Biocompatible Polyelectrolyte Complex Nanoparticles from Lactoferrin

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Biocompatible polyelectrolyte complex nanoparticles from lactoferrin and pectin as potential vehicles for antioxidative curcumin Jing-Kun Yan, Wen-Yi Qiu, Yao-Yao Wang, and Jian-Yong Wu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b01848 • Publication Date (Web): 28 Jun 2017 Downloaded from http://pubs.acs.org on July 4, 2017

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Journal of Agricultural and Food Chemistry

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Biocompatible Polyelectrolyte Complex Nanoparticles from

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Lactoferrin and Pectin as Potential Vehicles for Antioxidative

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Curcumin

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Jing-Kun Yan a,b,*, Wen-Yi Qiu a, Yao-Yao Wang a, Jian-Yong Wu b,*

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a

School of Food & Biological Engineering, Jiangsu University, Zhenjiang, 212013, China

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b

Department of Applied Biology & Chemical Technology, State Key Laboratory of Chinese

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Medicine and Molecular Pharmacology in Shenzhen, The Hong Kong Polytechnic University,

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Hung Hom, Kowloon, Hong Kong

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ABSTRACT: Polyelectrolyte complex nanoparticles (PEC NPs) were fabricated via

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electrostatic interactions between positively charged heat-denatured lactoferrin (LF) particles and

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negatively charged pectin. The obtained PEC NPs were then utilized as curcumin carriers. PEC

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NPs were prepared by mixing 1.0 mg/mL solutions of heat-denatured LF and pectin at a mass

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ratio of 1:1 (w/w) in the absence of NaCl at pH 4.50. PEC NPs that were prepared under

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optimized conditions were spherical in shape with a particle size of ~208 nm and zeta potential

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of ~−32 mV. Hydrophobic curcumin was successfully encapsulated into LF/pectin PEC NPs

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with high encapsulation efficiency (~85.3%) and loading content (~13.4%). The in vitro

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controlled release and prominent antioxidant activities of curcumin from LF/pectin PEC NPs

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were observed. The present work provides a facile and fast method to synthesize nanoscale food-

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grade delivery systems for the improved water solubility, controlled release, and antioxidant

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activity of hydrophobic curcumin.

ACS Paragon Plus Environment


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Curcumin (bis-α,β-unsaturated β-diketone, or diferuloylmethane) is a polyphenolic compound in

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turmeric (Curcuma longa) rhizomes. Curcumin has attracted significant attention worldwide as a

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functional ingredient for human health owing to its broad range of health benefits and biological

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activities, such as anticancer, antioxidant, antimicrobial, and anti-inflammatory.1-4 In many Asian

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countries for a long time, curcumin has been widely used as a food additive in sausage and

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canned foods, and as a sauce bittern coloring ingredient. Regardless of these potential functional

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properties, the poor water solubility and low bioavailability of curcumin has limited its

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applications in food and medicine.5 As for other poorly soluble drugs and nutraceutical

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ingredients, various delivery carriers or vehicles have been attempted to improve the

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bioavailability of curcumin, such as polymeric micelles,6 nanocomplexes,7 nanoemulsions,8

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liposomes,9 conjugates,10 and lipid nanoparticles (NPs).11 More effort is needed to develop

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alternative, more effective and tailor-made vehicles for curcumin delivery in different application

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systems.

INTRODUCTION

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Among the promising curcumin nanocarriers, the polyelectrolyte complexes (PECs) that

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are based on oppositely charged proteins and polysaccharides derived from food-grade

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ingredients, can remarkably improve the water solubility, stability, food compatibility, and

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bioavailability of curcumin. PEC can be formed simply by combining oppositely charged

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proteins and polysaccharides in aqueous solutions. The hydrophobic curcumin is bound to the

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protein probably by hydrophobic interactions plus van der Waals force and hydrogen bond

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interactions. The hydrophobic nature of proteins seems to play an important role in the

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effectiveness of proteins as carriers for curcumin. For instance, the effectiveness of β-

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lactoglobulin or soy protein isolate (SPI) for carrying curcumin was significantly increased by


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heat-induced denaturation and subsequent formation of aggregated particles.12,13 Previous studies

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have demonstrated that β-lactoglobulin,12 SPI,13,14 casein,15 zein,16 caseinate,17 lysozyme,18,19 and

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soybean Bowman-Birk inhibitor (BBI),20 are excellent carrier proteins for curcumin.

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Electrostatic interactions play a crucial role in the formation of colloidal PECs from

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proteins and polysaccharides and also affect the stability and properties.21 Various colloidal

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structures, such as soluble complexes, coacervates, NPs, filled hydrogel particles, and

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multilayered coatings, can be fabricated by controlling the Coulombic forces acting between the

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proteins and polysaccharides.22,23 Specifically, the strong interaction between proteins and

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anionic polysaccharides retains a steric stabilization of proteins and may also improve the

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stability of bioactive compounds encapsulated in the PECs.14 Meanwhile, these protein/

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polysaccharide complexes can have improved physicochemical and functional properties, thus

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offering great potential as carriers for drugs and bioactive agents. PECs formed with proteins and

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polysaccharides, have been widely studied including gelatin/chitosan,24 cationized gelatin/gum

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arabic,25 cationized gelatin/sodium alginate,26 ovalbumin/gum arabic,27 lactoferrin (LF)/anionic

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polysaccharides.28-30 These PECs can be favorable carriers for hydrophobic curcumin as

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demonstrated in two recent studies by Hu’s group that the core-shell (zein–pectin, zein–

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alginate/pectin) PEC NP delivery systems exhibited high curcumin loading efficiency, high

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particle yield, good water dispersibility, and enhanced bioactivities.31,32

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Pectin present in the cell walls of fruits and vegetables is a class of heteropolysaccharides

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that consist primarily of 1,4-linked α-D-galactopyranosyluronic acid residues interspersed with

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1,2-linked α-L-rhamnopyranose residues at varying frequencies (Scheme 1a).33 Pectin is a weak

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polyanion with a pKa of approximately 3.5.34 With its polyanionic property, pectin can easily

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form stable PECs with various positively charged proteins, such as gelatin,35 β-lactoglobulin,36,37



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whey protein,38 and low-density lipoprotein.39 LF is a polyfunctional iron-binding glycoprotein

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with a molecular weight of 80 kDa and comprises approximately 700 amino acid residues

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present abundantly in colostrum, breast milk, external secretions, and polymorphonuclear

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leukocytes (Scheme 1b).40 LF is a basic protein with an isoeletric point (pI) of 8.7, which is

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higher than those of most other common proteins (pI≈5). Consequently, LF is positively

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charged over a wide range of pH ranges.41 LF is also beneficial to human health with

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antimicrobial, anti-inflammatory, antitumor, antioxidant, and immunomodulatory activities.42

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Heat-denatured LF has been used to construct submicron-sized PECs via electrostatic

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complexation with anionic polysaccharides, such as pectin, alginate, carrageenan, N-succinyl

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chitosan, and galactomannan.29,30 Although Bengoechea et al. have reported the formation and

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characteristics of LF/pectin PEC at various conditions,28 to our best knowledge, no previous

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study has been reported on the fabrication and use of LF/pectin PEC as nanocarriers for

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curcumin to achieve improved solubility, bioavailability, and bioactivity.

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The present work was aimed at constructing PEC NPs with positively charged heat-

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denatured LF and negatively charged pectin in aqueous medium. The effects of pectin

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concentration, LF/pectin mass ratio, pH, and ionic strength on the Z-average diameter,

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polydispersity index (PDI), and zeta potential of the PEC NPs were evaluated with dynamic light

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scattering. The structural features of PEC NPs were characterized by various experimental and

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analytical methods. Subsequently, curcumin was incorporated in PEC NPs and the

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physicochemical properties, curcumin release profile, and antioxidant activity were evaluated.

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MARTERIALS AND METHODS

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Materials and chemicals. Pectin from citrus peel with galacturonic acid (≥74%, dried

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basis) and methoxyl groups (≥6.7%, dried basis) and its weight-average molecular weight was


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1.9×106 Da, 2,2-diphenyl-1-picrylhydrazyl (DPPH), 6-hydroxy-2,5,7,8-tetramethylchroman-2-

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carboxylic acid (Trolox), 2,2΄-azinobis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), and

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2,4,6-tris (2-pyridyl)-s-triazine were purchased from Sigma-Aldrich Chemical Co. (St. Louis,

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MO, USA). LF powder (80 kDa) and curcumin (with a purity of ≥95%) were obtained from

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Shanghai Yuanye Biotechnology Co. Ltd. (Shanghai, China). All other chemicals and solvents

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were of laboratory grade and used without further purification.

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Preparation of pectin/LF PEC NPs and curcumin-loaded PEC NPs. The pectin/LF

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PEC NPs were prepared according to a previous report,30 with minor modifications. Stock

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solutions (4.0 mg/mL) of LF and pectin were prepared separately by dissolving LF or pectin

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powder in deionized (DI) water. The solutions were stirred overnight to completely hydrate the

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LF and pectin powders. LF concentration was maintained at 1.0 mg/mL for the PEC NP

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formation tests and pectin was diluted to the desired concentration. The solution of native LF

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was heated in a water bath at 90 °C for 30 min to denature LF, yielding a cloudy suspension. The

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pH value of the heat-denatured LF was about 6.2. After cooling to room temperature, the pectin

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solution was slowly pipetted into the heat-denatured LF solution with continuous stirring at 25

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°C for 60 min. Afterwards, the PEC NPs formed were centrifuged at 4000 rpm for 20 min. The

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precipitate was lyophilized to yield the final PEC NPs. Experimental factors, such as pectin

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concentration, LF/pectin mass ratio, pH, and ionic strength during PEC NP formation, were

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determined and recorded.

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A stock solution of curcumin was prepared in absolute ethanol (1.0 mg/mL) for the

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preparation of curcumin-loaded PEC NPs. Subsequently, an equal volume (2.0 mL) of curcumin

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solution was added to the LF solution (2.0 mg/mL). After removal of free curcumin by

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centrifugation (3000 rpm, 10 min), curcumin-loaded PEC NPs were prepared according to the



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above procedure for LF/pectin PEC NP preparation. The scheme for the production of LF/pectin

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PEC NPs and the loading of curcumin on PEC NPs is shown in Figure 1.

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Characterization of blank and curcumin-loaded PEC NPs. The Z-average diameter

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(particle size), polydispersity index (PDI), zeta potential and dynamic light scattering (DLS) of

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the samples were measured with a Zetasizer Nano ZS (Malvern Instruments, UK). All

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measurements were taken at least thrice with 10 measurements each at 25 °C and the results were

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averaged. The IR spectra of samples were obtained with a Nexus 670 Fourier transform infrared

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(FTIR) spectrometer (Thermo Nicolet Co., USA) in the wavenumber range of 500–4000 cm−1

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with KBr pellets and referenced against that of air. The particle size and morphology of the blank

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and curcumin-loaded PEC NPs were observed by transmission electron microscopy (TEM)

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(Tecnai 12, Philips, 120 kV). For TEM analysis, a drop of the diluted sample solution was placed

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on a carbon-coated copper grid (300 meshes) and subsequently dried at room temperature for 30

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min. The crystalline and amorphous nature of curcumin, blank, and curcumin-loaded PEC NPs

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were analyzed by X-ray diffraction (XRD) (D8-Advance, Bruker, Co., Germany). XRD patterns

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were recorded with Cu Kα radiation (λ = 0.1541 nm) at 40 kV and 40 mA in the 2θ range of 5°–

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50° with a scanning rate of 4° min−1. The differential scanning calorimetry (DSC) thermograms

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of curcumin, blank, and curcumin-loaded PEC NPs were measured with a DSC 822e thermal

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analyzer (Mettler Toledo Co., Switzerland). Approximately 3.0 mg of samples were placed in

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aluminum pans, which were then hermetically sealed with aluminum lids. Afterward, thermal

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analysis was performed from 25 °C to 300 °C under a dry nitrogen atmosphere with a flow rate

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of 20 mL/min and a heating rate of 10 °C/min. The recorded DSC curve of a sealed empty pan

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was used as the reference.


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Encapsulation efficiency (EE) and loading content (LC) of curcumin. The EE (%) and

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LC (%) of curcumin-loaded PEC NPs were determined according to a reported method with

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minor modifications.26 Briefly, a given amount of the freeze-dried sample was dispersed in

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ethanol–water mixture (1:1, v/v) and vortexed for 5 min. The resultant mixture was centrifuged

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at 20,000 rpm for 10 min. The supernatant was collected and the curcumin concentration was

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determined at 427 nm with a UV/vis spectrophotometer (Cary 8454, Agilent Technologies,

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USA) and the calibration curve (R2 = 0.9994) of free curcumin. All measurements were

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performed thrice at 25 °C. EE (%) and LC (%) were calculated by,

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EE (%) = Curcumin encapsulated in PEC NPs×100/Total weight of curcumin

(1)

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LC (%) = Curcumin encapsulated in PEC NPs×100/Total weight of PEC NPs

(2)

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In vitro drug release studies. The in vitro drug release profile of curcumin from curcumin-

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loaded PEC NPs in solutions with two different pH values (4.5 and 7.4) was determined with a

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direct dispersion method as reported previously.43 A known amount of curcumin-loaded PEC

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NPs was dispersed in 30 mL of buffer solution and then divided into 30 Eppendorf tubes over a

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period of five days. The tubes were incubated at 37 °C under gentle agitation. At proper time

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intervals (0, 1, 2, 4, 8, 12, 24, 48, 72, 96, and 120 h), a tube was taken for measurement. The tube

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was centrifuged at 12,000 rpm for 10 min to pelletize the released drug. The pellets were

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dissolved in 4.0 mL of ethanol–water mixture (1:1, v/v) and curcumin concentration was

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determined as described above. The cumulative release (%) was quantified by,

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Cumulative release (%) = Released curcumin×100/ Total curcumin

(3)

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In vitro antioxidant activity. The in vitro antioxidant activities of LF/pectin PEC NPs,

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curcumin-loaded PEC NPs, curcumin in ethanol, and curcumin in water were evaluated by

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DPPH radical scavenging activity, Trolox equivalent antioxidant capacity (TEAC), and ferric-



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reducing ability of plasma (FRAP) assays. The control solution of curcumin in water was

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prepared as reported previously.44 Vitamin C (Vc) was used as an antioxidant reference. Details

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of the operation conditions and methods have been reported previously.45

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Statistical analysis. All experiments were conducted in triplicates and the results

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represented by mean ± standard deviation (SD). Statistical analysis of the experimental data was

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performed by Student’s t-test and analysis of variance (ANOVA) using OriginPro Software

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Version 8.0 (OriginLab Corp., MA, USA). P

Biocompatible Polyelectrolyte Complex Nanoparticles from Lactoferrin

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