Development of Î²-Carotene-Loaded Organogel-Based Nanoemulsion

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Development of beta-carotene-loaded organogel-based nanoemulsion with improved in vitro and in vivo bioaccessibility Yuting Fan, Luyu Gao, Jiang Yi, Yuzhu Zhang, and Wallace Yokoyama J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b02125 • Publication Date (Web): 11 Jul 2017 Downloaded from http://pubs.acs.org on July 12, 2017

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Journal of Agricultural and Food Chemistry

Development of beta-carotene-loaded organogel-based nanoemulsion with improved in vitro and in vivo bioaccessibility Yuting Fan1, Luyu Gao1, Jiang Yi1*, Yuzhu Zhang2, Wallace Yokoyama2 1

Department of Food Science and Engineering, College of Chemistry and Environmental Engineering,

Shenzhen University, Shenzhen 518060, China 2

Western Regional Research Center, ARS, USDA, Albany, California 94710, United States

*To whom correspondence should be addressed. Tel: 86-755-26557377. Fax: 86-755-26536141. E-mail: [email protected]

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ACS Paragon Plus Environment


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Abstract

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Beta-carotene (BC), a naturally occurring lipophilic carotenoid, is beneficial for human health.

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However, its water solubility and bioavailability is low. In this study, organogel-based nanoemulsion

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was successfully prepared to improve BC’s loading amount, solubility, and bioavailability. Corn oil

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was selected as oil phase for organogel due to the greatest release amount of BC. Tween 20 was

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optimized as the emulsifier based on the highest extent of lipolysis and BC bioaccessibility. The

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nanoemulsion was a better alternative than organogel according to both the extent of lipolysis and

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BC bioaccessibility. Cellular uptake of BC was significantly improved through organogel-based

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nanoemulsion, compared with BC suspension. Caveolae/lipid raft-mediated route was the main

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endocytosis pathway. Pharmacokinetics results confirmed that the in vivo bioavailability of BC in

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nanoemulsion was 11.5-fold higher than BC oil. The information obtained suggested that

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organogel-based nanoemulsion may be an effective encapsulation system for delivery of insoluble,

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and indigestible bioactive compounds.

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Keywords: beta-carotene, organogel-based nanoemulsion, bioaccessibility, bioavailability

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Introduction

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Beta-carotene (BC), a natural lipophilic carotenoid that mainly presents in vegetables and fruits, is a

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useful colorant and food supplement.1 BC has the highest pro-vitamin A activity of carotenoids and

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strong hydrophobic antioxidant activity. The prevalence of vitamin A deficiency was controlled and

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reduced among women and young children with BC fortified rice.2 BC treatment can prevent K562

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cells from oxidative damage induced by H2O2 at low concentration.3 In addition to antioxidant

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activity and pro-vitamin A activity, BC has been considered to have many other biological functions,

25

which is beneficial for human health. There is an inverse correlation between BC intake and risk of

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developing several chronic diseases, such as various types of cancers and cardiovascular diseases.4,

27

5

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However, BC is insoluble in water and its solubility in edible oils is low. In the literature, BC

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solubility is between 0.10-0.15% by weight in the bulk oils.6 And the solubility is negatively

30

correlated with the fatty acid length. Furthermore, BC is prone to isomerization, degradation,

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oxidation, resulting in the decrease and loss of antioxidant activity and pro-vitamin A activity.

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Various delivery systems are developed to protect and deliver BC, such as nanocomplex, Pickering

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emulsion, microemulsion, liposome, conventional emulsion, and nanoemulsion.7-11 Among these

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encapsulation systems, nanoemulsion may be a good alternative for its potential advantages

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(greater bioavailability of incorporated lipophilic functional compounds and higher stability to

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particle aggregation or flocculation). A nanoemulsion is defined to be a conventional emulsion that

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contains very small droplets with a diameter between 20 to 200 nm,12 and will be in the legislation

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of nanoparticle hat set the diameter at 100 nm and below. In fact, oil-in-water (o/w)

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nanoemulsion-based delivery systems have been widely used for improving the chemical stability 3



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and enhancing the bioavailability of BC. However, BC loading amount for nanoemulsion systems

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has reportedly been low.11 Even though BC loading amount can be increased through heat

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treatment or sonication because the oil solubility remarkably increased with the increase of

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temperature, the loading amount is still extremely low, greatly restricting the application.

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Organogels are liquid edible oils entrapped, solid-like, thermo-reversible structured gels. It was

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formed upon self-assembly of organogelators, like monoacylglycerols, fatty acids, and fatty

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alcohols.13 Organogels have received increasing interest from food industry recently for the

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potentials which can be used for a variety of purposes in health value-added foods. Organogels are

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suitable delivery systems, due to their ability to dissolve, stabilize, and deliver hydrophobic

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bioactive compounds.

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Organogel-based nanoemulsions have been developed to protect and deliver lipophilic

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nutraceuticals (such as curcumin, D-Limonene, and capsaicin).14-17 Results showed that the loading

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amount, bioavailability, and biological activity of encapsulated bioactive compounds had been

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significantly improved. However, little information is available on the establishment of BC-loaded

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organogel. The goal of this study was to prepare BC-loaded organogel-based nanoemulsion with

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high pressure homogenizer. The effect of oil types and emulsifiers on the lipolysis profile and BC

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bioaccessibility in organogel-based nanoemulsion system was investigated. Cellular uptake and

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pharmacokinetics in the rat were examined. The endocytosis routes were also exploited.

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Materials and Methods

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Materials

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Corn oil (C18:1 32.9%, and C18:2 53.57%) and coconut oil (C12:0 50.0%, C14:0 19.5%) were purchased

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from a local market (Shenzhen, China). Neobee 1053 (medium-chain triglycerides, MCT) (C8:0 4


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49.5%, C10:0 50.3%) was obtained from Stepan Company (Maywood, New Jersey, U.S.).

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beta-carotene (97%, BC), porcine pepsin, porcine bile extract (30% glycocholate, 40% taurocholate,

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7% taurodeoxycholate, 15% glycodeoxycholate and 5% hyodeoxycholate), sodium taurocholate,

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porcine pancreatin, Nystatin, 5-(N-Ethyl-N-isopropyl)amiloride (EIPA), phenylarsine oxide (PAO),

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sodium azide, and HPLC-grade solvents (methanol, ethanol, acetonitrile, dichloromethane,

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n-hexane) were purchased from Sigma-Aldrich (St. Louis, MO U.S.) and used without further

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purification. Tween 20, Tween 40, Tween 60, Tween 80, and span 20 were obtained from

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Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). Monostearin was purchased from Aladdin

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(Shanghai, China). Dulbecco’s modified Eagle’s medium (DMEM) (containing 4.5 g/L D-glucose and

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GlutaMAX™), penicillin and streptomycin (100×), fetal bovine serum (FBS), TrypLETM Select, Hanks’

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balanced salt solution (HBSS), and phosphate buffer solution (PBS)(10×) were purchased from

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GIBCO (Grand Island, NY, U.S.). Cells of the human colon carcinoma cell line (HTB-37) were

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obtained from ATCC (Manassas, VA, U.S.). Ultrapure water was used in all experiments.

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Methods

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Preparation of BC-Loaded organogel and organogel-based nanoemulsion

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The BC-loaded organogel was first prepared by mixing BC, oils (MCT, coconut oil, and corn oil),

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Span 20, and monostearin at weight ratio of (1:7:1:2). The mixture was then heated to 140 °C to

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dissolve BC complete under magnetic stirring (600 rpm). After that the mixture was cooled to room

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temperature to form BC-loaded organogel. The loading amount of BC in organogel was

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approximately 9.1% (w/w). Span 20 was added to increase the solubility of BC in oil carriers.

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BC-loaded organogel-based nanoemulsion was prepared by mixing BC loaded organogel as the

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oil phase, Tweens (Tween 20, Tween 40, Tween 60, and Tween 80) as the emulsifiers, and ultrapure 5



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water at weight ratio of (35:15:50) for 5 min (IKA, T25, Staufen, German)) first, following by

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homogenized with a high pressure homogenizer (AH-2010, ATS Engineering Inc, Canada) 5 times at

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800 bar (80 MPa). The loading of BC in the final emulsion formulation is 30.5 mg/mL.

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Particle diameter analysis

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The average particle sizes were determined by Zetasizer Nano ZSE (Malvern Instruments,

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Worcestershire, UK). The mean particle size and particle size distribution was calculated by

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intensity. BC-loaded organogel-based nanoemulsion samples were prepared by diluting the stock

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samples 1:100 with phosphate buffer (pH 7.0, 10mM) before detection. The refractive index values

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used for the instrumental analysis of oil droplets and dispersant were 1.45 and 1.33, respectively.

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All measurements were made in triplicate at room temperature.

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Lipolysis of BC-Loaded organogel and organogel-based nanoemulsion

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A protocol based on a previous method was used to evaluate the lipolysis profile and BC

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bioaccessibility in organogel and organogel-based nanoemulsion.11 In brief, 0.5g organogel or 1.43g

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nanoemulsions were digested with pancreatin and bile salt. Twenty mL of simulated intestinal fluid

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containing 150 mM NaCl and 10 mM CaCl2 at pH 7 was added to facilitate lipolysis and BC

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micellization. Final concentrations of bile extract and pancreatin were optimized to be 20mg/mL

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and 1mg/mL, respectively. The acidity increased immediately due to the lipase hydrolysis of the

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encapsulated oils and the pH was kept at 7 by the addition of 0.25 M NaOH dropwise with an

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autotitrator (TitraLab TiM840, Radiometer, Lyon, France) at 37 °C in a thermostatic water and

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stirred at 250 rpm. The volume of NaOH added over time (2h) was recorded. The rate and extent of

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lipolysis was calculated from the volume of NaOH added at different time points with the following

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formula.18 6


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Extent =

V × C × 100% 2 × M

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Where VNaOH is the volume of NaOH added. And CNaOH is 0.25 in this case. Moil was the average

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molecular weight of oils.

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An aliquot of the digesta was centrifuged at 10,000 rpm (4 °C) for 1 h (Thermo Scientific, Sorvall

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LYNX 6000) after lipolysis to obtain BC micellar phase, and the aqueous phase was filtered with a

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hydrophilic 0.22 μm filter (PTFE). The filtrate containing BC micellar phase was extracted and

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quantitated by HPLC as described below. BC bioaccessibility was calculated by the following

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formula: Bioaccessibility#$ =

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100 × W#$ & '()) W#$ & *+)

Cellular uptake

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Caco-2 Cells after 60-80 passages were used in this study. Cells were incubated in DMEM

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supplemented 10% FBS, 1× nonessential amino acid (NAA), and 1× penicillin and streptomycin at

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37 ˚C in a humidified atmosphere of 90 % humidity and with 5% CO2.19 Cells were seeded at a

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density of 10,000 cells/well in 12-well plates and the medium was changed every 48 h. After 5 d,

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the cell monolayers were observed with an optical microscope (Leica, IL, U.S.) to ensure that the

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confluence reached approximately 90%. After that, Caco-2 cell monolayers were washed with

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DMEM three times Then, BC in THF/DMSO (1:1, v/v) solution (as control), BC micelles (digested

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nanoemulsions prepared with corn oil) after in vitro digestion, and BC nanoemulsions diluted in

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DMEM to a final BC concentration of 5 μg/mL were added. No significant cytotoxicity was

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observed for all samples after 10-fold or more dilution.19 After 4 h incubation, the upper samples

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were removed and Caco-2 cell monolayers were washed in triplicates with pre-cooled PBS solution

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to stop cellular uptake. In order to remove surface BC, Caco-2 cells were further washed with 7



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pre-cooled PBS with 3.0 mg/mL sodium taurocholate in triplicates. Then, 1.0 mL 10% ethanol PBS

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solution was added to dissociate cell monolayers and cells suspensions were obtained with cell

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scrapers. All of the samples were stored at refrigerator (-80 °C) before BC extraction and analysis.

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Effects of inhibitors on cellular uptake of lipid-based nanoemulsions

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Four blocking reagents (Nystatin, EIPA, PAO, and sodium azide), with various inhibition

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mechanisms were used to analyze the specific mechanism involved in the cell uptake of BC-loaded

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organogel-based nanoemulsions.

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Caco-2 cells were pre-incubated with four blocking reagents at suitable concentrations (Table 1)

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for 30 min, respectively, and were incubated with nanoemulsions for 4 h at 37 °C for cell uptake

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experiment. For control, cells were incubated with PBS (pH 7.4) without inhibitors. The results

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were expressed as the inhibition percentage versus control.

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Protein determination

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Caco-2 cell protein was determined using the bicinchoninic acid (BCA) assay with bovine serum

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albumin as the standard.20

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BC extraction and determination

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BC was extracted from micelles with ethanol:n-hexane (1:2, v:v) three times21 and analyzed by

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HPLC as described in the next section. Micelles (0.2 mL) was de-emulsified by adding 1.0 mL

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ethanol, followed 1.5 mL n-hexane immediately in a 10 mL Pyrex® glass tube with screw cap, and

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the samples were vortexed for half minute. The top layers were kept to separate for 1-2 minutes.

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BC extraction was conducted in the absence of light, and heat.

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25 μL of 5 μg/mL trans-beta-Apo-8’-carotenal in ethanol was added to 0.5mL Caco-2 cell

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suspension as an internal standard. The cell suspensions were extracted three times with 8


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ethanol:n-hexane (1:2, v:v). Organic fractions were combined and the extract concentrated under

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a stream of nitrogen gas at 40 ˚C. Then, the BC extract was dissolved in 0.1 mL of

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methanol:dichloromethane (1:1, v:v) containing 0.1% BHT as antioxidant for HPLC analysis. The

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recovery of trans-beta-Apo-8’-carotenal from Caco-2 cells was at least 95%.

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High-Performance Liquid Chromatography (HPLC)

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An Agilent 1100 HPLC system equipped with a DAD UV-vis absorption detector (Agilent, Santa

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Clara, CA) was used to quantify BC content in micelles, Caco-2 cells and rat plasma.11, 19 A C30

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reverse-phase analytical column (YMC Carotenoid, 250×4.6 mm i.d., 5μm, YMC, Inc., Wilmington,

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NC) was used to separate the carotenoids with a flow rate of 1 mL min-1 at 25 ˚C. A reverse-phase

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C18 column (50×3.0 mm ID, 5 μm, YMC, Inc) was used as a guard column. The injection volume was

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20 μL and the detection wavelength was 450 nm. The chromatography conditions were as follows:

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Solvent A: methanol: acetonitrile: H2O (84:14:2, v:v:v), solvent B: dichloromethane. Gradient

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elution of each sample was achieved with the gradient 20- 55%B for 15 min, 55%B for 5 min, and

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55-20%B for 5 min. The standard curve of the absorption peak area versus BC concentration was

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plotted and fitted with a linear function.

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In Vivo Bioavailability Study

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Male SD rats weighing about 250 g were used for the in vivo pharmacokinetics analysis of BC. All

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animal experiments were performed according to the protocols approved by the Administrative

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Committee on Animal Research of Shenzhen University. Rats were acclimatized for 1 week and fed

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standard chow diet (Laboratory Rodent Chow). After 1 week, six rats randomly chosen were kept

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without food the night prior to the gavage. The animals were administered BC in corn oil (control)

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or BC nanoemulsion by oral gavage with a dose of 100 mg BC per kg body weight. At various time 9



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intervals (0, 0.5, 1, 2, 4, 8, 12, and 24 h), blood samples (500 μL) were withdrawn from the

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retro-orbital plexus under mild ether anesthesia and collected in tubes with heparin. Plasma was

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immediately separated by centrifuging the blood samples at 4000g for 10 min at 4 °C and stored at

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-80 ˚C for HPLC analysis.

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To 200 μL of plasma, 25 μL of 5 μg/mL trans-beta-Apo-8’-carotenal in ethanol was added and

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mixed for 10 s. The plasma was extracted with ethanol:n-hexane (1:2, v:v) three times. Organic

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fractions were combined and the extract concentrated under a stream of nitrogen gas at 40 ˚C.

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The BC extract was dissolved in 0.1 mL of methanol:dichloromethane (1:1, v:v) containing 0.1%

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BHT using HPLC.

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Statistical Analysis

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All experiments were performed in triplicates and were reported as mean±STD. The data were

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analyzed by the analysis of variance (ANOVA) with the SPSS 23.0 package (IBM, New York).

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Duncan’s multiple-range test was used to determine the significant differences of mean values.

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Differences with P < 0.05 was considered statistically significant.

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Results and discussion

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Effects of oils on lipolysis and bioaccessibility of BC-loaded organogel

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Oils are primary in determination the lipolysis and BC bioaccessibility in organogel.18 Three

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various oils (MCT, coconut oil, and corn oil) were chosen to assess the effect of fatty acid length

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and oils saturation degrees on the extent of lipolysis and the bioaccessibility of BC in organogel

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under in vitro digestion. As can be seen in Figure 1, MCT had the greatest extent of lipolysis

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(87.3%), following by coconut oil (64.6%) and corn oil (51.4%). An inverse correlation between the

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extent of lipolysis and fatty acid length was observed. There was also a negative relationship 10


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between lipolysis and oils saturation degrees. Whereas, BC in MCT had the lowest bioaccessibility

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(7.3%). BC bioaccessibility was in the following order: corn oil>coconut oil>MCT. Results clearly

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demonstrated that BC bioaccessibility was greater with corn oil than with coconut oil and MCT. The

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results suggested that the amount of BC released was positively proportional to the length and

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unsaturation degrees of oils as well as inversely correlated with the extent of lipolysis. Previous

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studies have also confirmed improved bioaccessibility of BC in nanoemulsion or vegetable and

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fruits with longer fatty acid chain.22, 23 Long length fatty acid facilitated the transfer from organogel

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to micelles. Compared to the poorly swollen micelles gained from the lipolysis of MCT and coconut

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oil, the more swollen micelles formed from long chain FA results in a higher BC solubilization

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capacity. Our previous study also confirmed that unsaturated fatty acid-rich oils were better than

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saturated fatty acid-rich oils in transferring BC from samples to micelles.23 Therefore, corn oil was

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chosen as the carrier oils for BC-loaded organogel-based nanoemulsion preparation.

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Effects of emulsifiers on lipolysis and bioaccessibility of BC-loaded organogel nanoemulsion

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The lipolysis profile and BC bioaccessibility in nanoemulsion can be affected largely by the

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interfacial composition of the nanoemulsion. Yu et al. found that organogel-based nanoemulsions

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encapsulated with Tween 20 showed higher curcumin bioaccessibility than that stabilized with

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modified starch and whey protein, even though no explanations were given.16 The BC

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micellarization

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protein>decaglycerolmonolaurate>soybean soluble polysaccharides.24 In this study, four nonionic

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surfactants were used to prepare BC-loaded organogel-based nanoemulsion and optimized in

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terms of BC bioaccessibility during lipolysis. As can be seen in Figure 2, BC-loaded organogel-based

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nanoemulsion stabilized with Tween 20 had the greatest extent of lipolysis (82.4%), following by

extent

in

emulsions

was

in

the

11


following

order:

whey


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Tween 40 (75.7%), Tween 60 (68.4%) and Tween 80 (66.5%) (Figure 2). This was mainly attributed

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to the different aliphatic chain length and hydrophilic/lipophilic balance (HLB) value. Recent studies

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showed that the extent of lipolysiss was positively correlated with the HLB of the surfactant and

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negatively correlated to the surfactant aliphatic chain length.25 The aliphatic chain lengths of

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Tween 20, 40, 60 and 80 were 12, 16, 18, and 18 carbons, respectively. As the aliphatic chain length

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of the Tweens increased, the HLB value decreased, leading to the decrease of lipolysis.

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Among all four samples, Tween 20 facilitated the highest BC bioaccessibility (62.5%), followed by

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Tween 40 (56.5%). No significant differences of BC bioaccessibility were found when Tween 60

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(47.2%) or Tween 80 (46.7%) was used, possibly due to the similar aliphatic chain length and HLB

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values. There is a positively correlated relationship between the extent of lipolysis and BC

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bioaccessibility. Therefore, Tween 20 was used as an emulsifier for BC-loaded organogel-based

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nanoemulsion preparation. The mean particle diameter, particle size distribution, and PDI value of

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BC-loaded organogel-based nanoemulsion stabilized with Tween 20 was analyzed with Zetasizer

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Nano ZSE. As can be seen in Figure 3, the mean particle size of BC-loaded nanoemulsion was 176.3

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nm and the PDI value was 0.174, suggesting a narrow particle size distribution.

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Comparison of the in vitro lipolysis profile and bioaccessibility of BC in organogel and

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organogel-based nanoemulsion

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The lipolysis profile, BC bioaccessibility after digestion, and the correlation between the extent

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of lipolysis and BC bioaccessibility were shown in Figure 4. Bulk oil had the lowest rate and extent

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of lipolysis (27.4%), while organogel had the greater extent of lipolysis (51.4%), suggesting

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organogel increased the lipolysis. This is mainly attributed to the increased interaction of lipase and

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oil after oil incorporated in organogel. BC nanoemulsion showed the greatest rate and extent of 12


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lipolysis (82.4%), mainly attributed to the smaller particle diameter (Figure 3) and higher lipid

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surface area to volume ratio in nano-droplet after emulsification.11 Higher lipid surface area

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resulted in greater extent of lipid hydrolysis by lipase.

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The bioaccessibility of BC in organogel was 33.3%, while the bioaccessibility of BC in oil was only

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4.3%, indicating BC bioaccessibility was increased by approximately 8-fold when loaded in

241

organogel, possibly due to the high solubility and loading amount of BC in organogel. Furthermore,

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BC bioaccessibility further increased to 62.5% after organogel-based nanoemulsion formation. The

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major increase of BC bioaccessibility in nanoemulsion was mainly attributed to the significantly

244

increased lipolysis. The lipid hydrolysis facilitated the released from oil droplets to micelles formed

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during digestion by bile acids monoglycerides, and phospholipids.26 In addition, a positive

246

relationship between extent of lipolysis and BC bioaccessibility was observed, suggesting increase

247

the extent of lipolysis should be an effective approach to improve the bioaccessibility of

248

encapsulated hydrophobic nutraceuticals in corn oil.

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Cellular uptake of BC nanoemulsion

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Caco-2 cells were used as models for simulating intestine absorption because it has many

251

functions of the small intestinal villus epithelium.27 The cellular uptake of BC in THF/DMSO

252

suspension, BC micelles after in vitro digestion, and BC-loaded organogel-based nanoemulsions

253

was studied. Cellular uptake of BC-loaded in nanoemulsion was appreciably higher than BC in

254

THF/DMSO suspension (control), indicating nanoemulsion-based delivery system can improved the

255

internalization of encapsulated BC. As can be observed in Figure 5, the cellular uptake of BC in

256

THF/DMSO suspension was 267.5 pmol/mg protein, similar to the previous results.19 After 4 h of

257

incubation, cellular uptakes of BC micelles and BC nanoemulsion was 1167.3 and 835.3 pmol/mg 13



258

protein, respectively, which were 4.4- and 3.1-fold greater than that for BC in THF/DMSO

259

suspension (control), respectively. Furthermore, BC micelles showed significantly higher BC

260

accumulation than BC nanoemulsion (p < 0.01) with Caco-2 cells. Similarly, Yu et al. showed that

261

the transport of curcumin in digested nanoemulsion was significantly higher than that in

262

nanoemulsion itself.16 The results suggested that the uptake of encapsulated BC may be

263

droplet-diameter-dependent. In this study, the mean particle diameter of BC micelles after

264

ultracentrifuge was approximately 10 nm, and much smaller than Tween 20 stabilized BC

265

nanoemulsion (176.3 nm). Smaller particle size resulted in higher in vitro bioaccessibility of

266

nanoencapsulated BC.28

267

Effects of inhibitors on cellular uptake of BC-loaded nanoemulsions

268

Inhibitors of four different endocytosis routes were used to determine the mechanism of uptake

269

of BC-loaded organogel-based nanoemulsion. The uptake of nanoemulsion was significantly

270

decreased by all four inhibitors (Figure 6). The toxicity of inhibitors at the concentration used on

271

Caco-2 cells was evaluated prior to cellular uptake experiments. All the cell viabilities were above

272

95%, indicating these inhibitors are nontoxic at the concentration used and the possible inhibition

273

of uptake are not due to cell toxicity.

274

The endocytosis of nano-droplets is an active transport mechanism and requires energy. In this

275

study sodium azide29 decreased lipid nanoemulsion particle intake by about 25% had an inhibitive

276

effect on intake (Figure 6) indicating that at least some intake is by an endocytosis mechanism.

277

5-(N-Ethyl-N-isopropyl)amiloride (EIPA) have been reported to be an effective inhibitor of

278

macropinocytosis30 In this study, cellular uptake of lipid-based nanoemulsion droplets was

279

decreased approximately 14% with EIPA, indicating macropinocytosis was involved in the 14


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internalization (Figure 5). Phenylarsine oxide (PAO) is an inhibitor of clathrin-mediated endocytosis

281

by interacting with vicinal dithiol-containing molecules.31 Larger than 50% of the internalization of

282

nanoemulsions was significantly inhibited by PAO, suggesting clathrin-mediated endocytosis may

283

also play a vital role in the uptake process. Nystatin is used to inhibit cholesterol-dependent uptake

284

by caveolin- and lipid-raft-mediated endocytosis.32 The results showed that Nystatin had highest

285

inhibition effects, indicating caveolae/lipid raft-dependent endocytosis may be the most important

286

manner in the internalization of nanoemulsions. Compared to the other two endocytosis routes,

287

macropinocytosis

288

microwave-produced solid lipid nanoparticles (SLNs) indicated that clathrin-mediated route was

289

the most preferred pathway.33, 34 The difference between these results may be attributed to the

290

differences in surface properties, particle size, and lipid condition. In this study, the mean particle

291

size was 176.3 nm, which was smaller than that of SLNs (273.4nm).34 Furthermore, the

292

zeta-potential of BC nanoemulsion was -10.7 mV, whereas the zeta-potential of SLNs was −18.3

293

mV.34 Our findings illustrate that endocytosis of nanoemulsions is complicated and may be the

294

consequence of the combined action of clathrin, lipid raft/caveolae, and macropinocytosis and lipid

295

raft/caveolae route played the most important role. Chai and He also demonstrated similar results

296

in the study of the transport mechanisms of SLNs and polymer nanoparticles, respectively.31, 35

297

In vivo bioavailability of BC

was

least

important.

However,

previous

research

of

uptake

of

298

In this study, rats were used as animal models to directly study the release and bioavailability of

299

BC-loaded nanoemulsion in vivo. Nanoemulsion-based delivery system were reported to increase

300

the bioavailability of encapsulated BC.11, 23 No BC was detected in rat plasma before the gavage

301

administration. As can be observed in Figure 7, the BC plasma concentrations in organogel-based 15



302

nanoemulsion were all remarkably higher than those of BC-oil sample at all various time intervals.

303

Both nanoemulsion and bulk oil showed a sharp increase of BC plasma concentration within 1h and

304

sustained decreases of BC plasma concentration were observed after 1 h, possibly due to the

305

metabolism of BC and transfer from blood to liver. The corresponding pharmacokinetics

306

parameters (Cmax, and AUC0-24) were calculated and shown in Table 2. The Cmax values of BC were

307

7.50 and 0.64 for nanoemulsion and bulk oil, respectively, indicating Cmax increased by 11.7-fold by

308

nanoemulsion. And the AUC0-24 values of BC were 64.77 and 5.63 for nanoemulsion and bulk oil,

309

respectively and an 11.5-fold increase was observed. The results clearly demonstrated that

310

organogel-based nanoemulsion could appreciably enhance the bioavailability of BC. The increase in

311

oral bioavailability may be mainly attributed to the increase of BC solubility in nanoemulsion,

312

compared to BC dispersed in bulk oil.16

313

In conclusion, organogel-based nanoemulsion with high BC loading amount was prepared and

314

evaluated. Corn oil was optimized for the highest BC bioaccessibility in organogel. Tween 20 was

315

chosen based on the greatest extent of lipolysis and BC bioaccessibility in organogel-based

316

nanoemulsion. Compared to bulk oil, organogel remarkably improved the bioaccessibility of

317

encapsulated BC and organogel-based nanoemulsion showed the highest BC bioaccessibility.

318

Cellular uptake of BC-loaded in nanoemulsion was appreciably higher than BC in THF/DMSO

319

suspension (control). Inhibition study suggested that lipid raft/caveolae route played the most

320

important role in endocytosis of nanoemulsion. Furthermore, in vivo rats experiment showed that

321

BC bioavailability of organogel-based nanoemulsion increased approximately 11.5-fold compared

322

to BC in bulk corn oil. The results obtained may provide some useful information for the application

323

of organogel-based nanoemulsion in loading, protecting, and delivering lipophilic nutraceuticals in 16


Page 16 of 33

Page 17 of 33


324

food, cosmetics and pharmacy industry.

325

Funding

326

This work was supported by the National Natural Science Foundation of China (No.31601512) and

327

Young Scholars' Scientific Research Startup Funding from Shenzhen University (No.2016010).

328

Notes

329

The authors declare no competing financial interest. Mention of trade names or commercial

330

products in this publication is solely for the purpose of providing specific information and does not

331

imply recommendation or endorsement by the U.S. Department of Agriculture. USDA is an equal

332

opportunity provider and employer.

333

17



334

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22


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Table 1: Inhibitors with Different Endocytosis Functions and the Concentrations. Endocytosis inhibitors

Inhibitor of

Concentrations

EIPA

macropinocytosis pathway

50 μM

PAO

clathrin-mediated endocytosis

10 μM

Nystatin

caveolae/lipid raft-dependent endocytosis

30 μM

Sodium azide

energy-dependent route

1 mg/mL

23



Page 24 of 33

Table 2: Pharmacokinetics Parameters of BC Bulk Oil and BC-Loaded Organogel-Based Nanoemulsion in Rats. AUC0-∞ Samples

Dose (mg/kg)

Cmax (μg/mL)

Tmax (h) (μg/mL·h)

BC oil

100

0.64±0.32

1

5.63±1.23

100

7.50±1.05

1

64.77±5.37

BC nanoemulsion

24


Page 25 of 33


Figure Captions Figure 1 Effects of oil types (MCT, coconut oil, and corn oil) on the lipolysis and BC bioaccessibility of BC-loaded organogel. Data are expressed as mean±STD. Figure 2 Effects of emulsifier types on the lipolysis and BC bioaccessibility of BC-loaded organogel-based nanoemulsions. Data are expressed as mean±STD. Figure 3 Particle size distribution of BC-loaded organogel-based nanoemulsion stabilized with Tween 20. Figure 4 Comparison of the rate and extent of lipolysis (A), in vitro bioaccessibility (B), and relationship between lipolysis and BC bioaccessibility (C) of BC corn oil, BC-loaded organogel, and BC-loaded organogel-based nanoemulsion. Data are expressed as mean±STD. Different letters (a-c) indicate a significant difference between two samples (P

Development of Î²-Carotene-Loaded Organogel-Based Nanoemulsion

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