Preparation of Hollow Biopolymer Nanospheres Employing Starch

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Preparation of hollow biopolymer nanospheres employing starch nanoparticle templates for enhancement of phenolic acid antioxidant activities Xiaojing Li, Man Li, Jing Liu, Na Ji, Caifeng Liang, Qingjie Sun, and Liu Xiong J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 03 May 2017 Downloaded from http://pubs.acs.org on May 4, 2017

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Journal of Agricultural and Food Chemistry

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Preparation of hollow biopolymer nanospheres employing starch nanoparticle

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templates for enhancement of phenolic acid antioxidant activities

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Xiaojing Li† Man Li† Jing Liu†† Na Ji† Caifeng Liang† Qingjie Sun*, † Liu Xiong *, †

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† College of Food Science and Engineering, Qingdao Agricultural University (Qingdao, Shandong

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Province, 266109, China)

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††Central laboratory, Qingdao Agricultural University (Qingdao, Shandong Province, 266109,

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China)

ACS Paragon Plus Environment


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ABSTRACT: Phenolic acids have been extensively studied because of their bioactive properties

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and disease prevention and control capacities. However, undesired odors and taste, low aqueous

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solubility, and thermal and ultraviolet (UV) light instability severely restrict their application. The

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aim of this work was to evaluate the enhancement in antioxidative activities of phenolic acids in

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hollow nanospheres and their stability in terms of their antioxidative activities under harsh

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conditions. For the first time, we have successfully fabricated hollow short linear glucan (SLG) @

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gum arabic (GA) nanospheres and hollow in situ SLG/GA hybrid nanospheres by removing the

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sacrificial starch nanoparticle templates through α-amylase treatment and Ostwald ripening. These

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two hollow nanospheres had a huge cavity area for the encapsulation of phenolic acids and their

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loading capacities were more than 20%. Furthermore, they can be used as nanoreactors to

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immobilize phenolic acids, enhance their antioxidative activities, and improve their stability when

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exposed to high salt concentrations, UV light, or heating treatments.

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KEYWORDS: nanocapsules; amylolysis; Ostwald ripening; antioxidative activities


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INTRODUCTION

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The typical attributes of hollow nanospheres have attracted great attention due to their large

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surface-to-volume ratio, low density, short solid-state diffusion lengths, and good surface

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permeability.1 The invention of hollow nanospheres is of significance in many energy- and

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environmental-related applications including lithium storage,2 supercapacitors,3 and dye-sensitized

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solar cell fields.4 In recent years, hollow nanospheres have also been considered for the

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development of drug delivery systems and nanoreactors.5-6

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Numerous efforts have contributed to the preparation of hollow structures. The most widely

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designed method is the sacrificial-core-based approach, which can be further categorized into the

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hard-core templating process, soft-core templating process, and the Ostwald ripening approach,

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and so on.7Among these approaches, the hard-core templating process is the most straightforward

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and effective method to prepare hollow structures that are closest to the original shape of the

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templates.8 Furthermore, the hollow nanostructure materials prepared through the hard-core

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templating process typically have a larger mesoporous volume than those prepared via soft-core

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templating or the Ostwald ripening processes, which is potentially favorable for high active

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ingredients/catalyst loading content.9

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Metal,10 metal oxides,11 and polymer nanoparticles12 are the most commonly used hard-core

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templates due to their uniform size and shape. However, the poor compatibility between the

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inorganic

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functionalization/modification of the template surface is the most widely used method to improve

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their compatibility, but it makes the synthesized method complicated. In addition, the removal of

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these templates was commonly via a fierce reaction, such as calcination or wet chemical etching,

template

and

shell

materials

has

limited


their

application.

The


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which is not environmentally friendly. The selection of suitable sacrificial hard-core templates is

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therefore an essential prerequisite for the synthesis of the desired hollow structures. Starch

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nanoparticles prepared through the retrogradation of short linear glucan (SLG) may have

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tremendous potential to be used as a hard-core template due to their non-toxicity, biocompatibility,

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and biodegradability.13 Additionally, they could easily be hydrolyzed by α-amylase under mild

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conditions. However, the preparation of starch nanoparticles for use as the hard-core template of

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hollow nanospheres, to our knowledge, has not been reported so far.

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Most shell materials are inorganic nanoparticles due to their strong adsorption capacities, yet

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the high price of inorganic nanoparticles has reduced the profitability of hollow nanoparticles.14 In

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recent years, polymer macromolecules have attracted wide attention as a novel type of shell

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material due to their electric charge, which enables the fabrication of hollow nanospheres via

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layer-by-layer self-assembly on the surface of nanostructure templates.15 However, very few

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studies have been devoted to investigating the potential biomacromolecules as shell materials.

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Gum arabic (GA) is a naturally anionic polysaccharide–protein conjugate16 and it can interact with

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starch chains to form a stable complex,17 depositing on the starch nanoparticles as a shell. The

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hydrophobic interior structure of GA could interact with hydrophobic drugs/active ingredients to

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embed more substances. In addition, its external hydrophilic structure could improve the solubility

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of drugs and active ingredients, and enhance bioavailability in body fluids.

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Phenolic acids, which are a group of natural antioxidants, are widely found in many

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vegetables and fruits.18 Phenolic acids have been extensively studied because of their bioactive

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properties and disease prevention and control capacities.19 At first, many scientists used them to

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substitute for chemical antioxidants and antimicrobials in foods.20 Afterward, evidence suggested


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that phenolic acids possess potentially protective effects against diseases caused by oxidative

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damage. Recently, the effective inhibitory effect of phenolic acids on cancer invasion and

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metastasis has also been widely investigated.21 However, undesired odors and taste, low aqueous

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solubility, and instability against UV light and heating severely restrict their application.22 These

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problems can be overcome by embedding technology and the widely used embedding materials

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include a laminar hydrogel hood,23 microspheres,24 and emulsions. 25

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Due to their ultra-fine size, nanomaterials have become the preferred choice for

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pharmaceutical and medical applications, especially for controlled and targeted drug release.26

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Nano-sized particles have been used as the encapsulating material for functional active ingredients

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(e.g. phenolic phytochemicals) or for drug delivery to decrease their instability and improve their

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bioavailability.27

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In this paper, for the first time, hollow SLG@GA nanospheres were fabricated through a

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green amylolysis technique by using natural GA and starch nanoparticles as the shell and

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sacrificial-core materials, respectively. These hollow nanospheres can embed and immobilize both

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hydrophilic and hydrophobic phenolic acids, hide their undesired odors and taste, and improve

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their stability in extreme environments. Furthermore, their porous nanoshells, formed by amylase

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hydrolysis, may allow free radicals access to the interior of the nanocapsules. The newly

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developed hollow nanospheres via this facile, green, and gentle enzymolysis technique could

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improve phenolic acid bioactivity for eliminating free radicals, and antioxidative stability, similar

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to nanoreactors.

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MATERIALS AND METHODS

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Materials. Waxy maize starch (98% amylopectin) was purchased from Tianjin Tingfung



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Starch Development Co., Ltd (Tianjin, China). Disodium hydrogen phosphate (Na2HPO4) and

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citric acid (C6H8O7) were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai,

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China). Pullulanase (E.C.3.2.1.41, 6000 ASPU/g, 1.15 g/mL, where ASPU is defined as the

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amount of enzyme that liberates 1.0 mg glucose from starch in 1 min at pH 4.4 and 60 °C) was

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supplied by Novozymes Investment Co. Ltd. (Bagsvaerd, Denmark). Porcine pancreas α-amylase

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(1400 units/mg protein) was from Sigma-Aldrich (Shanghai, China). Chlorogenic acid (CGA,

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95%) and ferulic acid (FA) were purchased from Shanghai Moqi Biotechnology Co., Ltd.

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(Shanghai, China). Gum Arabic (GA) was obtained from Tianjin Kaixin Chemical Industrial Co.,

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Ltd. (Tianjin, China). All other reagents were of analytical grade.

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Preparation of gum solution. The stock solution of GA was prepared by dissolving 2 g

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gum in 100 ml water and was stirred overnight at room temperature. The solution was centrifuged

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to remove the insoluble materials.

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Synthesis of gum Arabic coated short linear glucans hollow nanosphere. Short linear

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glucans (SLG) powder was prepared by debranching gelatinized waxy maize starch, inactivation

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pullulanase and freeze-dried.13 Then, SLG powder (20 g) was dissolved in 200 mL of distilled

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water by heating in a sealed tube at 120 °C for 30 min, and pH was maintained at 7. After cooling

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down to 25 °C, the solution was stored at 4 °C for 1 h and then centrifuged at 11,000 g for 2 min

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to obtain starch nanoparticles. Nanoparticles (10 g) were dispersed in GA solution (2% (w/v), 100

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mL) with vigorous stirring at 25 °C for 3 h. The suspensions were washed several times with

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distilled water to remove GA unadsorbed on the surface of SLG nanoparticles. The SLG@GA

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nanoparticles solution was hydrolyzed by α-amylase at 40 °C for 12 h to obtain hollow SLG@GA

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nanospheres. Another equal GA coated SLG solution was stored at 40 °C for 12 h to obtain


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control nanospheres. The suspensions were washed several times with distilled water until

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neutrality was achieved, and then vacuum freeze dried. SLG

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Synthesis of in-situ short linear glucans/gum arabic hollow nanospheres.

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powder was dissolved in deionized water (10%, w/v) by heating in a sealed tube at 120 °C for 30

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min. The solution (100 mL) was mixed with GA solution (2% (w/v), 100 mL) with vigorous

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stirring at 25°C for 4 h to obtain in-situ SLG/GA hybrid nanospheres solution. The suspensions

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were washed several times with distilled water to remove GA unadsorbed on the surface of

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SLG/GA hybrid nanoparticles. Then the suspension was hydrolyzed by α-amylase at 40 °C for 12

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h to obtain hollow in-situ SLG@GA hybrid nanospheres. Another equal in-situ SLG@GA hybrid

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nanospheres solution was stored at 40 °C for 12 h to get control nanospheres. The suspensions

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were washed several times with distilled water until neutrality was achieved, and then vacuum

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freeze dried.

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Transmission electron microscopy (TEM).

Transmission electron micrographs of

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nanoparticles were taken with a Hitachi (Tokyo, Japan) 7650 transmission electron microscope

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with an acceleration voltage of 80 kV. Nanoparticle suspensions (0.25%, w/w) were sonicated at

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25 °C. A drop of suspension was spread onto copper grids coated with carbon support film. The

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copper grids were left to stand for freeze-drying.

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Dynamic light scattering (DLS).

The average size and size distribution of the

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nanoparticles were determined by dynamic light scattering using a Malvern Zetasizer Nano

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(Malvern Instruments Ltd., UK) equipped with a He-Ne laser (0.4 mW, 633 nm) and a

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temperature-controlled cell holder. The measurements were performed in samples diluted in Milli

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Q water and analyzed at 25 °C. The mean intensity-weighted diameter was recorded.



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Determination of zeta potential of the nanoparticles. Nanoparticle suspensions (0.01%,

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w/v) were measured for their electrophoretic mobility by laser Doppler velocimetry using a

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Malvern Zetasizer Nano. The electrophoretic mobility of each sample was measured three times,

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and at least 12 runs were performed in each measurement.

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Nanoparticle dissociation test. To better understand the driving forces of GA/SLG

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hollow nanoparticle formation, and interaction forces between hollow nanoparticles and their

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internal polyphenol, dissociation behaviors were studied. Nanoparticle dispersions were mixed

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with an equal volume of each dissociating reagent (urea, SDS) at various concentrations. These

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mixed solutions were adjusted to pH 7 and stood overnight. The suspension turbidity was

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determined with a UV-Vis spectrophotometer at 600 nm.

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Iodine-binding short linear glucan. The content of iodine-binding SLG was determined

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by a UV-vis spectrophotometer (Persee UV-1810, Beijing, China). Samples were prepared

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according to the protocol used for apparent amylose content determination. The absorbance values

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of iodine-binding samples were determined using a UV-vis spectrophotometer at a wavelength of

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620 nm (the λ where apparent amylose content was determined).

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X-ray diffraction pattern (XRD). The X-ray diffraction pattern of samples was studied

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with an X-ray diffractometer (Bruker AXS Model D8 Discover). Before determination, the

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samples were equilibrated to 20% moisture content in a saturated relative humidity chamber for 24

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h at room temperature. The scanning range and rate were 5-40° (2θ) and 1.0 °/min, respectively.

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Differential scanning calorimeter (DSC).

The thermal properties of samples were

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investigated using a differential scanning calorimeter (DSC1, Mettler-Toledo, Schwerzenbach,

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Switzerland), as described by Sun et al. (2014). 13


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Polyphenols loading. Ferulic acid (FA) stock solution (0.5%, w/v) was prepared in

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aqueous ethanol (95%, v/v). Then the FA solution was diluted from the prepared stock by

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dissolving it in ethanol (20%, v/v). Chlorogenic acid (CGA) stock solution (0.05%, w/v) was

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prepared in deionized water. Hollow nanocapsules (10 mg) were redispersed in 10 mL of the FA

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or CGA solution with continually stirring at a constant rate in the dark at 25 °C. After 24 h, the

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mixtures were separated by centrifugation at 11,000 rpm, 25 °C for 30 min and the supernatant

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was collected. The nanocapsules filled with FA or CGA were washed three times with ethanol

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(20%, v/v) or deionized water to remove surface-absorbed FA or CGA. The FA (CGA)

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concentration in the original solution and in the supernatant solution was determined using a UV-

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vis spectrophotometer (Persee UV-1810, Beijing, China) at a wavelength of 280 nm (327 nm).

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The calibration curve of FA (CGA) was calculated based on the absorbance of the FA (CGA)

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solution at 280 nm (327 nm) versus concentration. The encapsulation efficiency (%EE) and

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loading capacity (%LC) were calculated from the following equations:

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% =

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% =

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DPPH radical scavenging activity of ferulic acid.

× 100%

(1)

× 100%

(2)

The DPPH radical scavenging

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activity was measured as described by Yi et al. (2015) with slight modification.28 Pure FA and

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freeze-dried SLG@GA nanospheres loaded with the same concentration of FA powders were

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dissolved in ultrapure water, and 5 mL of the solution was mixed with 5 mL ethanolic DPPH

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solution (0.2 mM). The mixture was incubated at room temperature for 30 min in the dark.

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Ethanolic DPPH solution (5 mL) mixed with 5 mL ultrapure water served as the control. After the

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incubation, the absorbance was measured at 517 nm. The DPPH radical scavenging activity was



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calculated as shown in Eq3: !!" #$%&'()*() % =

+,- ,. ,/

× 100%

(3)

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in which Ab is the absorbance of the control (ethanolic DPPH solution (5 mL) mixed with 5 mL

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ultrapure water instead of the sample solution) and As represents the absorbance of the test sample.

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The DPPH radical scavenging activities of FA and encapsulated FA were also determined

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after treatments of light, salt, and temperature. The nanoparticle solutions undergone the

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ultraviolet radiation treatment were exposed to ultraviolet light (20 w) for 30 min. nanoparticles in

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0.5 M NaCl solutions were incubated at 25 °C for 10 min to determine the effect of salt. In

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addition, they were incubated at 80 °C for 30 min and then cooled to room temperature to evaluate

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the stability. Pure FA solution was used as a control for all three treatments.

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·OH radical scavenging activity of chlorogenic acid. The ·OH radical scavenging

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activity was measured by a Fenton-type reaction. FeCl2 was dissolved in phosphate buffered

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solution (0.2 mol/L, pH 7.4). Then 0.5 mL FeCl2 solution (0.75 mmol/L) mixed with equal 1, 10-

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phenanthroline (0.75 mmol/L). After homogeneously mixing H2O2 solution was added to obtain a

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final concentration of 0.01%. Pure chlorogenic acid solutions or encapsulated chlorogenic acid

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nanoparticle suspension with different concentrations were added, and then the mixtures were

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incubated at 37 °C for 60 min in the dark. After the incubation, the absorbance was measured at

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536 nm. The ·OH radical scavenging activity was calculated as shown in Eq4:

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, ,

· 1" 2%3*$%4 #$%&'()*() % = , . ,5 × 100% 6

(4)

5

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in which As is the absorbance of the test sample, A0 represents the absorbance of control (ultrapure

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water instead of the sample solution), and A1 is the absorbance of the blank (ultrapure water

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instead of the sample solution and H2O2 solution).


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The ·OH radical scavenging activities of CGA and encapsulated CGA were also determined

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after treatments of light, salt, and temperature. The sample was treated in the extreme environment

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following the method of section of DPPH radical scavenging activity of ferulic acid. Pure CGA

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solution was used as a control for all three treatments.

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Statistical analysis.

Each measurement was carried out using at least three fresh,

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independently prepared samples. The data were subjected to statistical analysis using SPSS 17.0

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(SPSS Inc., Chicago, United States), analyzed using analysis of variance (ANOVA), and

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expressed as mean values ± standard deviations. Differences were considered at a significant level

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of 95% (p

Preparation of Hollow Biopolymer Nanospheres Employing Starch

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