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Polyelectrolyte complex nanoparticles from chitosan and acylated rapeseed cruciferin protein for curcumin delivery Fengzhang Wang, Yijie Yang, Xingrong Ju, Chibuike C Udenigwe, and Rong He J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b05083 • Publication Date (Web): 16 Feb 2018 Downloaded from http://pubs.acs.org on February 16, 2018
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Journal of Agricultural and Food Chemistry
Polyelectrolyte complex nanoparticles from chitosan and acylated rapeseed cruciferin protein for curcumin delivery Fengzhang Wang1, Yijie Yang1, Xingrong Ju1, Chibuike C. Udenigwe2, Rong He1*
1
College of Food Science and Engineering/Collaborative Innovation Center for Modern Grain
Circulation and Safety/Key Laboratory of Grains and Oils Quality Control and Processing, Nanjing University of Finance and Economics, Nanjing 210023, China 2
School of Nutrition Sciences, Faculty of Health Sciences, University of Ottawa, 451 Smyth Road,
Ottawa, Ontario, K1H 8M5, Canada
*Corresponding authors: Email:
[email protected] (R. He)
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ABSTRACT: Curcumin is a polyphenol that exhibits several biological activities, but its low
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aqueous solubility results in low bioavailability. To improve curcumin bioavailability, this study has
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focused on developing polyelectrolyte complexation method to form layer-by-layer assembled
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nanoparticles, for curcumin delivery, with positively charged chitosan (CS) and negatively charged
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acylated cruciferin (ACRU), a rapeseed globulin. Nanoparticles (NPs) were prepared from ACRU
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and CS (2:1) at pH 5.7. Three samples with weight of 5%, 10% and 15% of curcumin, respectively,
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in ACRU/CS carrier were prepared. To verify the stability of the NPs, encapsulation efficiency and
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size of the 5% Cur-ACRU/CS NPs were determined at intervals of five days in one month period.
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Fourier transform infrared spectroscopy (FTIR), X-ray diffraction and differential scanning
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calorimetry confirmed the electrostatic interaction and hydrogen bond formation between the carrier
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and core. The result showed that hollow ACRU/CS nanocapsules (ACRU/CS NPs) and curcumin
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loaded ACRU/CS nanoparticles (Cur-ACRU/CS NPs) were homogenized spherical with average
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sizes of 200-450 nm and zeta potential of +15 mV. Encapsulation and loading efficiencies were 72%
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and 5.4%, respectively. In vitro release study using simulated gastro (SGF) and intestinal fluids (SIF)
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showed controlled release of curcumin in 6 h of exposure. Additionally, the Cur-ACRU/CS NPs are
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nontoxic to cultured Caco-2 cells and the permeability assay indicated that Cur-ACRU/CS NPs had
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improved permeability efficiency of free curcumin through the Caco-2 cell monolayer. The findings
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suggest that ACRU/CS NPs can be used for encapsulation and delivery of curcumin in functional
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foods.
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KEYWORDS: Rapeseed protein; Curcumin; Chitosan; Nanoencapsulation; Polyelectrolyte
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complexes
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INTRODUCTION
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Many attempts have been made to develop new delivery systems for bioactive compounds. Presently,
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nanoparticles have been widely used in delivery systems and show great potential in medical,
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biological and pharmaceutical applications1. Several mechanisms can be used to prepare
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nanoparticles, such as ionic cross-linking, covalent cross-linking, polyelectrolyte complexation, and
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self-assembly of hydrophobically modified polysaccharides among others2. Polyelectrolyte
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complexes are formed from strong electrostatic interactions between oppositely charged polymers as
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well as other forces such as hydrogen bonding and hydrophobic interactions. This method does not
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involve the use of organic solvents and cross-linking agents, empowering it an ideal nanocarrier
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preparation method due to its low cost and low energy requirements and effectiveness3.
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Curcumin is a hydrophobic polyphenolic compound isolated from Curcuma longa that exhibits
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strong antioxidant, anti-inflammatory, antimicrobial and anticancer activities4-6. Nevertheless, its low
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aqueous solubility, chemical instability, poor oral bioavailability and rapid clearance limit its
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application in therapeutics and the food industry7. A variety of nanoparticles has been studied as
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potential vehicles for delivering curcumin through different routes in the body. In recent years, food
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grade proteins, such as zein8, kafirin9, legumin and casein10 are popularly used for curcumin
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nanoencapsulation. However, low loading efficiency and fast release of the encapsulated compound
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are still observed. To address this challenge, acylated cruciferin and chitosan were used in this study
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to encapsulate curcumin to improve its oral bioavailability.
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Cruciferin, a natural storage protein derived from rapeseed, is an ideal material for delivery of
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nutrients due to its well-balanced essential amino acids composition, good emulsifying and gelling
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properties11, 12. Cruciferin is composed of six subunits with an isoelectric point (pI) of around 7.2 and
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a molecular weight of 300 kDa. Although cruciferin is not completely soluble in water, its structure
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can be modified (e.g. by succinic acid anhydride treatment) to improve its solubility. There is a
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dearth of information on the use of rapeseed proteins in delivering bioactive compounds. In this
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study, polyelectrolyte method was used for encapsulation and this involved static interaction between
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positively charged chitosan and negatively charged cruciferin to form a shell to encapsulate
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curcumin.
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Several studies have utilized polysaccharides such as cyclodextrin13, starch14, cellulose15,
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hyaluronic acid16 and calcium carbonate17 to produce nanoparticles. Chitosan, a positively charged
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polyelectrolyte, has been widely used to encapsulate biomaterials destined for delivery because of its
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unique chemical properties such as biodegradability, biocompatibility, low toxicity and mucosal
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adhesion, which favor the absorption and bioavailability of the encapsulated materials18-20.
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The objective of this study was to prepare hybrid nanoparticles by polyelectrolyte method using
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chitosan and modified cruciferin as the shell and curcumin as the core, for enhancing the
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bioavailability of curcumin. Formulations of both hollow nanoparticles and curcumin-loaded
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nanoparticles were characterized in terms of particle size distribution, zeta potential, loading and
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encapsulation efficiency, and morphology. The encapsulation process was monitored by
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spectroscopy and calorimetry techniques to identify the driving forces in nanoparticle formation. In
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addition, in vitro release study was conducted in simulated gastrointestinal conditions to evaluate the
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controlled release of free curcumin from the ACRU/CS carrier. Caco-2 cells were also used to
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evaluate cellular uptake, cytotoxicity and transport efficiency of the nanoparticles.
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MATERIALS AND METHODS
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Materials. Curcumin (85% purity) was purchased from DingBei Biological Company (Nanjing,
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China). Chitosan (99% purity, with medium molecular weight) and succinic acid anhydride were
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purchased from Sigma Aldrich (St. Louis, MO, USA). Rapeseed protein was extracted from Brassica
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napus, which was a gift from Donghai Oil Industry (Nantong, China). Caco-2 cells were provided by
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Food Technology Department at Nanjing Agricultural University, China. Dulbecco’s modified Eagle
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medium (DMEM) and fetal bovine serum (FBS) were purchased from Life Technologies (Carlsbad,
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CA, USA). Phosphate buffer (PBS, pH 7.2-7.4), Hank’s balanced salt solution (HBSS) and MTT
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(3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetra-zolium bromide) were purchased from Solarbio
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Science and Technology Co., Ltd. (Beijing, China).
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Preparation of cruciferin. Cruciferin, a globular rapeseed protein, was extracted using the
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previously reported method21 with some modifications. Briefly, an aqueous slurry of defatted
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rapeseed meal (1: 10, w/v) was adjusted to pH 11 with 1 M NaOH, stirred for 4 h and centrifuged at
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12,000 rpm for 20 min at 4°C. The supernatant was passed through an ultrafiltration membrane with
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a molecular weight cut-off (MWCO) of 100 kDa using an ultrafiltration system (UFSC 40001, U.S.)
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under constant nitrogen passage of 60 psi. Then the retentate (>100 kDa) and the permeate (
