Polyelectrolyte Complex Nanoparticles from Chitosan and Acylated

Feb 16, 2018 - This method does not involve the use of organic solvents and cross-linking agents, empowering it as an ideal nanocarrier preparation me...
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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 is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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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*

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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 (