. The low cellular uptake efficiency of C3G has been reported in

527

a previous work regarding ATC extract from blueberry, where only 0.19%–0.24% of

528

the initial C3G level is detected in Caco-2 cells after 2 h.8 This result is closely

529

associated with the poor lipophilic properties (log P ≈ 0.39) of C3G, which restrict its

530

partition into cell membrane.7 Furthermore, the great number of free hydroxyl groups

531

on C3G delays its retention at the aqueous–membrane interface by forming hydrogen

532

bond with the polar groups of lipid molecules.8

533

As depicted in Figure 5A, the cellular uptake of nano-C3G was 2.55-, 3.35-, 3.25-,

534

and 3.55-fold that of free-C3G at 5.00, 10.00, 20.00, and 30.00 μg/mL, respectively.

535

The cellular uptake of nano-C3G was generally twofold to threefold that of free-C3G

536

at the same incubation time (Figure 5B). These results suggest that nanogel

537

encapsulation enhances the C3G absorption in Caco-2 cells. Various “degradable”

538

nanocarriers have been designed to improve the cellular uptake of polyphenols. For

539

example, the uptake efficiency of quercetin by Caco-2 cells increased from ~1%, ~2%,

540

and ~12% to ~12.5%, ~15%, and ~17.5%, respectively, at 1, 2, and 4 h of incubation

541

time, after being incorporated into zein–chitosan nanoparticles.40 Similarly, Xue et al.41

542

suggested that the cellular uptake of nano-Cur in zein–sodium caseinate nanoparticles

543

is approximately twofold that of the free Cur. Nanocarriers are proposed to be directly 26


Page 26 of 62

Page 27 of 62


544

internalized into cells through endocytosis,42,43 which is perhaps the major route for the

545

composite nanogel to enhance the cellular uptake of loaded C3G. Therefore, related

546

experiments are performed subsequently to investigate the role of endocytosis on nano-

547

C3G absorption.

548

The cellular accumulation of C3G in Caco-2 cells drops off remarkably when they

549

are maintained at 4 °C (P < 0.001) or treated with sodium azide (P < 0.01) (Figure 6),

550

a metabolic inhibitor which blocks ATP production in cells,24 suggesting that C3G-

551

bearing nanogel is internalized through active and energy-dependent endocytosis. The

552

endocytosis of nanocarriers by enterocyte can be further divided into clathrin-related

553

route, caveolae-related route, macropinocytosis, and clathrin- and caveolae-

554

independent routes.17,42 As illustrated in Figure 6, inhibitors cause diverse effects on

555

the cellular accumulation of nano-C3G. Chlorpromazine, which blocks the assembly of

556

clathrin at the cell membrane, is used to inhibit clathrin-related endocytosis.44

557

Chlorpromazine treatment led to a one-third reduction in the cellular uptake of nano-

558

C3G (P < 0.01 vs. control). Therefore, the endocytosis of C3G-bearing nanogel is

559

confirmed to be mediated by the clathrin-related route. The clathrin-related route is the

560

“classic pathway” for Caco-2 cell entry, which occurs prevalently to cationic chitosan

561

nanoparticles,45 anionic protein nanoparticles,43 and nonionic micelles.24 MβCD can

562

bind to cholesterol in the plasma membrane, thereby disrupting the integrity of caveolae

563

and lipid rafts.42 The accumulation of nano-C3G in Caco-2 decreased significantly (P

564

< 0.05 vs. control) in the presence of MβCD, indicating that caveolae-mediated route

565

was also involved. Different from clathrin- and caveolae-mediated pathways, 27



Page 28 of 62

566

macropinocytosis

567

Extracellular particles triggering the macropinocytosis of cells are internalized via

568

plasma membrane ruffling that forms macropinosomes with a diameter above 1 mm.

569

EIPA, a sodium-proton exchange blocking agent derived from amiloride,47 inhibited

570

the cellular accumulation of nano-C3G significantly (P < 0.05 vs. control), directly

571

demonstrating the endocytosis of C3G-bearing nanogel via macropinocytosis pathway.

572

Particles with a diameter above 150 nm can be internalized by macropinocytosis

573

pathway,48 and the DZ of the C3G-bearing nanogel in this work was 185.67 nm (Figure

574

1A). By contrast, quercetin, an inhibitor of clathrin- and caveolae-independent

575

pathways, caused no remarkable changes in nano-C3G uptake, demonstrating that the

576

endocytosis of C3G-bearing nanogel was not through clathrin- and caveolae-

577

independent routes. In summary, the clathrin-, caveolae-related routes and

578

macropinocytosis pathway contributed to the endocytosis of C3G-bearing nanogel. Soy

579

protein isolate (SPI) nanoparticles improves the absorption of VB12 via multiple

580

pathways,

581

macropinocytosis routes,43 which is consistent with our present work. By contrast, Liu

582

et al. revealed that soybean Bowman-Birk inhibitor nanoparticles are internalized into

583

the cytoplasm via only clathrin-mediated endocytosis.12 These results suggest that

584

endocytosis routes involved in the internalization of “biodegradable” nanocarriers are

585

different according to their structure feature.

is

including

a

dynamin-independent

clathrin-

and/or

bulk

internalization

caveolae-mediated

process.46

endocytosis

and

586

Monolayer permeability and transcytosis pathways. Caco-2 monolayers were

587

established in this work to estimate the intestinal permeability of free- and nano-C3G. 28


Page 29 of 62


588

Only wells with a TEER value above 400 Ω·cm2 were utilized in this work to ensure

589

the integrity and confluence of monolayers.49 The permeation rate of free- and nano-

590

C3G at 20.00 μg/mL concentration was then determined in the AP-to-BL direction. As

591

shown in Figure 7A, the fraction of free-C3G transported across the Caco-2 monolayers

592

increased linearly with prolonged incubation time, reaching a value of 2.05% after 3 h.

593

The work by Yi et al. (2006) reported that the transported fraction of C3G in blueberry

594

extracts across the Caco-2 monolayers is 3%–4% in average after 2 h of incubation.8

595

By contrast, the transport efficiency of C3G in the digested jambo fruit were only

596

approximately 0.3%, 0.7%, and 0.8% after 0.5, 1, and 2 h of incubation, respectively.10

597

This discrepancy suggests that other components in the food matrix would appreciably

598

affect the intestinal permeation of C3G. Herein, the Papp value of free-C3G was

599

calculated to be (0.67 ± 0.05) × 10-6 cm/s (Figure 7B), which was below 1 × 10-6 cm/s,

600

a common cutoff to suggest a high permeation ability.50 The intestinal barrier is

601

therefore considered a limiting factor for the oral bioavailability of free-C3G.

602

By contrast, 4.31% of initial nano-C3G was detected in the BL compartment after 3

603

h (Figure 7A), and the Papp value of nano-C3G was 2.16-fold that of free-C3G (P
0.05 for both vs. control;

719

P < 0.05 for free-C3G while P < 0.01 for nano-C3G vs. oxidative group). 34


Page 34 of 62

Page 35 of 62


720

SOD catalyzes the harmful superoxide radicals to H2O2 and molecular oxygen,55 and

721

CAT and GSH-px further convert H2O2 to water. GSH-px is also responsible for the

722

scavenging of the lipid peroxides by reducing GSSH to GSH.56 They are important

723

antioxidant enzymes in normal organisms to protect cells against oxidative damage.

724

The levels of antioxidant enzymes were evaluated herein to confirm whether the

725

protective effects of C3G are associated with the modulation of endogenous antioxidant

726

defense system.

727

The SOD, CAT, and GSH-px levels in Caco-2 cells decreased remarkably after

728

exposure to 700 μM of H2O2 (P < 0.001 for SOD and GSH-px and P < 0.01 for CAT

729

vs. control) (Figures 12B, C, and D), suggesting that the endogenous antioxidant system

730

was disrupted by the accumulated oxidative stress. Pretreating Caco-2 cells with free-

731

C3G of 2.50 μg/mL led to a 42.24% increase in SOD activity, and the protective effect

732

of free-C3G became significant at elevated concentration (5.00 μg/mL) (P < 0.01 vs.

733

oxidative group) (Figure 12B). The SOD activity in Caco-2 cells pre-incubated with

734

2.50 and 5.00 μg/mL of nano-C3G was 42.39 and 43.83 U/mg, respectively, which

735

were higher than that of the oxidative group (P < 0.05 for 2.50 μg/mL and P < 0.01 for

736

5.00 μg/mL). Significant difference between free- and nano-C3G groups was observed

737

at 2.50 μg/mL. The CAT activities in Caco-2 cells pretreated with free- and nano-C3G

738

of 2.50 μg/mL were 1.38- and 1.49-fold that of the oxidative group (Figure 12C),

739

respectively, but their protective effects were insignificant. Free- and nano-C3G groups

740

of 5.00 μg/mL exhibited significantly higher CAT activity than the oxidative group (P

741

< 0.05 for free-C3G and P < 0.01 for nano-C3G), whose levels were 22.23 and 23.59 35



742

U/mg, respectively. Similarly, the protective effects of free- and nano-C3G on GSH-px

743

activity were also concentration-dependent. As depicted in Figure 12D, pre-incubation

744

with free- and nano-C3G of 2.50 μg/mL improved the GSH-px level in Caco-2 cells to

745

1.43- and 1.82-fold that of the oxidative control (P < 0.05 for nano-C3G), respectively.

746

No significant differences were observed in GSH-px level between the control group

747

and those pretreated with 5.00 μg/mL C3G regardless of its form, indicating that the

748

GSH-px activity was recovered to normal level. Moreover, nano-C3G was more

749

effective in restoring GSH-px activity than free-C3G at this concentration (P < 0.05).

750

In the present work, C3G can alleviate the H2O2-induced oxidative stress by

751

decreasing the ROS level, inhibiting MDA accumulation, and recovering endogenous

752

antioxidant enzymes. This result has been further confirmed by Qin et al.,4 who reported

753

that C3G can reduce the ROS and MDA level while improving GSH content in H2O2-

754

treated MKN-28 cells in a concentration-dependent manner. In a recent work, amyloid-

755

beta (1-40)-induced damage model was utilized to investigate the antioxidant

756

mechanisms of C3G.57 These authors revealed that, apart from reducing ROS

757

generation and improving activities of SOD and GSH-px, C3G also protects cells

758

against oxidative damage by regulating the Nrf2 pathway and apoptosis-related genes,

759

such as Bcl-2 and Bax. The protective effects of C3G against oxidative stress induced

760

by H2O2 were also observed in pancreatic β-cells, where C3G reduced the levels of

761

intracellular ROS and MDA, regulated ERK and p38 phosphorylation, and inhibited

762

MAPK signaling.58 These results suggest that C3G as a kind of ATCs has good research

763

value for its strong antioxidant activity. 36


Page 36 of 62

Page 37 of 62


764

Information about the effect of nanoencapsulation on the antioxidant activity of C3G

765

is still currently limited. Herein, we revealed that nano-C3G is more effective in

766

recovering the cell viability and activities of SOD and GSH-px than C3G at certain

767

concentrations. Two main factors account for the improved antioxidant activity of C3G

768

in nanogels. First, as discussed above, nanoencapsulation promoted the cellular

769

absorption of C3G by multiple energy-dependent endocytosis pathways. Moreover,

770

C3G in the internalized nanogels would be released in a sustainable manner,14 which is

771

favorable for maintaining its chemical stability in the cytoplasm by avoiding extensive

772

metabolism. Similarly, in a recent work, C3G bound with WPI-Glu conjugates are more

773

effective in blocking ROS generation in Caco-2 models than the free one because the

774

conjugates are speculated to inhibit its chemical degradation during the pre-incubation

775

process.4 Improved free radical-scavenging stability of C3G is also realized by

776

nanoliposome encapsulation.15 However, these experiments were performed by in vivo

777

DPPH and ABTS assays rather than cell models. The specific mechanism regarding the

778

promoting effects of nanoencapsulation on the cellular antioxidant activity of C3G still

779

requires further investigation.

780

In summary, C3G-bearing composite nanogel with a particle size below 200 nm was

781

fabricated in this work. It presented good pH- and NaCl-stability and contributed to the

782

chemical stability of C3G against various environmental stresses. The nanogel was

783

more resistant to proteolysis than many previously reported nanocarriers. The cellular

784

uptake of nano-C3G was significantly higher than free-C3G, and the endocytosis of

785

nano-C3G appeared to be regulated by clathrin- and caveolae-related routes and 37



786

macropinocytosis pathway. The Papp value of nano-C3G was more than twofold that of

787

free-C3G. The transcytosis of nano-C3G across the Caco-2 monolayers occurred

788

through clathrin-related route and macropinocytosis pathway. CRE, ER, Golgi complex,

789

microtubules, and lysosomes appeared to be involved in its intracellular trafficking.

790

Nano-C3G was also more effective in attenuating the oxidative damage induced by

791

H2O2 compared with free-C3G owing to its improved cellular absorption. The

792

biodegradable nanogel fabricated herein is suitable for the delivery of C3G as

793

functional ingredients with enhanced chemical stability, intestinal absorption and

794

cellular antioxidant capacity.

795

ASSOCIATED CONTENTS

796

Supporting information

797

Cytotoxicity of free- and nano-C3G on Caco-2 cells. Effect of free- and nano-C3G on

798

the TEER values of Caco-2 monolayers during the intestinal permeation study.

799

AUTHOR INFORMATION

800

Corresponding authors

801

*Tel:

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

802

*Tel:

86-25-84392191. Fax: 86-25-84392191. E-mail: [email protected] (C.L.).

803

Funding

804

This work was supported by the National Postdoctoral Program for Innovative Talents

805

(BX201700101), the China Postdoctoral Science Foundation Funded Project

806

(2017M621668), the Natural Science Foundation of Jiangsu Province (BK20180298),

807

and the National Natural Science Foundation of China (31801555; 31601435). 38


Page 38 of 62

Page 39 of 62


808

Notes

809

The authors declare no competing financial interest.

810

REFERENCES

811

1.

812

potential health benefits of haskap (Lonicera caerulea L.): Role of cyanidin-3-O-

813

glucoside. J. Funct. Foods 2018, 44, 24-39.

814

2.

815

nonacylated anthocyanins. J. Food Sci. 2006, 71, C504-C512.

816

3.

817

loaded nanocomplexes prepared with chitosan hydrochloride and carboxymethyl

818

chitosan. Food Hydrocolloid. 2018, 74, 23-31.

819

4.

820

Y.; Liu, G., Maillard-reacted whey protein isolates enhance thermal stability of

821

anthocyanins over a wide pH range. J. Agric. Food Chem. 2018, 66, 9556-9564.

822

5.

823

liposomal encapsulation on degradation of anthocyanins of black carrot extract by

824

adding ascorbic acid. Food Funct. 2017, 8, 1085-1093.

825

6.

826

Botting, N. P.; Kay, C. D., Human metabolism and elimination of the anthocyanin,

827

cyanidin-3-glucoside: a C-13-tracer study. Am. J. Clin. Nutr. 2013, 97, 995-1003.

828

7.

829

scheme: Classifying nutraceuticals according to factors limiting their oral

Rupasinghe, H. P. V.; Arumuggam, N.; Amararathna, M.; De Silva, A., The

Sadilova, E.; Stintzing, F. C.; Carle, R., Thermal degradation of acylated and

Ge, J.; Yue, P.; Chi, J.; Liang, J.; Gao, X., Formation and stability of anthocyanins-

Qin, X. G.; Yuan, D.; Wang, Q.; Hu, Z. Z.; Wu, Y.; Cai, J.; Huang, Q. R.; Li, S.

Guldiken, B.; Gibis, M.; Boyacioglu, D.; Capanoglu, E.; Weiss, J., Impact of

Czank, C.; Cassidy, A.; Zhang, Q. Z.; Morrison, D. J.; Preston, T.; Kroon, P. A.;

McClements, D. J.; Li, F.; Xiao, H., The nutraceutical bioavailability classification

39



830

bioavailability. Annu. Rev. Food Sci. T. 2015, 6, 299-327.

831

8.

832

blueberry extracts by Caco-2 human intestinal cell monolayers. J. Agric. Food Chem.

833

2006, 54, 5651-5658.

834

9.

835

anthocyanin molecules within a ferritin nanocage increases their stability and cell

836

uptake efficiency. Food Res. Int. 2014, 62, 183-192.

837

10. Peixoto, F. M.; Fernandes, I.; Gouvea, A. C. M. S.; Santiago, M. C. P. A.; Borguini,

838

R. G.; Mateus, N.; Freitas, V.; Godoy, R. L. O.; Ferreira, I. M. P. L. V. O., Simulation

839

of in vitro digestion coupled to gastric and intestinal transport models to estimate

840

absorption of anthocyanins from peel powder of jabuticaba, jamelao and jamb fruits. J.

841

Funct. Foods 2016, 24, 373-381.

842

11. Peinado, I.; Lesmes, U.; Andres, A.; McClements, D. J., Fabrication and

843

morphological characterization of biopolymer particles formed by electrostatic

844

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

845

26, 9827-9834.

846

12. Liu, C.; Cheng, F.; Yang, X., Fabrication of a soybean bowman–birk inhibitor (bbi)

847

nanodelivery carrier to improve bioavailability of curcumin. J. Agric. Food Chem. 2017,

848

65, 2426-2434.

849

13. Chen, F. P.; Li, B. S.; Tang, C. H., Nanocomplexation between curcumin and soy

850

protein isolate: influence on curcumin stability/bioaccessibility and in vitro protein

851

digestibility. J. Agric. Food Chem. 2015, 63, 3559-3569.

Yi, W. G.; Akoh, C. C.; Fischer, J.; Krewer, G., Absorption of anthocyanins from

Zhang, T.; Lv, C.; Chen, L.; Bai, G.; Zhao, G.; Xu, C., Encapsulation of

40


Page 40 of 62

Page 41 of 62


852

14. Liu, Z. H.; Hu, Y. F.; Li, X.; Mei, Z. X.; Wu, S.; He, Y.; Jiang, X. W.; Sun, J. X.;

853

Xiao, J. B.; Deng, L. H.; Bai, W. B., Nanoencapsulation of cyanidin-3-O-glucoside

854

enhances protection against uvb-induced epidermal damage through regulation of p53-

855

mediated apoptosis in mice. J. Agric. Food Chem. 2018, 66, 5359-5367.

856

15. Liang, T. S.; Guan, R. F.; Wang, Z.; Shen, H. T.; Xia, Q. L.; Liu, M. Q.,

857

Comparison of anticancer activity and antioxidant activity between cyanidin-3-O-

858

glucoside liposomes and cyanidin-3-O-glucoside in Caco-2 cells in vitro. RSC Adv.

859

2017, 7, 37359-37368.

860

16. Yameen, B.; Choi, W. I.; Vilos, C.; Swami, A.; Shi, J. J.; Farokhzad, O. C., Insight

861

into nanoparticle cellular uptake and intracellular targeting. J. Control. Release 2014,

862

190, 485-499.

863

17. Chai, G. H.; Xu, Y. K.; Chen, S. Q.; Cheng, B. L.; Hu, F. Q.; You, J. A.; Du, Y.

864

Z.; Yuan, H., Transport mechanisms of solid lipid nanoparticles across caco-2 cell

865

monolayers and their related cytotoxicology. ACS Appl. Mater. Inter. 2016, 8, 5929-

866

5940.

867

18. Jones, O. G.; Decker, E. A.; McClements, D. J., Comparison of protein-

868

polysaccharide nanoparticle fabrication methods: Impact of biopolymer complexation

869

before or after particle formation. J. Colloid Interf. Sci. 2010, 344, 21-29.

870

19. Visentini, F. F.; Sponton, O. E.; Perez, A. A.; Santiago, L. G., Formation and

871

colloidal stability of ovalbumin-retinol nanocomplexes. Food Hydrocolloid. 2017, 67,

872

130-138.

873

20. Dai, Q.; Zhu, X.; Abbas, S.; Karangwa, E.; Zhang, X.; Xia, S.; Feng, B.; Jia, C., 41



874

Stable nanoparticles prepared by heating electrostatic complexes of whey protein

875

isolate-dextran conjugate and chondroitin sulfate. J. Agric. Food Chem. 2015, 63, 4179-

876

4189.

877

21. Davidov-Pardo, G.; Joye, I. J.; Espinal-Ruiz, M.; McClements, D. J., Effect of

878

maillard conjugates on the physical stability of zein nanoparticles prepared by liquid

879

antisolvent coprecipitation. J. Agric. Food Chem. 2015, 63, 8510-8518.

880

22. Feng, J.; Wu, S.; Wang, H.; Liu, S., Improved bioavailability of curcumin in

881

ovalbumin-dextran nanogels prepared by Maillard reaction. J. Funct. Foods 2016, 27,

882

55-68.

883

23. Feng, J.; Xu, H.; Zhang, L.; Wang, H.; Liu, S.; Liu, Y.; Hou, W.; Li, C.,

884

Development of nanocomplexes for curcumin vehiculization using ovalbumin and

885

sodium alginate as building blocks: Improved stability, bioaccessibility, and

886

antioxidant activity. J. Agric. Food Chem. 2019, 67, 379-390.

887

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

888

improving curcumin oral absorption: In vitro and in vivo. Colloid. Surface. B 2015,

889

133, 108-119.

890

25. Wagoner, T.; Vardhanabhuti, B.; Foegeding, E. A., Designing whey protein-

891

polysaccharide particles for colloidal stability. Annu. Rev. Food Sci. T. 2016, 7, 93-116.

892

26. Zhou, M. Y.; Hu, Q. B.; Wang, T. R.; Xue, J. Y.; Luo, Y. C., Effects of different

893

polysaccharides on the formation of egg yolk LDL complex nanogels for nutrient

894

delivery. Carbohyd. Polym. 2016, 153, 336-344.

895

27. Ge, J.; Yu, X. Y.; Wang, S.; Chi, J. P.; Liang, J.; Sun, Y.; Gao, X. L.; Yue, P. X., 42


Page 42 of 62

Page 43 of 62


896

Nanocomplexes composed of chitosan derivatives and beta-Lactoglobulin as a carrier

897

for anthocyanins: Preparation, stability and bioavailability in vitro. Food Res. Int. 2019,

898

116, 336-345.

899

28. Feng, J.; Cai, H.; Wang, H.; Li, C. Y.; Liu, S. B., Improved oxidative stability of

900

fish oil emulsion by grafted ovalbumin-catechin conjugates. Food Chem. 2018, 241,

901

60-69.

902

29. Swer, T. L.; Mukhim, C.; Bashir, K.; Chauhan, K., Optimization of enzyme aided

903

extraction of anthocyanins from Prunus nepalensis L. LWT-Food Sci. Technol. 2018,

904

91, 382-390.

905

30. Klimaviciute, R.; Navikaite, V.; Jakstas, V.; Ivanauskas, L., Complexes of dextran

906

sulfate and anthocyanins from vaccinium myrtillus: Formation and stability. Carbohyd.

907

Polym. 2015, 129, 70-78.

908

31. Li, J.; Song, H.; Dong, N.; Zhao, G., Degradation kinetics of anthocyanins from

909

purple sweet potato (Ipomoea batatas L.) as affected by ascorbic acid. Food Sci.

910

Biotechnol. 2014, 23, 89-96.

911

32. Xu, X.-J.; Fang, S.; Li, Y.-H.; Zhang, F.; Shao, Z.-P.; Zeng, Y.-T.; Chen, J.; Meng,

912

Y.-C., Effects of low acyl and high acyl gellan gum on the thermal stability of purple

913

sweet potato anthocyanins in the presence of ascorbic acid. Food Hydrocolloid. 2019,

914

86, 116-123.

915

33. Yang, P.; Yuan, C.; Wang, H.; Han, F.; Liu, Y.; Wang, L.; Liu, Y., Stability of

916

anthocyanins and their degradation products from cabernet sauvignon red wine under

917

gastrointestinal pH and temperature conditions. Molecules 2018, 23, 1-20. 43



918

34. He, J.; Wallace, T. C.; Keatley, K. E.; Failla, M. L.; Giusti, M. M., Stability of

919

black raspberry anthocyanins in the digestive tract lumen and transport efficiency into

920

gastric and small intestinal tissues in the rat. J. Agric. Food Chem. 2009, 57, 3141-3148.

921

35. Correa-Betanzo, J.; Allen-Vercoe, E.; McDonald, J.; Schroeter, K.; Corredig, M.;

922

Paliyath, G., Stability and biological activity of wild blueberry (Vaccinium

923

angustifolium) polyphenols during simulated in vitro gastrointestinal digestion. Food

924

Chem. 2014, 165, 522-531.

925

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

926

gastric digestion of emulsions using composite whey protein-cellulose nanocrystal

927

interfaces. Colloid. Surface. B 2017, 158, 137-146.

928

37. McClements, D. J., Theoretical analysis of factors affecting the formation and

929

stability of multilayered colloidal dispersions. Langmuir 2005, 21, 9777-9785.

930

38. Toro-Uribe, S.; Ibanez, E.; Decker, E. A.; McClements, D. J.; Zhang, R.; Lopez-

931

Giraldo, L. J.; Herrero, M., Design, fabrication, characterization, and in vitro digestion

932

of alkaloid-, catechin-, and cocoa extract-loaded liposomes. J. Agric. Food Chem. 2018,

933

66, 12051-12065.

934

39. Oliveira, H.; Perez-Gregório, R.; de Freitas, V.; Mateus, N.; Fernandes, I.,

935

Comparison of the in vitro gastrointestinal bioavailability of acylated and non-acylated

936

anthocyanins: Purple-fleshed sweet potato vs red wine. Food Chem. 2019, 276, 410-

937

418.

938

40. Ma, J.-J.; Yu, Y.-G.; Yin, S.-W.; Tang, C.-H.; Yang, X.-Q., Cellular uptake and

939

intracellular antioxidant activity of zein/chitosan nanoparticles incorporated with 44


Page 44 of 62

Page 45 of 62


940

quercetin. J. Agric. Food Chem. 2018, 66, 12783-12793.

941

41. Xue, J.; Zhang, Y.; Huang, G.; Liu, J.; Slavin, M.; Yu, L., Zein-caseinate composite

942

nanoparticles for bioactive delivery using curcumin as a probe compound. Food

943

Hydrocolloid. 2018, 83, 25-35.

944

42. Sahay, G.; Alakhova, D. Y.; Kabanov, A. V., Endocytosis of nanomedicines. J.

945

Control. Release 2010, 145, 182-195.

946

43. Zhang, J.; Field, C. J.; Vine, D.; Chen, L., Intestinal uptake and transport of vitamin

947

B-12-loaded soy protein nanoparticles. Pharm. Res.-Dordr 2015, 32, 1288-1303.

948

44. Jin, Y.; Song, Y.; Zhu, X.; Zhou, D.; Chen, C.; Zhang, Z.; Huang, Y., Goblet cell-

949

targeting nanoparticles for oral insulin delivery and the influence of mucus on insulin

950

transport. Biomaterials 2012, 33, 1573-1582.

951

45. Ma, Z. S.; Lim, L. Y., Uptake of chitosan and associated insulin in Caco-2 cell

952

monolayers: A comparison between chitosan molecules and chitosan nanoparticles.

953

Pharm. Res.-Dordr 2003, 20, 1812-1819.

954

46. Kerr, M. C.; Teasdale, R. D., Defining macropinocytosis. Traffic 2009, 10, 364-

955

371.

956

47. Nakase, I.; Niwa, M.; Takeuchi, T.; Sonomura, K.; Kawabata, N.; Koike, Y.;

957

Takehashi, M.; Tanaka, S.; Ueda, K.; Simpson, J. C.; Jones, A. T.; Sugiura, Y.; Futaki,

958

S., Cellular uptake of arginine-rich peptides: Roles for macropinocytosis and actin

959

rearrangement. Mol. Ther. 2004, 10, 1011-1022.

960

48. Mailander, V.; Landfester, K., Interaction of nanoparticles with cells.

961

Biomacromolecules 2009, 10, 2379-2400. 45



962

49. Elendran, S.; Muniyandy, S.; Lee, W. W.; Palanisamy, U. D., Permeability of the

963

ellagitannin geraniin and its metabolites in a human colon adenocarcinoma Caco-2 cell

964

culture model. Food Funct. 2019, 10, 602-615.

965

50. Artursson, P.; Palm, K.; Luthman, K., Caco-2 monolayers in experimental and

966

theoretical predictions of drug transport. Adv. Drug Deliver. Rev. 2012, 64, 280-289.

967

51. Miller, S. G.; Carnell, L.; Moore, H. P. H., Post-Golgi membrane traffic: brefeldin

968

A inhibits export from distal Golgi compartments to the cell surface but not recycling.

969

J. Cell Biol. 1992, 118, 267-283.

970

52. Bayer, N.; Schober, D.; Prchla, E.; Murphy, R. F.; Blaas, D.; Fuchs, R., Effect of

971

bafilomycin A1 and nocodazole on endocytic transport in HeLa cells: Implications for

972

viral uncoating and infection. J. Virol. 1998, 72, 9645-9655.

973

53. Matias, A.; Nunes, S. L.; Poejo, J.; Mecha, E.; Serra, A. T.; Amorim Madeira, P.

974

J.; Bronze, M. R.; Duarte, C. M. M., Antioxidant and anti-inflammatory activity of a

975

flavonoid-rich concentrate recovered from Opuntia ficus-indica juice. Food Funct.

976

2014, 5, 3269-3280.

977

54. Schieber, M.; Chandel, N. S., ROS function in redox signaling and oxidative stress.

978

Curr. Biol. 2014, 24, R453-R462.

979

55. Gill, S. S.; Tuteja, N., Reactive oxygen species and antioxidant machinery in

980

abiotic stress tolerance in crop plants. Plant Physiol. Bioch. 2010, 48, 909-930.

981

56. Bhabak, K. P.; Mugesh, G., Functional mimics of glutathione peroxidase:

982

Bioinspired synthetic antioxidants. Accounts Chem. Res. 2010, 43, 1408-1419.

983

57. Meng, L.-s.; Li, B.; Li, D.-n.; Wang, Y.-h.; Lin, Y.; Meng, X.-j.; Sun, X.-y.; Liu, 46


Page 46 of 62

Page 47 of 62


984

N., Cyanidin-3-O-glucoside attenuates amyloid-beta (1-40)-induced oxidative stress

985

and apoptosis in SH-SY5Y cells through a Nrf2 mechanism. J. Funct. Foods 2017, 38,

986

474-485.

987

58. Lee, J. S.; Kim, Y. R.; Song, I. G.; Ha, S. J.; Kim, Y. E.; Baek, N. I.; Hong, E. K.,

988

Cyanidin-3-glucoside isolated from mulberry fruit protects pancreatic beta-cells against

989

oxidative stress-induced apoptosis. Int. J Mol. Med. 2015, 35, 405-412.

47



990

FIGURE CAPTIONS

991

Figure 1. Size distribution (A), surface morphology (bar: 200 nm) (B), FTIR spectra

992

(C), pH stability (D), and NaCl stability (E) of the composite nanogel.

993

Figure 2. Degradation of free- and nano-C3G during thermal treatment at 60 °C and

994

pH 4.0 (A) or 7.0 (B) and the corresponding t1/2 values (C). *, **, and *** represent P

995

< 0.05, 0.01, and 0.001, respectively, compared with the corresponding free-C3G group.

996

Figure 3. Degradation of free- and nano-C3G in the presence of 0.05% (A) or 0.10%

997

(B) ascorbic acid at 40 °C and pH 4.0 and the corresponding t1/2 values (C). *, **, and

998

*** represent P < 0.05, 0.01, and 0.001, respectively, compared with the corresponding

999

free-C3G group.

1000

Figure 4. The retention rate of free- and nano-C3G (A), release of TCA-soluble

1001

nitrogen (B), and changes in the particle size and surface charge of C3G-bearing

1002

nanogel (C) as a function of digestion time.

1003

Figure 5. Effects of C3G concentration (5.00, 10.00, 20.00, and 30.00 μg/mL) (A) and

1004

incubation time (0.5, 1, 2, and 4 h) (B) on the cellular uptake of free- and nano-C3G. *

1005

and ** represent P < 0.05 and 0.01, respectively, compared with the corresponding free-

1006

C3G group.

1007

Figure 6. Effects of various endocytosis inhibitors on the cellular uptake of nano-C3G.

1008

*, **, and *** represent P < 0.05, 0.01, and 0.001, respectively, compared with the

1009

control.

1010

Figure 7. (A) Transport of free- and nano-C3G across the Caco-2 monolayers over time.

1011

(B) Papp values of free- and nano-C3G. ** represents P < 0.01 compared with the free48


Page 48 of 62

Page 49 of 62


1012

C3G group.

1013

Figure 8. Effects of various endocytosis (A) and intracellular trafficking inhibitors (B)

1014

on the transport of nano-C3G across the Caco-2 monolayers. *, **, and *** represent

1015

P < 0.05, P < 0.01, and P < 0.001, respectively, compared with the control.

1016

Figure 9. Schematic diagram of the endocytosis and transcytosis mechanisms of nano-

1017

C3G in Caco-2 cell models.

1018

Figure 10. Effect of free- and nano-C3G (2.50 and 5.00 μg/mL) on the viability of

1019

Caco-2 cells. *** represents P < 0.001, compared with the control; ## and ### represent

1020

P < 0.01 and P < 0.001, respectively, compared with the oxidative group; ΔΔ represent

1021

P < 0.01, compared with free-C3G group of the same concentration.

1022

Figure 11. Qualitative (a) and quantitative analyses (b) of the effect of free- and nano-

1023

C3G (2.50 and 5.00 μg/mL) on the ROS levels in Caco-2 cells. *** represents P < 0.001,

1024

compared with the control; #, ##, and ### represent P < 0.05, P < 0.01, and P < 0.001,

1025

respectively, compared with the oxidative group.

1026

Figure 12. Effect of free- and nano-C3G (2.50 and 5.00 μg/mL) on MDA, SOD, CAT,

1027

and GSH-px levels in Caco-2 cells. *, **, and *** represent P < 0.05, P < 0.01, and P

1028

< 0.001, respectively, compared with the control; #, ##, and ### represent P < 0.05, P

1029

< 0.01, and P < 0.001, respectively, compared with the oxidative group. Δ represent P

1030

< 0.05, compared with free-C3G group of the same concentration.

49



Figure 1

50


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Figure 2

51



Figure 3

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Figure 4

53



Figure 5

54


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Figure 6

55



Figure 7

56


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Figure 8

57



Figure 9

58


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Figure 10

59



Figure 11

60


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Figure 12

61



TOC graphic

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Enhanced chemical stability, intestinal absorption, and intracellular

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