Development of oral delivery systems with enhanced antioxidant and

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Food and Beverage Chemistry/Biochemistry

Development of oral delivery systems with enhanced antioxidant and anticancer activity: Coix seed oil and #-carotene co-loaded liposomes Chunqing Bai, Jingxia Zheng, Li Zhao, Lili Chen, Hua Xiong, and David Julian McClements J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b04879 • 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 oral delivery systems with enhanced antioxidant and

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anticancer activity: Coix seed oil and β-carotene co-loaded liposomes

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Chunqing Bai1,3, Jingxia Zheng2, Li Zhao1, LiLi Chen1, Hua Xiong2*, and David Julian

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McClements3*

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1National

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Science and Technology Normal University，Nanchang 330013，China

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2State

R&D Branch Center for Freshwater Fish Processing，College of Life Science，Jiangxi

Key Laboratory of Food Science and Technology, Nanchang University, Nanchang

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330047, China

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3Department

of Food Science, University of Massachusetts, Amherst, MA 01003, USA

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Corresponding Authors:

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Hua Xiong, State Key Laboratory of Food Science and Technology, Nanchang University,

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Nanchang 330047, China. Tel: (86) 13607911660. E-mail: [email protected]

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David Julian McClements, Department of Food Science, University of Massachusetts, Amherst,

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MA

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[email protected]

01003,

USA.

Tel:

(413)

545-1019.

Fax:

1

ACS Paragon Plus Environment

(413)

545-1262.

E-mail:


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ABSTRACT

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Fortifying food and beverage products with combinations of bioactive agents is a major

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initiative within the food industry because of their potentially additive or even synergistic

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benefits on human health. Coix seed oil (CSO) has been reported to possess anticancer activity,

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whereas β-carotene (βC) is a natural antioxidant that may also exhibit anticancer activity.

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However, both of these bioactives are insoluble in water and have poor oral bioavailability. The

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aim of this study was to overcome these obstacles by encapsulating both βC and CSO into

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liposomes (L-βC-CSO). The effect of different combinations of these two bioactive agents on the

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physiochemical properties, stability, release, antioxidant, and anticancer activity of the liposomes

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was then determined. Increasing the CSO level decreased the βC entrapment efficiency, increased

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the particle size, reduced the polydispersity, and raised the magnitude of the surface potential of

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the bioactive-loaded liposomes. Moreover, the βC and CSO levels affected their orientation

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within the lipid bilayer, which also influences the physiochemical properties, stability, and in

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vitro release behavior of the system. Compared to liposomes containing single bioactive types,

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the combined systems exhibited higher bioavailability, increased anticancer and antioxidant

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activity. These results suggest that the combined bioactive-loaded liposomes could be an

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efficient formulation for potential applications in functional foods and supplements.

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Keywords: β-carotene; Coix seed oil; Liposomes；DPPH；Anticancer

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1. INTRODUCTION

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The development of nutraceutical-fortified foods and beverages containing multiple kinds of

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bioactive agents are being investigated for their potential additive or even synergistic health

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benefits, i.e., the combination of bioactives works better than the individual ones.1 β-carotene

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(βC), a representative of the carotenoid family, is well known for its potential health promoting

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functions such as antioxidant and anticancer activities, as well as its ability to reduce the risk of

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heart disease and cataract colorectal adenomas. 2 Coix seed oil (CSO) has also been reported to

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possess a wide spectrum of biological activities, including stomachic, diuretic, antiphlogistic,

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anodynic, antispasmodic, and antitumor effects. 3 Food and beverage products fortified with both

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βC and CSO could therefore be of interest to consumers. However, both of them are

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water-insoluble compounds that also have poor chemical stability and low bioaccessibility.

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These challenges must therefore be overcome when developing commercial food products.

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In recent years, colloidal delivery systems (CDS) have attracted growing attention due to

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their ability to overcome these obstacles. Various types of CDS have been developed to

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encapsulate different types of bioactives and to incorporate them into different food matrices,

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such as microcapsules, microspheres, emulsions, microemulsions, micelles, and liposomes.4-6

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Liposomes are particularly attractive CDS for oral ingestion due to their high biocompatibility,

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low toxicity, and low immunogenicity. Moreover, they can be designed to increase the

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bioavailability of hydrophobic bioactive agents in the human gut by controlling their

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composition and structure.

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Hydrophobic bioactive agents are typically embedded within the hydrophobic domains

61

formed by the tails of the phospholipid molecules in the bilayers. According to the literature, the

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orientation of the bioactives within the bilayers depends on the molecular and physiochemical 3



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properties of both the bioactive agents and phospholipids, which may impact their

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gastrointestinal fate.

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stability, and in vitro release characteristics of carotenoids loaded into liposomes.8 The

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encapsulation, retention, and release of the carotenoids trapped within the liposomes was found

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to depend on the type of carotenoid used, which was attributed to differences in their molecular

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orientations in the bilayer. In the current study, we examined the behavior of two bioactive

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agents with different molecular characteristics within liposomes. Although both βC and CSO are

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strongly hydrophobic, their molecular structures are appreciably different. We hypothesized that

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this would impact the physiochemical properties of liposomes containing different ratios of βC

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and CSO.

7,8

Previously, researchers have studied the loading capacity, storage

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In addition, researchers have reported that the biological activity of certain types of

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bioactive agent can exhibit enhanced behavior when used in combination.9,10 The magnitude of

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the effect depends on the levels of the two bioactive agents used, with certain ratios being

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synergistic and others being either additive or antagonistic. 11,12 For this reason, the impact of the

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ratio of βC-to-CSO in the liposomes on their potential bioactivity was examined in the current

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study. Previously, researchers have examined either βC-loaded or CSO-loaded liposomes, but the

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best of our knowledge, there are no previous reports on the utilization of these two bioactives in

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combination. 7,13 Consequently, liposomes containing different ratios of βC and CSO (L-βC-CSO)

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were prepared and their properties were compared to those of liposomes containing either βC

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(L-βC) or CSO (L-CSO).

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2. MATERIALS AND METHODS

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2.1. Materials

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Egg yolk phosphatidylcholine (EPC, with 98% purity) was purchased from Shenyang

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Biotechnologies Co., Ltd. (Shenyang, China). Cholesterol was purchased from the Shanghai

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Chemical Reagent Co., Ltd. (Shanghai, China). CSO was kindly supplied by Hecheng Sanxian

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Biotechnologies Co., Ltd. (Guangzhou, China). βC was purchased from Sigma-Aldrich (USA).

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Caco-2 cells were obtained from the Institute of Biochemistry and Cell Biology, Shanghai

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Institutes for Biological Sciences (Shanghai, China). All other chemicals used were of analytical

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grade.

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2.2. Liposome Preparation

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Empty, single-bioactive, and mixed-bioactive loaded liposomes were prepared. L-βC-CSO

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loaded liposomes with different mass ratios of βC to CSO (1:1, 1:5 and 1:10) were prepared by a

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modified ethanol injection method. 5 Briefly, egg yolk lecithin (40 mg), cholesterol (8 mg), CSO

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(0.24 mg, 1.2 mg, 2.4 mg), and 0.24 mL βC dichloromethane solution (1 mg/mL) were dissolved

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in 4 mL ethanol and mixed thoroughly. The organic solution obtained was then injected slowly

98

into 10 mL phosphate buffer solution (PBS, 20 mM, pH 6.8) maintained at 45 °C with magnetic

99

stirring. After stirring for 20 min, the liposomal suspensions were transferred to a shaker and

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then oscillated for another 20 min at 45 °C. Rotary evaporation at 42 °C was then carried out to

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remove dichloromethane and ethanol. After adjusting the volume of the samples to 10 mL with

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PBS, the samples were sonicated at 50% amplification strength for 1 min with a 1 s on/1 s off

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pulse sequence to obtain homogeneous liposomes (Sonics & Material Vibra Cell, 400 W, 20 kHz,

104

UK). L-βC, L-CSO (0.24 mg CSO), and unloaded liposomes (L) were also prepared for

5



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comparison using the same method except without the addition of CSO or/and βC. All samples

106

were stored in a refrigerator (4 °C) overnight before characterization and further use.

107

2.3. Encapsulation Efficiency Determination

108

The encapsulation efficiency (EE) was determined according to Tan’s method with a slight

109

modification.8 Before calculate the EE, the amount of free and trapped βC were determined by

110

extraction using an organic solvent and then quantification using a UV-visible spectrophotometer.

111

Briefly, collected samples (2 mL) were added to 4 mL N-hexane and then vortexed vigorously

112

for 1 min at ambient temperature before being subjected to centrifugation at 5000 rpm for 10 min.

113

After being extracted another two times with N-hexane, the supernatant was collected, and the

114

absorbance was measured at 450 nm using N-hexane as a blank. The content of free βC (Cfree)

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was then calculated using a standard curve.

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The residual liposomal suspension after extraction was then mixed with 3 mL ethanol. After

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being demulsified using the sonicator for 30 min at ambient temperature, 4 mL of N-hexane was

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added. The extraction process and analysis were then carried out according to the above method

119

for free βC to obtain the loaded βC in the liposomes (Cloaded). Each experiment was carried out in

120

triplicate. The EE of βC was calculated using the following equation:

121

122

EE (%) 

Cloaded 100 C free  Cloaded

(1)

2.4. Measurement of Particle Size and Zeta Potential

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The average particle diameter (AD) and polydispersity index (PDI) of the samples were

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measured at 25 °C using dynamic light scattering (Nano ZS90, Malvern Instruments, Worcester,

125

UK). 1 mL samples were diluted with 10 mL of phosphate buffer solution (20 mM, pH 6.8) to

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avoid multiple scattering. The zeta-potential (ZP) was also measured using the same instrument.

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All measurements were repeated three times.

128

2.5. Morphological Characterization

129

The microstructure of L-βC-CSO (1:1) and L-βC samples was imaged using transmission

130

electron microscopy (TEM, H-600, Hitachi, Japan). Samples were placed onto a carbon-coated

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copper grid and dried for 1 min. After removing the excess sample with filter paper, the grid was

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stained with 1% sodium phosphotungstate solution for another 2 min. Excess liquid was then

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removed using filter paper and the grid was air-dried again at room temperature. The microscopy

134

of liposomes was then imaged at an accelerating voltage of 75 kV.

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2.6. Antioxidant Activity Assay

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The antioxidant activity of the samples was determined using the DPPH radical-scavenging 14

137

assay according to a method described previously with some slight modifications.

138

aliquot of each sample (2 mL) was added to 1 mL of 0.4 mmol/L DPPH ethanol solution. After

139

vortexing thoroughly, the mixture was placed in the dark for 40 min at room temperature

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followed by centrifugation for 10 min at 3000 rpm. The absorbance of the supernatant was then

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measured at 525 nm using a UV-visible spectrophotometer (UV-1600, Meipuda Co., Shanghai,

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China). For control samples, distilled water was used instead of liposomes to mix with the DPPH

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solution and otherwise treated in the same manner as for the liposomes. The percentage of the

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DPPH-scavenging activity was calculated using the following equation:

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DPPH (%)  (1 -

At ) 100 Ac

Briefly, an

(2)

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Here: At is the absorbance of 2 mL liposomes and 1 mL DPPH in absolute ethanol and Ac is the

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absorbance of 2 mL liposomes and 1 mL absolute ethanol. 7



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2.7. Storage Stability

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To investigate the storage stability of the liposomes, fresh prepared samples sealed in glass

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test tubes were stored at both 4 °C and 25 °C for 10 weeks in the dark. The retention rate of βC

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was determined weekly by measuring the total amount of βC in the samples (Ctotal storage) and the

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amount of βC initially prepared (Ctotal

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described in section 2.3, and the retention rate was calculated according to the following

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equation. Each experiment was carried out in triplicate.

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156 157

Retention rate (%) 

Ctotal storage Ctotal initially

initially).

The Ctotal was obtained by demulsification as

100

(3)

2.8. In vitro Release Studies Simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) were prepared according 15

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to Zhao’s method.

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(0.008 L/L), and pepsin (3.2 g/L). The SIF (pH 7.4) consisted of sodium hydroxide (1.81 g/L),

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potassium dihydrogen phosphate (8.09 g/L), pancreatin (4.76 g/L), and bile salts (5.16 g/L).

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The SGF (pH 1.2) contained sodium chloride (2 g/L), hydrochloric acid

The in vitro release properties of βC from L-βC and L-βC-CSO was evaluated using Tan’s 8

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method.

10 mL of preheated liposomal suspension was added to 30 mL SGF or SIF that had

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been preheated to 37 °C. The mixture was then adjusted to pH 1.2 or 7.4, respectively, followed

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by shaking at 100 rpm in a shaking incubator at 37 °C for 24 h. At appropriate time intervals, a

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certain amount of mixture was withdrawn and transferred into a 10 mL tube, and an equivalent

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volume of corresponding fresh dissolution media was added to replace it. The released βC was

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extracted and analyzed by the method for the determination of free βC as described in section 2.3.

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The cumulative amount of βC released as a function of time was calculated using the following

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equation: 8


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Release rate (%) 

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C free after incubation  C free initially Ctotal initially

100

(4)

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Here: Cfree initially represents the amount of free βC for the initially prepared liposomes, Cfree after

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incubation

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amount of total βC for the initially prepared liposomes.

represents the amount of free βC determined after incubation, and Ctotal

initially

is the

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To gain insight into the release mechanism of liposomes in simulated gastrointestinal fluid,

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the zero-order, first-order, Higuchi, and Korsmeyr–Peppas models were applied for the release

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data analysis.

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

Zero-order model: Mt/M∞=kt

(5)

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

First-order model: ln(1-Mt/M∞)=-kt

(6)

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

Higuchi model：Mt/M∞= kt1/2

(7)

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

Korsmeyr-Peppas model：Mt/M∞= ktn

(8)

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Here: Mt/M∞ is the fractional solute release, t is the release time; k is the rate constant, and n is

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the release exponent characteristic of the release mechanism. 12,16

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2.9. MTT Assay

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The anticancer efficiency of L-CSO, L-βC and L-βC-CSO (1:1, 1:5, 1:10) against Caco-2

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(CBCAS,

Shanghai,

China)

cells

was

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3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay. All samples

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were filtered through 0.22 μm millipore membranes and then diluted with sterile phosphate

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buffer solution to adjust the concentration of the βC in the range of 0-12 μg/mL. Caco-2 cells

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were cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco Laboratories,

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Gaithersburg, MD, USA) containing 10% fetal bovine serum (FBS), and incubated at 37 °C in a 9


evaluated

using

the


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CO2 incubator containing 5% CO2 and 95% air until the logarithmic growth phase. Tumor cells

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were then seeded at a concentration of 104 cells per well in 96-well plates and allowed to grow

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for 24h. For the cytotoxicity, the culture medium was removed and replaced with a mixture of

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DMEM and diluted liposomal suspensions at a volume ratio of 1:1, followed by cultivation for

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24 h, 48 h, and 72 h, respectively. Culture medium replaced by DMEM was regarded as the

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control. At the end of incubation, the cells were washed with fresh culture medium (without

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FBS), and then incubated with serum-free medium containing MTT (0.5 mg/mL) for another 4 h.

198

The absorbance of formazan crystals produced by the live cells were measured at 570 nm using

199

an enzyme-linked immunoassay (Perkin Elmer, San Jose, CA, USA). The cell inhibitory rate is

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expressed as a percentage of the control value.

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2.10. Statistical Analysis

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All experiments were performed at least three times and the data were expressed as the mean

203

± standard deviation (SD). The results were analyzed statistically for significance (p≤ 0.05) using

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SPSS 18.0 software (IBM Corp., NY, USA).

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3. RESULTS

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3.1. Entrapment Efficiency, Particle Size and Particle Charge

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The entrapment efficiency, average diameter and zeta potential of L, L-βC, L-CSO and

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L-βC-CSO are summarized in Table 1. The βC entrapment efficiency of all liposomal

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formations were higher than 80%. The values for L-βC-CSO (1:1, 1:5, 1:10, βC:CSO, w:w) were

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83.8 ± 1.3%, 81.3 ± 1.3, and 80.8 ± 1.1%, respectively, which were slightly lower than that of

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L-βC (86.0 ± 2.0%). As for the particle dimensions, the average size of L-βC (129.1 ± 2.2 nm)

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and L-CSO (141.1 ± 3.6 nm) were slightly larger than that of the unloaded liposomes (119.0 ±

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4.9 nm). The addition of CSO resulted in larger particles, with the average diameters of

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L-βC-CSO (1:1, 1:5, 1:10, βC /CSO, w/w) being 156.7±5.9, 181.0±2.3, and 193.1±6.8 nm,

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respectively. The PDI values of all liposomes were less than 0.30, indicating that they had a

216

fairly narrow distribution of particle sizes. The absolute value of zeta potentials for the L-βC

217

(-28.9±1.3 mV) and L-CSO (-32.8±2.3 mV) systems were both higher than that of the unloaded

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liposomes (-21.7±2.7 mV) but lower than that of the combined loaded ones (except L-βC-CSO

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1:1). Moreover, the zeta-potential became more negative as the ratio of CSO present increased.

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3.2. Morphological Characterization

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The microstructure of L-βC and L-βC-CSO (1:1) samples were examined by TEM (Figure.

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1). The images showed that they were relatively small and consisted of concentration bilayer

223

rings. The particle diameters of the samples observed in the images were in the range of 100-200

224

nm, which is consistent with the DLS results. In addition, the liposomes in the L-βC-CSO (1:1)

225

sample appeared to have a bigger size than those in the L-βC sample.

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3.3. DPPH Radical-Scavenging Activity

227

The DPPH-scavenging activity of fresh prepared L, L-βC, L-CSO and L-βC-CSO (1:1, 1:5,

228

1:10) samples was determined to evaluate their antioxidant activity (Figure 2). The

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DPPH-scavenging activity of the unloaded liposomes was around 33%, which was significantly

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lower than that of the bioactive-loaded liposomes. However, there were differences between the

231

free radical scavenging activities of the bioactive-loaded samples. The L-CSO exhibited better

232

antioxidant activity than L-βC. The co-loaded liposomes had higher antioxidant activity than the

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single-loaded liposomes. What’s more, the antioxidant activity of L-βC-CSO increased as the

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ratio of CSO increased: 51.9±6.6%, 66.7±2.5%, 73.5±3.7% for 1:1, 1:5, 1:10 (βC/CSO, w/w),

235

respectively. 11



236

3.4. Evaluation of Storage Stability

237

The βC retention rate of liposomal formations during storage at 4 °C and 25 °C was

238

monitored (Figure 3). βC retention decreased in all samples over time by an amount that

239

depended on system composition. Interestingly, more βC was retained in the L-βC system than

240

in the L-βC-CSO system. After ten weeks storage at 25°C, the retention rate of βC in L-βC-CSO

241

(1:10) decreased to 10.7%, while that in L-βC was still 49.2%. The amount of βC retained in the

242

L-βC-CSO systems after 10 weeks’ storage was in the order: 1:1>1:5>1:10, suggesting that the

243

addition of CSO promoted βC degradation. This trend was more significant for samples stored at

244

25 °C than at 4 °C.

245

3.5. In vitro Release Studies

246

The release of βC from L-βC and L-βC-CSO (1:1, 1:5, 1:10) samples in SGF and SIF was

247

measured over time (Figure 4). In both types of simulated gastrointestinal fluids, the cumulative

248

release of βC increased fairly rapidly during the first 9 h, and then became fairly steady

249

afterwards. The final values of the cumulative release decreased with increasing CSO level in the

250

SGF, but decreased in the SIF. It should be noted that foods typically only spend around 2 hours

251

in the stomach and 2 hours in the small intestine, which suggests that only a small fraction would

252

be released.

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3.6 MTT Assay

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In this experiment, the weight ratio of βC-to-CSO on the anticancer activity of the liposomes

255

was examined using the MTT assay. The cell growth inhibitory effect of L-CSO, L-βC, and

256

L-βC-CSO formulations against Caco-2 cell were determined (Figure 5). The concentrations of

257

βC and CSO in the samples after they were diluted and mixed with DMEM are presented in

258

Table 3. The results of the MTT assay demonstrated that the inhibition rate depended on system 12


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composition and incubation time (Figure 5). More dead cells were observed after incubation at

260

higher liposome levels and at longer incubation times. For the lowest level of liposomes, the

261

Caco-2 cell growth inhibition rates of L-CSO, L-βC and L-βC-CSO (1:1, 1:5, 1:10, βC:CSO,

262

w:w) after 24h incubation were 4.6±1.9%, 4.2±1.6%, 5.3±1%, 8.8±1.7% and 11.3±1.6%,

263

respectively. Whereas, the values increased to 20.5±0.8%, 26.2±1%, 28.6±1.3%, 22.3±1.3% and

264

20.7±0.9% for the highest level. This trend was even more obvious after incubation for 48 h and

265

72 h. Interestingly, when the volume ratio of liposomes-to-DMEM was less than 1:3, a higher

266

CSO content resulted in better Caco-2 cell growth inhibition with L-βC-CSO (1:10) showing the

267

highest inhibition value. However, when the liposome concentration was higher (≥1:3), the

268

L-βC-CSO (1:1) exhibited the highest inhibition activity.

269

4. DISCUSSION

270

This research focused on the development of liposomes as delivery systems because of their

271

potential benefits for food and beverage applications. 7,17 CSO and βC were examined because of

272

their potential beneficial health effects.

273

for encapsulation of either CSO or βC individually.

274

and bioavailability could be enhanced using liposomal formulations. In addition, other studies

275

have shown an additive or even synergistic increase in the biological activity of bioactive agents

276

when used in combination. For instance, sage extract and zein hydrolysates encapsulated in

277

liposomes were shown to exhibit synergistic antioxidant effects. 9 Combinations of metformin

278

and chlorin co-encapsulated in liposomes were reported to have improved therapeutic effects

279

compared to single-loaded ones. 10 For these reasons, we examined the synergist activity of CSO

280

and βC in liposome-based delivery systems. The ratio of the two bioactives employed is known

281

to be a critical parameter in determining the extent of any synergistic, additive or antagonistic

18

Previously, liposomes have been shown to be suitable 8,13

These studies showed that their stability

13



11,12

282

behavior.

Consequently, we examined combined formulations with different βC to CSO

283

ratios. The physiochemical properties, structure, release behavior, anticancer activity, and

284

antioxidant efficacy of single- and mixed-formulations were compared.

285

Initially, the encapsulation efficiency, particle size, and zeta-potential of all liposomal

286

formations were determined. All bioactive-loaded liposomes were larger than the unloaded

287

liposomes, suggesting that incorporation of the bioactives altered the liposome structure. 5, 13 The

288

same concentration of bioactive agent was used for preparing the L-CSO and L-βC systems,

289

however, the particle size of the former was larger than that of the latter (Table 1). In addition,

290

both single-loaded liposomes were smaller than the multiple-loaded ones. Moreover, the size of

291

the liposomes in the mixed systems increased as the CSO ratio increased. These size differences

292

may reflect differences in the manner in which the hydrophobic bioactives are integrated into the

293

phospholipid bilayers.

294

The bioactive composition also impacted other characteristics of the liposomes. An increase

295

in CSO ratio led to a reduction in βC encapsulation efficiency, an increase in negative charge,

296

and a decrease in polydispersity. The encapsulation properties of liposome suspensions are

297

known to depend on the amount and dimensions of hydrophobic domains inside them. 5 CSO and

298

βC are both hydrophobic substances that will tend to be solubilized within the lipid bilayers of

299

the liposomes. The observed decrease in βC encapsulation efficiency with increasing CSO level

300

for the L-βC-CSO system may therefore have been because the CSO occupied some of the

301

available space within the bilayers and so fewer carotenoids could be solubilized. The non-polar

302

carotenoids may orient with their long isoprenoid chains perpendicular to the lipid acyl chain

303

thus far from the surface. 8 The CSO consists mainly of triacylglycerols, but there is likely be

304

some residual free fatty acids present as well due to hydrolysis. The fact that the molecules in the 14


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305

CSO contain some polar groups may mean that they are arranged differently in the bilayer

306

structures of the liposomes. In particular, the polar groups may be located near the surfaces of the

307

liposomes while the non-polar chains lie parallel to the hydrocarbon chains in the phospholipids.

308

According to the literature, the pKa values of free fatty acids are around pH 5.

309

preparation pH of 6.8 the carboxyl groups of the free fatty acids would be deprotonated and

310

contribute to the negative charge on the surfaces of the liposomes. While, the decreased

311

zeta-potential as the result of the increase in CSO ratio indicated that more free fatty acids were

312

incorporated into the liposomes, which further confirmed more CSO was encapsulated in

313

liposomes.19 In addition, the absolute value of zeta potential can reflect the stability of liposomes,

314

high absolute value of zeta potential indicates high electric charge on the surface of the drug-

315

loaded liposomes, which can cause strong repellent forces among particles to prevent

316

aggregation and enhance the stability of liposomes. The absolute zeta-potential value for all

317

L-βC-CSO was higher than 30 mV, suggesting their good stability.

318

19

Thus, at the

The oxidation of lipids and proteins within foods and inside the human body may impact 20

319

various diseases, including cancer, cardiovascular and neurodegenerative diseases.

320

Consequently, there has been interest in fortifying food products with bioactive agents that

321

exhibit antioxidant activity. In this study, the antioxidant activity of L-βC-CSO was determined

322

using the DPPH assay, which is based on the free radical scavenging ability of this reagent. A

323

high free radical scavenging ability is assumed to be correlated to a high antioxidant activity. The

324

DPPH-scavenging activity of the unloaded liposomes was greater than 30%, even though it

325

contained no encapsulated bioactive agents. This effect may be attributed to the presence of

326

phospholipids, which have previously been reported to have free-radical scavenging activity in

327

the DPPH assay. 21

The DPPH scavenging activity increased after incorporation of either βC or 15



Page 16 of 37

328

CSO into the liposomes. However, the activity of L-βC was somewhat lower than that of the

329

L-CSO. The scavenging activity of the L-βC system can be attributed to the electron-rich

330

conjugated system of the encapsulated carotenoids, which has 11 conjugated double bonds.

331

Conversely, the scavenging activity of the L-CSO system can be attributed to the high level of

332

unsaturated fatty acids (>70%) in coix seed oil. Although they are themselves oxidatively

333

unstable, many in vitro and in vivo studies indicate that unsaturated fatty acids have free radical

334

scavenging activity, especially ω-3 ones.

335

radical-scavenging capacities were all higher than that of the single-loaded ones. Moreover, the

336

antioxidant activity increased as the CSO ratio in the combined systems increased. Hence, the

337

combination of βC and CSO may be a good choice for preparing antioxidant products.

20,22

As for the combined-loaded ones, the

338

Liposomes not only provide a physical–chemical barrier for encapsulated compounds

339

against pro-oxidant elements, they also make them water-dispersible in aqueous food

340

formulations. However, liposomes are thermodynamically unstable and tend to aggregate and

341

fuse during storage leading to the leakage of incorporated bioactive agents. In this section, we

342

therefore examined the effect of CSO addition on the storage stability of L-βC-CSO at 4 and 25

343

°C. The amount of βC retained in the L-βC and L-βC-CSO samples decreased throughout the

344

10-week storage period. Visual observation of the liposome suspensions indicated that their

345

yellow color faded over time, suggesting that βC degraded,

346

studies.

347

increased, indicating that the CSO accelerated βC degradation. Due to its high level of

348

unsaturated fatty acids CSO is known to be highly susceptible to oxidation.

349

may have accelerated the degradation of the carotenoid through a co-oxidation mechanism.

350

The rate of βC degradation in all systems was faster at the higher storage temperature, which is

23

7

which is consistent with previous

Interestingly, the βC retention ratio decreased as the level of CSO in the liposomes

16


3

Consequently, it 16

Page 17 of 37


24

351

consistent with the known impact of temperature on oxidation.

This results suggests that it

352

would be advantageous to store the liposomes under refrigerated conditions to extend their shelf

353

life.

354

We also examined the impact of the different liposome formulations on carotenoid

355

bioaccessibility. Previously, we observed that CSO leaked out of liposomes at a relatively slow

356

rate when they were incubated in either simulated intestinal fluids (SIF) or simulated gastric

357

fluids (SGF). Other researchers have also observed the sustained release of βC from liposomes. 8

358

In addition, studies have shown that the bioavailability of encapsulated hydrophobic bioactives

359

depends on the lipid phase surrounding them when they are encapsulated in emulsion-based

360

delivery systems.

361

the nature of the delivery system used, including their composition, structure, particle size,

362

permeability, and stability. 7,8 Although both liposomes and emulsion are colloidal systems, their

363

composition and structure are appreciably different. Moreover, the position of carotenoids in the

364

lipid bilayer probably changes as the ratio of CSO and βC changes, which may in turn alter their

365

bioaccessibility. We therefore examined the relationship between the bioactive composition and

366

bioaccessibility in the liposomal systems.

25-27

In general, the bioaccessibility of hydrophobic nutraceuticals depends on

367

The results indicated that all samples showed burst release in the initial stages, but a steady

368

release thereafter (Figure 4). The burst release stage may be due to bioactive agents that are free

369

or located on the surfaces of the bilayer membranes. By contrast, the steady release stage may be

370

due to the slow release of bioactive agents from inside the liposomes. In general, the release of

371

βC from both liposome systems was slower during incubation in SGF than in SIF, which is

372

consistent with previous research.

373

(hydrolysis) of phospholipid and oil molecules in the liposomes in the small intestine, combined

8

This may have occurred because of enzymatic degradation

17



374

with penetration of bile salts into the bilayer structures, thereby leading to swelling or disruption

375

of the liposomal vesicles and leakage of the βC.

376

Interestingly, the release of βC from the combined-system (L-βC-CSO) was slower than that

377

from the single-system (L-βC) in SGF, but faster in the SIF. This effect became more

378

pronounced as the level of CSO present increased. The impact of the bioactives on the

379

composition and structure of the lipid bilayer may have influenced the release behavior of the

380

encapsulated carotenoids under different conditions.

381

molecule is likely to be located deep within the hydrophobic core of the phospholipid bilayer

382

with an orientation perpendicular to the bilayer plane. 7 Conversely, the CSO molecules may be

383

inserted parallel to the bilayer close to the membrane surface. In this sense, the incorporation of

384

CSO would increase the bilayer surface viscosity and decease its permeability. As a result, the

385

increased viscosity may have altered the efficiency of liposome disruption and coalescence in the

386

stomach, leading to a decrease in bioactive leakage.

387

help prevent the bioactive substances from leaking out. This may be why an increase in CSO

388

ratio inhibited the leakage of the carotenoid in the SGF. Conversely, in the SIF, the presence of

389

the CSO may have promoted swelling or disruption of the liposomes, increasing the access of

390

digestive enzymes and bile salts to the lipid bilayer, resulting in considerable βC leakage. 30

29

7,28

The long highly hydrophobic βC

In addition, the low permeability would

391

For better understanding the effect of CSO on the release behavior of βC from liposomes,

392

four different models were used to elucidate the possible release mechanism involved. Table 2

393

represents the kinetic model parameters for the release of the carotenoid from different liposomal

394

formulations. All formulations were fitted well by the first-order model (R2>0.95) when

395

incubated in SGF, with the release constant k of L-βC being higher than that of L-βC-CSO.

396

Moreover, the higher the CSO to βC ratio, the lower the release constant was. As to liposomes 18


Page 18 of 37

Page 19 of 37


397

incubated in SIF, Higuchi model showed high correlation coefficient of 0.94-0.97, indicating that

398

diffusion controlled release mechanisim was the prime release mehanism.

399

Korsmeyr–Peppas model was the most suitable for describing βC release kinetics during

400

incubation in the SIF. However, the release constant k of L-βC was lower than that of L-βC-CSO,

401

and increased as the amount of CSO increased. The n parameter in the Korsmeyr-Peppas model

402

provides insights into the release mechanism. As shown in Table 2, the values of n for both

403

L-βC and L-βC-CSO (1:1) were lower than 0.43, indicating that the release mechanism

404

corresponded to Fickian diffusion.

405

(1:10) was between 0.43 and 0.85, suggesting their release was largely governed by particle

406

swelling and diffusion. In this sense, the incorporation of CSO had a positive effect on the

407

bioaccessibility of βC by altering the release character of the liposomal bilayers.

32

31

In addition, the

By contrast, the value for L-βC-CSO (1:5) and L-βC-CSO

408

CSO has been proved to be an ideal anti-tumor drug by thousands of clinical trials and

409

animal experiments. On the other hand, βC as a natural antioxidant exhibits good anticancer

410

activity. In recent years, CSO and βC has been wildly used in food and pharmacy-related

411

industry for the treatment or prevention of cancers. However, they are commonly administered

412

separately, as to the antitumor effect when co-delivered in the same liposomes is still unknown.

413

According to literatures, the ratio of combinations may affect the drug efficiency, which means

414

certain ratios of drug combinations may be synergistic while other ratios may be additive or even

415

antagonistic.

416

obtain optimum antitumor efficiency. In addition, exposure time and liposomes concentration

417

were another two important parameters that affect the efficiency. The potential anticancer

418

activity of CSO and βC co-loaded liposomes was assessed by determining their ability to alter

419

the viability of Caco-2 cells. Cell viability was measured as a function of and formulation

11,12

In this sense, finding the synergistic ratio of them is of much importance to

19



420

concentration and incubation time using the MTT assay. Cells exposed to CSO- or βC-single

421

delivery liposomes were also evaluated for comparison, and those added with DMEM were used

422

as a control group. The blank liposomes exhibited no toxicity (data not shown), whereas cell

423

viability was reduced for all the bioactive-loaded liposomes. Moreover, cell inhibition tended to

424

increase with in increasing incubation time and formulation concentration for all systems, which

425

agrees with previous studies on liposomes loaded with antitumor drugs. 11

426

In general, the inhibitory effect of L-βC was stronger than that of L-CSO, whereas the

427

combined-loaded systems exhibited stronger inhibition than the single-loaded ones. However,

428

the extent of the effects depended on the overall formulation concentrations. At relatively low

429

liposome-to-DMEM ratios (1:81 or 1:27), the inhibitory effect tended to increase with increasing

430

CSO level, but at relatively high ratios (1:3 or 1:1), there appeared to be a maximum in inhibition

431

at an intermediate CSO level. This suggests that too high liposome concentrations are

432

detrimental to their inhibitory effect on Caco-2 cell growth. This may have occurred because

433

their relatively high viscosity reduced the rate at which they interacted with the cell surfaces.

434

In conclusion, this study has shown that the antioxidant and anticancer activities of

435

bioactive-loaded liposomes can be increased by combining two different bioactive agents within

436

the same system. In this case, a highly unsaturated triacylglycerol oil (CSO) and a carotenoid

437

(βC) were used as the two bioactive agents. The presence of CSO slightly decreased the

438

encapsulation efficiency of βC in the liposomes, which was attributed to its ability to compete for

439

the available hydrophobic domains within the phospholipid bilayers. On the other hand,

440

incorporation of CSO led to a higher negative charge and a more uniform particle distribution.

441

The combined system (L-βC-CSO) showed good DPPH scavenging activity, with the βC

442

inhibiting CSO oxidation during storage. The release of βC from the combined-bioactive 20


Page 20 of 37

Page 21 of 37


443

liposomes (L-βC-CSO) was slower than from the single-bioactive ones (L-βC) in SGF, but faster

444

in SIF. These characteristics may be useful for developing colloidal delivery systems that retain

445

and protect bioactives in the stomach but release them in the small intestine, where they can be

446

absorbed. The anticancer activity of the combined-bioactive liposomes was higher than that of

447

the single-bioactive ones, but the degree of inhibition depended on the CSO level in the

448

liposomes. The utilization of combinations of β-carotene and coix seed oil in liposomes may

449

therefore be suitable for improving their bioavailability and bioactivity. However, further

450

research is needed to demonstrate that these systems are economically feasible and robust

451

enough for food applications. Moreover, in vivo studies are required to elucidate whether similar

452

effects are seen under more realistic conditions. Finally, the molecular and physicochemical

453

origins of the observed effects still need to be elucidated.

454

ACKNOWLEDGEMENTS

455

This research was financially supported by projects of the National Natural Science

456

Foundation of China (31560465), Jiangxi Provincial Natural Science Foundation of China

457

(20161BAB204190), Jiangxi Provincial Department of Education Youth Project (GJJ150803),

458

and the Program of China Scholarship Council (No.201608360170).

459

ORCID

460

Chunqing Bai: 0000-0002-2429-0562

461

David Julian McClements: 0000-0002-9016-1291

462

Notes

463

The authors declare no competing financial interest.

21



464

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Figure captions

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566

Figure 1. TEM images of the L-βC: A, B; L-βC-CSO (1:1): C, D

567

Figure 2. DPPH scavenging activity of un-L, L-βC, L-CSO and L-βC-CSO (1:1, 1:5, 1:10).

568

Columns with different letters are significantly different with each other at P

Development of oral delivery systems with enhanced antioxidant and

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