Enhancement of curcumin bioavailability by encapsulation in

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Enhancement of curcumin bioavailability by encapsulation in Sophorolipid-coated nanoparticles: An in vitro and in vivo study David Julian McClements, Shengfeng Peng, Ziling Li, Liqiang Zou, Wei Liu, and Chengmei Liu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b05478 • Publication Date (Web): 29 Jan 2018 Downloaded from http://pubs.acs.org on January 29, 2018

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Journal of Agricultural and Food Chemistry

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Enhancement of curcumin bioavailability by encapsulation in

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Sophorolipid-coated nanoparticles: An in vitro and in vivo study

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Shengfeng Peng a, Ziling Liab, Liqiang Zou a, Wei Liu *a, Chengmei Liu a, David Julian

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McClements *c.

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a

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330047, Jiangxi, PR China

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b

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

School of Life Science, Jiangxi Science and Technology Normal University, Nanchang,

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330013, Jiangxi, PR China

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c

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Massachusetts, Amherst, MA 01003, USA

Biopolymers and Colloids Laboratory, Department of Food Science, University of

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*Corresponding authors

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E-mail: [email protected]; Fax: +86 791 88334509; Tel: + 86 791 88305872x8106.

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E-mail: [email protected]; Fax: +1 413 545 1262; Tel: +1 413 545 1019.

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ABSTRACT

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There is great interest in developing colloidal delivery systems to enhance the water-

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solubility and oral bioavailability of curcumin, which is a hydrophobic nutraceutical

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claimed to have several health benefits. In this study, a natural emulsifier was used to form

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sophorolipid-coated curcumin nanoparticles. The curcumin was loaded into sophorolipid

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micelles using a pH-driven mechanism based on the decrease in curcumin solubility at

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lower pH values. The sophorolipid-coated curcumin nanoparticles formed using this

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mechanism were relatively small (61 nm) and negatively charged (-41 mV). The

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nanoparticles also had a relatively high encapsulation efficiency (82%) and loading

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capacity (14%) for curcumin, which was present in an amorphous state. Both in vitro and

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in vivo studies showed that the curcumin nanoparticles had an appreciably higher

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bioavailability than free curcumin crystals (2.7-3.6-fold), which was mainly attributed to

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their higher bioaccessibility. These results may facilitate the development of natural

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colloidal systems that enhance the oral bioavailability and bioactivity of curcumin in food,

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dietary supplement, and pharmaceutical products.

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Keywords:

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biosurfactant; sophorolipid

curcumin;

bioavailability;

nanoparticles;

bioaccessibility;

stability;

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INTRODUCTION

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Nutraceuticals are bioactive components found in foods, which are claimed to have

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certain health benefits when ingested orally . Curcumin is a polyphenolic nutraceutical

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typically isolated from the herb turmeric, which is a member of the ginger family . It has

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been reported to exhibit a broad range of biological activities that may be beneficial to

43

human health, including anticancer, anti-inflammatory, antimicrobial, and antioxidant

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activities . However, the utilization of curcumin as a nutraceutical ingredient in functional

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food and beverage products is currently limited by its poor water-solubility, chemical

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stability, and oral bioavailability characteristics . These challenges can often be overcome

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by encapsulating curcumin within colloidal delivery systems that contain small particles

48

dispersed within an aqueous medium, such as emulsions , nanoemulsions , micelles ,

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liposomes

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performance of each colloidal delivery system depends on the composition and structure

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of the particles it contains. Consequently, they must be carefully designed to exhibit the

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functional attributes required for a specific application. These functional attributes include:

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physical and chemical stability under different environmental conditions; rheological

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characteristics; optical properties; flavor profile; and gastrointestinal fate (including

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bioavailability) .

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1

2

2

3

4-5

8-10

, biopolymer microgels , and polymer nanoparticles 4

5

11-14

6-7

. The functional

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The food industry is increasingly moving towards formulating its products from natural ingredients, rather than synthetic ones

16-17

. Consequently, it would be beneficial to 3


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create curcumin delivery systems from natural components. Synthetic surfactants are often

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used to facilitate the formation and/or increase the stability of the particles in colloidal

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delivery systems. They do this by adsorbing to particle surfaces and generating strong

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repulsive forces between the particles, such as steric or electrostatic repulsion . Synthetic

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surfactants can often be replaced with natural alternatives (“biosurfactants”), which can be

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isolated from animal, plant, or microbial sources

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potential application of sophorolipids as biosurfactants for forming curcumin-loaded

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colloidal delivery systems. Sophorolpids are surface-active glycolipids composed of a

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polar sophorose group and a non-polar fatty acid group, which can be produced by

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microbial fermentation . In addition, a recently developed method of forming curcumin-

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loaded nanoparticles is utilized, which is based on changes in the solubility of curcumin

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with pH

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molecule with a low water-solubility . However, at a sufficiently high pH, some of the

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hydroxyl groups on curcumin become deprotonated, leading to a negative charge that

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increases the water-solubility. Consequently, curcumin can be loaded into the hydrophobic

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core of surfactant micelles by mixing an alkaline solution of curcumin with an acidic

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solution of surfactant micelles. When the two solutions are mixed, the pH is reduced, which

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causes the curcumin to become more hydrophobic and move into the surfactant micelles

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(Fig. 1). Previous studies have shown that polyphenols such as curcumin and rutin can be

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loaded into synthetic and natural surfactant micelles using the pH-driven method

18

16-17

. In this research, we focus on the

19

9, 20-21

. At relatively low pH, the curcumin has no charge and is a highly hydrophobic 15

9, 20-21

. Some 4



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of the advantages of this loading method are that it simple to perform, does not require

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heating or organic solvents. To the best of our knowledge, this is the first study of the

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preparation of sophorolipid-coated curcumin nanoparticles using the pH-driven method.

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The main objectives of the current study are to determine whether curcumin

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nanoparticles could be successfully produced from sophorolipids using the pH-driven

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method, to characterize the physicochemical properties of the nanoparticles formed, and to

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assess their potential for increasing curcumin bioavailability using both in vitro and in vivo

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methods. The results of this study may lead to the development of novel all natural

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curcumin delivery systems that can be incorporated into functional foods, dietary

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supplements, and pharmaceutical preparations.

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MATERIALS AND METHODS

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Materials. Powdered curcumin (composed of 76.4% curcumin, 17.3% demethoxycurcumin

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and 3.8% bisdemethoxycurcumin) was purchased from the Aladdin Industrial Corporation

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(Shanghai, China). Sophorolipid was bought from the Boliante Chemical Company (Xian, China).

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Ethanol, phosphoric acid, sodium hydroxide and other reagent chemicals were all of analytical

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grade.

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Nanoparticle preparation. Curcumin nanoparticles were prepared using the pH-

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driven method described in our previous study with some slight modifications . Briefly, a

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series of solutions were prepared by dissolving different amounts of the biosurfactant in 20

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5


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mM phosphoric acid to obtain sophorolipid concentrations of 2, 4, 8, 12 and 16 mg/mL.

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Powdered curcumin was then weighed into a 30 mM sodium hydroxide solution and stirred

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for 5 min to reach a final curcumin concentration of 2.0 mg/mL. This curcumin level was

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used because it was fully soluble within the initial alkaline solution, as well as within the

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sophorolipid micelles formed after the following step. The alkaline curcumin solutions

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were then added to similar volumes of acidic sophorolipid solutions while stirring

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continuously at 500 rpm on a magnetic stir-plate. The resulting mixtures were incubated

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for 30 min at room temperature and then centrifuged at 10,000 g for 10 min to remove any

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insoluble curcumin particles. The curcumin samples used for the X-ray Diffraction and

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storage stability studies were in a powdered form, which was produced by lyophilization

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using a freeze-drier (Alpha 2–4 LD plus, Martin Christ Gefriertrocknungsanlagen GmbH,

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Osterode am Harz, Germany).

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Nanoparticle characterization. The particle size distribution and electrical

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characteristics (z-potential) of the curcumin nanoparticles were measured at 25°C using a

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combined dynamic light scattering (DLS) – electrophoresis instrument (Nicomp 380 ZLS,

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Santa Barbara, CA, USA).

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measured at an angle of 90º after the nanoparticle suspensions had been diluted 4-fold with

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water to avoid multiple scattering effects.

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standard deviation based on at least three samples with each sample being measured in

116

triplicate.

The time-dependent fluctuations in light intensity were

All data were calculated as the mean and

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Microstructure images of the nanoparticle suspensions were obtained using an Atomic

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Force Microscope (AFM). An aliquot of nanoparticle suspension was placed on to a freshly

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cleaved mica substrate, and then images of the samples were acquired using an AFM

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(Agilent 5500, Agilent Technologies, Santa Clara, CA, USA) with a silicon cantilever of

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force constant of 0.58 N m operated in tapping mode at room temperature. -1

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The X-Ray Diffraction (XRD) patterns of pure curcumin powder and powdered

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sophorolipid-coated curcumin nanoparticles were recorded using a D8 Advance X-ray

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diffractometer (Bruker, Germany). The divergence slit was set at 1°, and the receiving

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slit was set at 0.1 mm for the incident beam. The scan rate was 2° per min over a 2q angle

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range of 5° to 40°.

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The encapsulation efficiency (EE) and loading capacity (LC) of the curcumin

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nanoparticles were determined according to the procedures described in our previous study

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non-encapsulated curcumin. The supernatant was then removed and diluted with

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anhydrous ethanol. The absorbance at 420 nm was determined using a UV-Vis

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spectrophotometer (Pgeneral T6, China) and the concentration of loaded curcumin was

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calculated from a calibration curve. The EE and LC were calculated using the following

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expressions:

. Briefly, nanoparticle suspensions were centrifuged at 10,000 g for 10 min to remove any

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EE (%) = mL,C / mIC × 100

(1)

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LC (%) = mL,C / mP × 100

(2) 7


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Here mL,C is the mass of curcumin trapped within the nanoparticles, mI,C is the initial mass of

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curcumin present in the system, and mP is the total mass of the nanoparticles (curcumin +

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sophorolipids). The total mass of the nanoparticles was determined by freeze drying the

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centrifuged nanoparticle suspension and then weighing the resulting powder. The mass of

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curcumin loaded in the nanoparticles was determined by rehydrating the nanoparticles in

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an aqueous solution and then measuring the absorbance using a UV-vis spectrophotometer.

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The initial mass of curcumin present in the system was calculated from the known

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quantities of the nutraceutical added initially, taking into account the various dilution steps.

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Physical stability of nanoparticles

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Influence of pH and ionic strength: A series of nanoparticle suspensions with pH

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values ranging from 2.0 to 8.0 were prepared by adding different amounts of either HCl or

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NaOH solution. A series of nanoparticle suspensions with different ionic strengths was

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prepared by incorporating different levels of salt into them: 10, 20, 50, 100, 200 or 1000

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mM NaCl. The resulting solutions were then stirred for 1 hour at ambient temperature and

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any changes in particle size and charge were measured using the methods described earlier.

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Storage stability: The influence of storage temperature and time on the stability of the

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nanoparticle suspensions was measured to provide some insights into their potential long-

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term stability. Powdered (lyophilized) curcumin nanoparticles were stored at 25 ºC, and

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aqueous suspensions containing curcumin nanoparticles were stored at 4 ºC or 25 ºC for

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30 days. The particle size, charge, and encapsulation efficiency were then record at 8



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different time intervals during storage.

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In vitro bioavailability. The in vitro bioavailability of curcumin was determining by

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passing the samples through a static simulated gastrointestinal tract (GIT), and then

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measuring the amounts of curcumin that had degraded and that had been solubilized in the

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mixed micelle phase.

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Simulated gastrointestinal tract (GIT). The static GIT method used in this study

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consisted of simulated mouth, stomach, and small intestine phases, and has been described

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previously .

165

method that has recently been developed .

9

This method is fairly similar to the international consensus INFOGEST 22

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Oral phase: 7.5 mL of a test sample were mixed with 7.5 mL of simulated saliva fluid

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containing mucin (30 mg/mL) and various salts, prepared as described elsewhere . The

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resulting mixtures was then adjusted to pH 6.8 and shaken at 90 rpm for 10 min at 37 °C

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to mimic oral conditions.

23

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Gastric stage: A simulated gastric fluid was prepared that contained NaCl (2 mg/mL),

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HCl (7 mg/mL), and pepsin (3.2 mg/mL), which was then heated to 37 °C. 15 mL of the

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simulated gastric fluid was then added to 15 mL of the bolus solution resulting from the

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oral phase. The resulting mixtures were then adjusted to pH 2.5 and shaken at 100 rpm for

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2 h to mimic gastric conditions.

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Small intestine phase: Samples from the simulated gastric phase were adjusted to pH

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7.0 with 2 M NaOH. Simulated small intestinal fluid containing pancreatin (24 mg/mL, 2.5 9


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mL), bile extract solution (50 mg/mL, 3.5 mL) and saline solution (0.5 M CaCl and 7.5 M

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NaCl, 1.5 mL) were then added into the reaction vessel. The pH of the sample was then

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maintained constant at pH 7.0 by addition of 50 mM NaOH solution using an automatic

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titration unit (pH stat).

2

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In vitro bioavailability determination. The in vitro bioavailability of curcumin was

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estimated by measuring changes in its concentration and location after passing through the

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simulated GIT. It was assumed that the overall bioavailability is mainly limited by its

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chemical degradation (transformation) and solubility in the mixed micelle phase

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(bioaccessibility), rather than by its uptake by the epithelium cells (absorption). Thus, the

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in vitro bioavailability (BA) = bioaccessibility (B*) ´ transformation (T*).

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transformation is defined as the fraction of curcumin that remains in an active state after

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passage through the GIT. The raw digesta of each sample was centrifuged at 40,000 g for

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30 min at 4°C to remove any insoluble matter. The supernatants were collected and

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assumed to be the mixed micelle fraction, in which the bioactive agent is solubilized in a

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form suitable for absorption. The solubilized curcumin was diluted with methanol and

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assayed using high performance liquid chromatography (1260 HPLC, Agilent

193

Technologies, Santa Clara, CA, USA) equipped with a UV-visible detector. Curcumin was

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separated on a Sunfire C18 column (250 mm × 4.6 mm, 5 μm; Waters Corporation, Milford,

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MA, USA), using a mobile phase consisting of 0.1% (v/v) acetic acid and acetonitrile

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(45:55 v/v) at a flow rate of 1.0 mL min , with detection by UV absorption at 420 nm.

The

−1

10



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The transformation and bioaccessibility of curcumin were then calculated using the following equations:

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Transformation (%) = C / C × 100 (3)

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Bioaccessibility (%) = C /C × 100 (4)

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Here, C and C are the concentrations of curcumin in the mixed micelle fraction and

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in the overall digesta at the end of the simulated GIT model, and C is the concentration of

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curcumin initially added (taking into account the various dilution steps). This latter value

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is therefore a measure of the total amount of curcumin that would be present in the small

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intestine phase if there were no losses due to chemical degradation. It should be noted that

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a simple in vitro GIT model cannot accurately simulate the complex processes occurring

207

within a living gastrointestinal tract, but it is useful for rapidly screening different samples

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and for identifying important physicochemical mechanisms.

D,C

M,C

M,C

I,C

D,C

D,C

I,C

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In vivo bioavailability. The in vivo bioavailability of curcumin was evaluated by oral

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administration to 12 male Sprague Dawley (SD) rats weighing between 260 and 300 g. The

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rats were randomly divided into two groups (n=6). Group 1 was administrated 100 mg/kg

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body weight crystalline curcumin and group 2 was administrated 100 mg/kg body weight

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curcumin nanoparticles by oral gavage. Aqueous suspensions (10 mg/mL) of crystalline

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curcumin were prepared by dispersing powdered curcumin into 1.0% sodium

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carboxymethyl cellulose (as a stabilizer). Aqueous suspensions (10 mg/mL) of curcumin

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nanoparticles were prepared by dispersing lyophilized sophorolipid-coated curcumin 11


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nanoparticles in distilled water. A total of 0.5 mL blood sample was collected from the

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retro-orbital plexus of each rat at different times (0.5, 1, 2, 4 and 8 h) into heparinized

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microcentrifuge tubes (containing 20 μL of 1000 IU heparin/mL of blood). These samples

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were then immediately centrifuged at 4000 g for 10 min at 4 °C to isolate the plasma, which

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was then stored at -80 °C until analysis by LC–MS/MS. According to previous studies

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curcumin is mainly present in a conjugated form (curcumin glucuronide) when it is

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absorbed through the intestinal cells in rats. So, the concentration of curcumin and

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curcumin glucuronide in rat plasma were analyzed for.

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glucuronide could be detected in the rat plasma, and so this value was used to determine

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the in vivo oral bioavailability.

24-26

,

However, only curcumin

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Plasma (100 µL) was mixed with 200 µL acetonitrile by vortexing and centrifuged at

228

10000 g for 5 min, at 4 °C. Aliquots of the extracts were injected onto a C18 column

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(Zorbax Eclipse Plus C18 column, 100mm×2.1mm, I.D., 3.5 μm, Agilent, USA) kept at

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40 °C. The mobile phase consisted of two components: A, acetonitrile and B, 0.1% formic

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acid. The gradient profile was as follows: 0-1min, 80%B→20%B; 1-3min, 20%; 3-3.5min,

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20%B→80%B. The flow rate was 0.3 ml/min. Curcumin and curcumin glucuronide were

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analyzed using a 6410 QQQ MS/MS system (Agilent Technologies, USA) equipped with

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an electrospray ionization source (ESI), operating in positive mode. The mass spectrometer

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ion source parameters were as follow: gas temperature, 350℃; gas flow, 10 L/min;

236

nebulizer gas, 40 psi; spray voltage 4000 kV. Nitrogen served as a nebulizer and collision 12



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gas. Curcumin and curcumin glucuronide were determined using the multiple reaction

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monitor mode as follows: CUR, m/z 369 > 285, m/z 369 >177. CUR-G, m/z 545 > 369,

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m/z 545 >177.

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Statistical analysis:

All measurements were replicated at least three times. The

241

results were then expressed as means ± standard deviations. Data were subjected to

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statistical analysis using SPSS software, version 18.0 (SPSS Inc., Chicago, IL, USA). The

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Student-Newman-Keuls test was performed to check significant comparisons and P < 0.05

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was considered statistically significant.

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RESULTS AND DISCUSSION

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Potential impact of fabrication method on curcumin stability: Curcumin has been

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reported to chemically breakdown upon prolonged storage in aqueous alkaline conditions

248

3, 5

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mechanism used to fabricate the curcumin nanoparticles. Nevertheless, a previous study

250

by our group indicated that only 0.39 ± 0.27% of curcumin was degraded when it was

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dissolved in an aqueous alkaline solution (pH 12.0) for 10 min . The pH-driven loading

252

method used in the current study only involved holding curcumin at pH 12.0 for 5 min, and

253

so the degradation of curcumin during this procedure is expected to be relatively small.

, and so it was possible that some of it may have degraded when using the pH-driven

9

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Effect of biosurfactant concentration: Initially, experiments were carried out to

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determine the optimum sophorolipid concentration required to form the curcumin 13


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nanoparticles. Consequently, the impact of sophorolipid concentration on the mean particle

257

diameter, polydispersity index, z-potential, and encapsulation efficiency of the curcumin

258

nanoparticles was determined (Table 1, A).

259

For the fresh samples (A), the mean particle diameter decreased from around 114 to

260

61 nm when the sophorolipid concentration increased from 1 to 4 mg/mL, but then it

261

remained relatively constant at higher sophorolipid levels. The polydispersity index (PDI)

262

of most of the samples was relatively small (< 0.2), indicating that they had fairly narrow

263

particle size distributions.

264

sophorolipid levels had higher PDI values, indicating that they had broad distributions.

265

The electrical characteristics of the nanoparticles also depended on biosurfactant

266

concentration, with the z-potential changing from around -41 to -21 mV when the

267

sophorolipid level increased from 1 to 8 mg/mL. The encapsulation efficiency increased

268

from around 41 to 89% as the sophorolipid concentration increased from 1 to 8 mg/L.

However, some of the samples containing intermediate

269

An understanding of the physicochemical basis of the pH-driven loading mechanism

270

is required to interpret the observed results. At a sufficiently high pH, curcumin has a

271

negative charge and is soluble in water, but when the pH is reduced it loses its charge and

272

becomes more hydrophobic . In the presence of biosurfactant micelles, there are two

273

possibilities for the hydrophobic curcumin molecules to reduce their thermodynamically

274

unfavorable contact with water: (i) they can form crystals; or, (ii) they can be solubilized

275

within the hydrophobic interior of the micelles (Fig. 1). At relatively low biosurfactant

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14



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levels, there may not be enough biosurfactant micelles present to completely solubilize all

277

of the curcumin, and so some curcumin crystals are formed. Alternatively, the rate of

278

crystal formation may exceed the rate of solubilization, so that some curcumin crystals are

279

generated before solubilization can occur. At relatively high biosurfactant levels, there may

280

be enough sophorolipid micelles available to solubilize all of the curcumin molecules

281

present, and/or the solubilization rate may exceed the crystallization rate, thereby leading

282

to fewer curcumin crystals being formed. The curcumin crystals are likely to be larger than

283

the curcumin-swollen micelles, and are likely to have different electrical characteristics,

284

which would account for the observed decrease in mean particle diameter and change in z-

285

potential with increasing biosurfactant concentration (Table 1). During the solubilization

286

process, it is likely that some of the original sophorolipid micelles incorporated curcumin

287

and swelled, whereas others dissociated so that they could release biosurfactant molecules

288

that could then cover the increased surface area of the curcumin-swollen micelles. The

289

PDI was relatively small at low sophorolipid levels because the system mainly contained

290

only large curcumin aggregates, and was relatively small at high sophorolipid levels

291

because the system mainly contained curcumin-loaded micelles. On the other hand, the

292

PDI was relatively large at intermediate sophorolipid levels because the system contained

293

a mixture of small micelles and large curcumin crystals.

294

The observed increase in encapsulation efficiency with increasing sophorolipid

295

concentration may also be ascribed to a similar physicochemical mechanism. At relatively 15


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low biosurfactant levels, more curcumin crystals are formed in the aqueous phase because

297

of the low micelle solubilization capacity or slow solubilization rate. Consequently, these

298

relatively large crystals would be removed during the centrifugation step, thereby leading

299

to a low measured encapsulation efficiency. Conversely, at high biosurfactant levels, most

300

of the curcumin will be solubilized within the biosurfactant micelles leading to the

301

formation of sophorolipid-coated curcumin nanoparticles. These nanoparticles are

302

relatively small and are not removed by centrifugation, thereby leading to a higher

303

measured encapsulation efficiency.

304

Effect of rehydration on nanoparticle stability: For commercial applications, it is

305

often advantageous to have a powdered form of a nutraceutical ingredient, as this improves

306

handling, transport and storage, and allows it to be incorporated into a broader range of

307

products. For this reason, the impact of converting the aqueous suspension of curcumin

308

nanoparticles into a powder using freeze drying (lyophilization) was assessed. The

309

physiochemical properties of the curcumin nanoparticles were then measured after the

310

powder had been rehydrated to establish the influence of lyophilization on their

311

performance (Table 1, B).

312

As with the freshly prepared systems, the mean particle diameter of the curcumin

313

nanoparticles arising from the rehydrated powders decreased steeply as the sophorolipid

314

concentration increased from 1 to 4 mg/mL, but then remained fairly constant at higher

315

biosurfactant levels. At sophorolipid concentrations of 4 mg/mL or higher, the rehydrated 16



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curcumin nanoparticles rapidly dispersed in water and had physicochemical characteristics

317

similar to those of the original systems (Table 1). At lower sophorolipid concentrations,

318

the rehydrated curcumin nanoparticles had much larger particle sizes and polydispersity

319

indices than the original systems, suggesting that appreciable particle aggregation occurred

320

during the drying and/or rehydration stages. Indeed, large precipitates could even be

321

observed by eye in these samples. At low biosurfactant concentrations, there may have

322

been more large curcumin crystals present, which were more prone to aggregation than the

323

smaller curcumin nanoparticles. In addition, the encapsulation efficiency of the curcumin

324

was much lower in the rehydrated samples than in the original systems at low biosurfactant

325

levels. This result can be attributed to the fact that any large curcumin crystals would have

326

been removed during the centrifugation step, and so there would be a smaller amount of

327

water-dispersible curcumin remaining in the final nanoparticle suspensions.

328

From a practical point of view, it is important to create delivery systems using the

329

lowest amount of surfactant possible, so as to reduce costs, potential toxicity, and any

330

undesirable off-flavors. For this reason, a biosurfactant concentration of 4 mg/mL was used

331

in the remainder of the studies, since it led to relatively small highly-charged nanoparticles

332

with a high encapsulation efficiency.

333

Characterization of curcumin nanoparticles: A variety of analytical techniques

334

were employed to provide additional information about the nature of the sophorolipid-

335

coated curcumin nanoparticles formed using the pH-driven method. As discussed in the 17


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336

previous section, the curcumin nanoparticles were relatively small (61 nm) and highly

337

negative charged (-41 mV) when formed using a sufficiently high sophorolipid level (4

338

mg/mL). In addition, they had a relatively high encapsulation efficiency (82.2 ± 0.7%) and

339

loading capacity (13.7 ± 0.2%). In other words, over 82% of the curcumin added to the

340

system was trapped inside the nanoparticles, and each nanoparticle consisted of around 14%

341

curcumin and 86% sophorolipid. The encapsulation efficiency and loading capacity values

342

for our nanoparticles were in good agreement with those reported for other systems in

343

previous studies. For instance, the encapsulation of curcumin in Pluronic P123 micelles

344

loaded using a heating method resulted in an EE of 46% and LC of 4.4% . Curcumin

345

encapsulation in copolymer mPEG-PCL micelles using a nanoprecipitation method yielded

346

an EE of 89% and LC of 21% . Curcumin encapsulation in casein micelles using the pH-

347

driven method led to an EE of 81% and LC of 4%. The biosurfactant used in our study

348

therefore had similar or better encapsulation characteristics as other synthetic and natural

349

surfactants used previously. At the same time, the pH-driven method is relatively fast,

350

simple, inexpensive, and does not require the use of organic solvents or sophisticated

351

processing equipment, which would be advantageous for commercial applications.

27

28

352

Additional information about the microstructure of the curcumin nanoparticles was

353

obtained using AFM. The curcumin nanoparticles appeared as smooth spheres that were

354

evenly distributed throughout the images (Fig. 2), with dimensions consistent with those

355

determined by dynamic light scattering. The physical state of the curcumin within the 18



Page 20 of 41

356

sophorolipid-coated nanoparticles was investigated using X-ray diffraction. For the sake

357

of comparison, the X-ray diffraction patterns of pure curcumin, pure sophorolipid, and

358

sophorolipid-coated curcumin nanoparticles were determined (Fig. 3). For pure curcumin

359

powder, diffraction peaks were detected at 2θ values ranging from 5° to 30°, indicating a

360

highly crystalline structure . Conversely, no peaks were observed for the pure sophorolipid,

361

indicating that the pure biosurfactant was not in a crystalline form. In addition, no peaks

362

were observed for the powdered sophorolipid-coated curcumin nanoparticles, which

363

suggested that the curcumin was in an amorphous form in these systems. This result

364

suggests that encapsulation of curcumin within the hydrophobic interior of nanoparticles

365

inhibited its tendency to crystallize. Studies in the pharmaceutical industry suggest that

366

delivering solid bioactive agents in an amorphous (rather than crystalline) form increases

367

their oral bioavailability . Consequently, the nanoparticle delivery systems developed in

368

our study may be particularly suitable for the oral delivery of curcumin in a highly

369

bioavailable form.

29

30

370

Stability of curcumin nanoparticles: Knowledge of the impact of pH and ionic

371

strength on the physicochemical properties and stability of curcumin nanoparticles is

372

important because they may experience different solution conditions when added to food

373

products or when they travel through the human GIT. The nanoparticle suspensions were

374

therefore incubated at different pH values (1.5 to 8) for 30 minutes, and then their visual

375

appearance and particle size were determined. From pH 3 to 8, there was no obvious change 19


Page 21 of 41


376

in the visual appearance or mean particle diameter of the nanoparticle suspensions (Fig.

377

4A), suggesting that they were relatively stable to aggregation in this pH range. However,

378

the nanoparticle suspensions became turbid and the mean particle diameter increased

379

steeply when the pH was reduced to 2.0 and 1.5. The aggregation of curcumin nanoparticles

380

at low pH values can be explained by the pK values of the hydrophilic sugar residues on

381

the sophorolipid molecules. The curcumin nanoparticles had a relatively high negative

382

charge (-30 mV) at neutral pH, which is mainly due to charged carboxylic acid groups (-

383

COO ) on the sugar residues of the sophorolipid. Typically, carboxylic groups have pK

384

values around pH 3.5, which means that lose their charge (-COOH) when the pH is around

385

or below this value . Consequently, at pH 2 and below, the negative charge on the curcumin

386

nanoparticles was insufficient to generate an electrostatic repulsion strong enough to

387

overcome any attractive interactions (such as van der Waals), and so nanoparticle

388

aggregation occurred.

a

-

a

31

389

The influence of ionic strength on the stability of the curcumin nanoparticles was

390

determined by incubating them in solutions containing different NaCl levels (Fig. 4B). At

391

< 500 mM NaCl, the curcumin nanoparticles were relatively stable to aggregation without

392

any changes in their visual appearance and mean particle diameter, which suggested that

393

the electrostatic repulsion between the nanoparticles was still strong enough to overcome

394

any attractive interactions. Conversely, at 500 mM NaCl and above, the nanoparticles were

395

highly unstable to aggregation, as seen by changes in visual appearance and the increase in 20



Page 22 of 41

396

mean particle size. This phenomenon can be attributed to the ability of the cationic counter-

397

ions in salt (Na ) to accumulate around the anionic curcumin nanoparticles and screen the

398

electrostatic repulsion between them . As described earlier, the reduced electrostatic

399

repulsion would then not be strong enough to overcome the attractive interactions between

400

the nanoparticles, thereby leading to aggregation.

+

32

401

The ability of a delivery system to remain stable during long-term storage under

402

different conditions is important for many commercial applications. Consequently, the

403

stability of the curcumin nanoparticles was determined by measuring changes in their

404

appearance, particle size, surface potential, and encapsulation efficiency during storage.

405

Aqueous suspensions of curcumin nanoparticles were stored at 4 and 25 °C, whereas

406

powdered curcumin nanoparticles were stored at 25 °C and then rehydrated prior to

407

analysis. There was little change in the appearance of the nanoparticle suspensions when

408

they were stored at 4 °C in aqueous form or at 25 °C in powdered form for one month (Fig.

409

5A). Moreover, the mean particle diameter, z-potential, and encapsulation efficiency of the

410

curcumin nanoparticles changed little when stored under the same conditions (Fig. 5B-D).

411

However, the nanoparticle suspensions turned form yellow to brown, and large flocs

412

became visible to the eye, when they were stored at 25 °C in aqueous form for one month.

413

In addition, the mean particle diameter increased from around 54 to 114 nm, the z-potential

414

changed from around -41 to -19 mV, and the encapsulation efficiency deceased from

415

around 82 to 59%. Taken together, these results indicate that storage conditions have a 21


Page 23 of 41


416

major impact on the stability of the curcumin nanoparticles. The systems rapidly degrade

417

when stored in aqueous form at ambient temperature, but their shelf-life can be extended

418

by reducing the storage temperature or converting them into a powder. The origin of the

419

instability mechanism is currently unknown, but it could be due to chemical degradation

420

of curcumin in aqueous solutions, which is known to lead to color changes and precipitation.

421

3

422

In Vitro Bioavailability of Curcumin: The in vitro bioavailability of curcumin in the

423

sophorolipid-coated nanoparticles was evaluated by passing them through a simulated

424

gastrointestinal tract (GIT), and then measuring the total amount of curcumin remaining,

425

as well as the fraction that was present in the mixed micelle phase. These results were then

426

used to calculate the transformation (T*), bioaccessibility (B*), and in vitro bioavailability

427

(BA) of curcumin. The transformation was defined as the percentage of curcumin

428

remaining in the overall system after passage through the simulated GIT, whereas the

429

bioaccessibility was defined as the fraction of curcumin in the small intestine phase that

430

was solubilized in the mixed micelles and therefore available for absorption. As shown in

431

Fig. 6, the fraction of curcumin remaining was appreciably higher when it was in the free

432

crystal form (88.3%) than when it was in the nanoparticle form (48.0%). Curcumin

433

degradation under simulated GIT conditions occurs mainly due to exposure to aqueous

434

neutral or alkaline environments, but may also be partly due to digestive enzymes (such as

435

pepsin) . The surface area of curcumin exposed to the aqueous phase would be much larger 33

22



Page 24 of 41

436

for the nanoparticles than for the free crystals due to the much smaller dimensions of the

437

nanoparticles. Consequently, the curcumin in the nanoparticles may have been more

438

susceptible to degradation than the curcumin in the larger crystals. Conversely, the

439

bioaccessibility of curcumin in the free crystals (9.1%) was significantly lower than that in

440

the nanoparticles (61.3%). A number of physicochemical phenomena may account for this

441

large difference in bioaccessibility . First, the water-solubility of hydrophobic materials is

442

known to increase as their particle size decreases, thereby leading to a higher local

443

concentration of curcumin around the nanoparticles than around the crystals. Consequently,

444

the driving force for solubilization into the mixed micelles would be greater for the

445

nanoparticles. Second, the surface area of particles increases as their dimensions decrease,

446

which would have led to a faster dissolution rate of the curcumin from the nanoparticles

447

than the larger crystals. Third, the curcumin within the nanoparticles was in an amorphous

448

form, which is known to be more soluble than the crystalline form

449

of curcumin solubilized by the mixed micelle phase after passage through the simulated

450

GIT is a measure of the amount available for absorption, and can therefore be used as a

451

measure of in vitro bioavailability . The amount of bioavailable curcumin was much higher

452

(2.7-fold) in the nanoparticles (294 ± 20 µg/mL) than in the crystals (80.1 ± 2.1 µg/mL).

453

This effect can mainly be attributed to the much higher bioaccessibility of the curcumin in

454

the nanoparticles (since the chemical stability in the nanoparticles was actually lower).

455

Overall, these results suggest that the in vitro bioavailability of curcumin can be increased

34

30, 34

. The absolute amount

33

23


Page 25 of 41


456

appreciably by delivering it in the form of sophorolipid-coated nanoparticles, which is

457

mainly due to a large increase in bioaccessibility.

458

In Vivo Bioavailability of Curcumin: In vitro measurements provide valuable

459

information about the physicochemical mechanism that impact the bioavailability of

460

nutraceuticals, but they cannot accurately model the complexity of a living GIT. For this

461

reason, the in vivo bioavailability of the different forms of curcumin was determined by

462

oral administration to rats at a dose of 100 mg/Kg. The impact of delivery system type on

463

the pharmacokinetics was determined by measuring the serum curcumin concentration

464

over time after administration (Fig. 7). Numerous studies have reported that curcumin

465

undergoes metabolism after oral administration. For instance, in vivo studies with rats

466

and mice have shown that curcumin undergoes metabolic O-conjugation to curcumin

467

glucuronide and curcumin sulfate, as well as reduction to tetrahydrocurcumin,

468

hexahydrocurcumin, octahydrocurcumin, and hexahydrocurcuminol

469

metabolites formed, curcumin glucuronide is the major one found in the plasma after oral

470

administration of curcumin in rats . In our study, the actual curcumin concentration in the

471

blood was too low to determine and so the concentration of curcumin glucuronide was used

472

to evaluate the in vivo bioavailability of the ingested curcumin delivery systems. A number

473

of important pharmacokinetic parameters were then calculated from these curves, including

474

the maximum plasma concentration (C ), the time to reach this maximum (T ), and the

475

area under the curve from 0 to 8 hours (AUC ). Oral administration of the curcumin in the

35-36

. Among the

24

max

max

0-8 h

24



Page 26 of 41

476

form of nanoparticles led to an appreciably higher and more prolonged level of curcumin

477

in the blood of the rats (C = 2.74 µg/mL; T = 1 h; AUC = 6.51 µg h/mL), than in the

478

form of free crystals (C

479

differences were statistically significant (p < 0.01). The AUC

480

approximately 3.6-fold greater when it was delivered as nanoparticles than as free crystals.

481

This enhancement in bioavailability is in good agreement with the results from the in vitro

482

model, and highlights the potential of the curcumin nanoparticles as nutraceutical delivery

483

systems. In this study, we used sophorolipid-coated curcumin nanoparticles to improve the

484

in vivo bioavailability characteristics of curcumin.

485

have also been reported to improve the bioavailability of curcumin

486

and co-workers utilized different kinds of emulsifier to prepare curcumin nanosuspensions

487

and reported that the oral bioavailability of curcumin increased between 1.2- and 3.7-times

488

compared to free curcumin depending on the type of emulsifier used . Rice bran protein

489

nanoparticles have also been utilized to encapsulate curcumin and the oral bioavailability

490

of the curcumin was shown to increase around 9.2-times compared to free curcumin .

491

Compared to these delivery systems, the main advantages of sophorolipid-coated curcumin

492

nanoparticles are that they are simple to prepare, do not require organic solvents, and can

493

be fabricated from natural ingredients.

max

max

max

= 0.47 µg/mL; T

max

0-8 h

= 1 h; AUC

= 1.43 µg h/mL). These

0-8 h

0-8 h

value of curcumin was

Numerous other delivery systems 37-39

. For instance, Wang

39

40

494

In summary, it was shown that curcumin could be loaded into sophorolipid micelles

495

using a simple pH-driven method based on the decrease in its water-solubility at lower pH 25


Page 27 of 41


496

values. Under basic conditions, curcumin has a negative charge that makes it water-soluble

497

and so it can be mixed with an aqueous suspension of sophorolipid micelles. When the pH

498

is reduced, the curcumin loses its charge and so its water-solubility decreases, which causes

499

it to move into the hydrophobic core of the surfactant micelles. As a result, the micelles

500

swell in size leading to the formation of sophorolipid-coated curcumin nanoparticles.

501

Experiments showed that these nanoparticles were relatively small and negatively charged,

502

had a relatively high encapsulation efficiency, and that the curcumin was in amorphous

503

form. Both in vitro and in vivo studies showed that encapsulating curcumin in sophorolipid-

504

coated nanoparticles greatly increased its oral bioavailability, which was mainly attributed

505

to their ability to increase the solubilization of the curcumin in the mixed micelle phase. In

506

summary, a simple method of producing curcumin nanoparticles has been developed that

507

may be useful for the development of nutraceutical delivery systems suitable for

508

application in functional foods, supplements, and pharmaceuticals.

509

510

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Zhang, Z. P.; Zhang, R. J.; Zou, L. Q.; Chen, L.; Ahmed, Y.; Al Bishri, W.; Balamash, K.; McClements, D. J.,

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based on liposomes decorated with chitosan and sodium alginate. Food chemistry 2016, 196, 396-404. 33. Zou, L.; Zheng, B.; Zhang, R.; Zhang, Z.; Liu, W.; Liu, C.; Xiao, H.; McClements, D. J., Food-grade nanoparticles for encapsulation, protection and delivery of curcumin: comparison of lipid, protein, and phospholipid nanoparticles under simulated gastrointestinal conditions. RSC Advances 2016, 6 (4), 31263136. 34. McClements, D. J., Crystals and crystallization in oil-in-water emulsions: Implications for emulsionbased delivery systems. Advances in Colloid and Interface Science 2012, 174, 1-30. 35. Ireson, C.; Orr, S.; Jones, D. J.; Verschoyle, R.; Lim, C.-K.; Luo, J.-L.; Howells, L.; Plummer, S.; Jukes, R.; Williams, M., Characterization of metabolites of the chemopreventive agent curcumin in human and rat hepatocytes and in the rat in vivo, and evaluation of their ability to inhibit phorbol ester-induced prostaglandin E2 production. Cancer Research 2001, 61 (3), 1058-1064. 36. Asai, A.; Miyazawa, T., Occurrence of orally administered curcuminoid as glucuronide and glucuronide/sulfate conjugates in rat plasma. Life Sciences 2000, 67 (23), 2785-2793. 37. Yu, H.; Huang, Q., Improving the oral bioavailability of curcumin using novel organogel-based nanoemulsions. Journal of Agricultural and Food Chemistry 2012, 60 (21), 5373-9. 38. Hu, L.; Jia, Y.; Niu, F.; Jia, Z.; Yang, X.; Jiao, K., Preparation and enhancement of oral bioavailability of curcumin using microemulsions vehicle. Journal of Agricultural and Food Chemistry 2012, 60 (29), 7137-41. 39. Wang, Y.; Wang, C.; Zhao, J.; Ding, Y.; Li, L., A cost-effective method to prepare curcumin nanosuspensions with enhanced oral bioavailability. Journal of colloid and interface science 2017, 485, 9198. 40. Liu, C.; Yang, X.; Wu, W.; Long, Z.; Xiao, H.; Luo, F.; Shen, Y.; Lin, Q., Elaboration of curcumin-loaded rice bran albumin nanoparticles formulation with increased in vitro bioactivity and in vivo bioavailability. Food Hydrocolloids 2017.

621 622

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623


FUNDING STATEMENT

624

We appreciate the financial support by the National Science Foundation of China (No.

625

21766018, 31601468). This material was also partly based upon work supported by the

626

National Institute of Food and Agriculture, USDA, Massachusetts Agricultural Experiment

627

Station (MAS00491) and USDA, AFRI Grants (2014-67021 and 2016-25147). Shengfeng

628

Peng thanks the Chinese Scholarship Council for funding to support his work (No.

629

201706820021).

630 631

30



Page 32 of 41

632

Figure captions

633

Fig. 1 Schematic of sophorolipid-coated curcumin nanoparticle fabrication using the pH-driven

634

loading mechanism. An acidic sophorolipid micelle solution is mixed with a basic curcumin

635

solution, which causes the curcumin molecules to become more hydrophobic and move into the

636

hydrophobic core of the surfactant micelles.

637

Fig. 2 Atomic forces microscopy image of sophorolipid-coated curcumin nanoparticles.

638

Fig. 3 XRD spectra of pure curcumin, pure sophorolipid, and powdered sophorolipid-coated

639

curcumin nanoparticles.

640

Fig. 4 Effect of (A) pH and (B) NaCl concentration on the mean particle size and appearance of

641

sophorolipid-coated curcumin nanoparticles.

642

Fig. 5 Physicochemical stability of sophorolipid-coated curcumin nanoparticles stored in the form

643

of aqueous suspensions at 4 and 25 °C, or powders at 25 °C for one month: (A) change of

644

appearance, (B) change of encapsulation efficiency, (C) change of the average diameter, (D) change

645

of the zeta potential.

646

Fig. 6 The transformation and bioaccessibility of free curcumin and sophorolipid-coated curcumin

647

nanoparticles after pass through gastrointestinal tract.

648

Fig. 7 Bioavailability of free curcumin and sophorolipid-coated curcumin nanoparticles. (Inset)

649

Pharmacokinetics parameters of two curcumin samples. AUC, area under the plasma

650

concentration−time curve from 0 h to 8 h; Cmax, peak concentration; Tmax, time to reach peak

651

concentration. 31


Page 33 of 41


652

Table 1. Impact of sophorolipid concentration on the physicochemical characteristics of (A) freshly

653

prepared curcumin nanoparticles and (B) rehydrated powdered curcumin nanoparticles. The

654

curcumin concentration was 1 mg/mL. Sophorolipid Mean

Polydispersity

z-potential

Encapsulation

diameter (nm)

index

(mV)

efficiency (%)

1

114 ± 13

0.160 ± 0.015

ab

-39.6 ± 1.9

a

40.9 ± 1.3

b

2

101.8 ± 9.8

0.276 ± 0.035

c

-40.9 ± 1.2

a

64.9 ± 2.4

c

4

60.8 ± 3.7

a

0.295 ± 0.045

c

-41.2 ± 2.0

a

82.2 ± 0.7

de

6

59.5 ± 0.9

a

0.153 ± 0.023

ab

-26.4 ± 1.1

b

89.3 ± 1.6

e

8

60.9 ± 0.8

a

0.089 ± 0.017

a

-20.50 ± 0.56

88.6 ± 1.3

e

1

400 ± 34

d

0.709 ± 0.029

d

-40.6 ± 1.8

a

25.8 ± 3.9

a

2

290 ± 15

c

0.657 ± 0.015

d

-39.9 ± 2.4

a

59.1 ± 7.8

c

4

58.8 ± 2.8

a

0.222 ± 0.006

bc

-41.1 ± 0.9

a

79.2 ± 6.1

d

6

57.9 ± 0.4

a

0.077 ± 0.024

a

-26.77 ± 1.63

b

88.7 ± 0.3

e

8

59.8 ± 1.1

a

0.089 ± 0.008

a

-19.00 ± 1.98

c

89.9 ± 1.1

e

concentration (mg/mL)

A

B

b

b

c

655

32



Page 34 of 41

Fig. 1

33


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Fig. 2

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Fig. 3

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Fig. 4

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Fig. 5

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Fig. 6

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Fig. 7

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GRAPHIC FOR TABLE OF CONTENTS

40


Enhancement of curcumin bioavailability by encapsulation in

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