Preparation of bioactive polysaccharide nanoparticles with enhanced

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Bioactive Constituents, Metabolites, and Functions

Preparation of bioactive polysaccharide nanoparticles with enhanced radical scavenging activity and antimicrobial activity Yang Qin, Liu Xiong, Man Li, Jing Liu, Hao Wu, Hongwei Qiu, Hongyan Mu, Xingfeng Xu, and Qingjie Sun J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b00388 • Publication Date (Web): 12 Apr 2018 Downloaded from http://pubs.acs.org on April 12, 2018

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Journal of Agricultural and Food Chemistry

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Preparation of bioactive polysaccharide nanoparticles with enhanced radical scavenging activity and antimicrobial activity

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Yang Qin1†

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Liu Xiong1† Man Li† Jing Liu‡ Hao Wu† Hongwei Qiu†

Hongyan Mu†

Xingfeng Xu† Qingjie Sun*

4 †

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College of Food Science and Engineering, ‡Central Laboratory, Qingdao Agricultural University

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(Qingdao, Shandong Province, 266109, China)

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1

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*Correspondence

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[email protected]), College of Food Science and Engineering, Qingdao Agricultural University,

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Equally-contributing author author

(Tel:

86-532-88030448,

Fax:

86-532-88030449,

266109, 700 Changcheng Road, Chengyang District, Qingdao, China.

ACS Paragon Plus Environment

e-mail:


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ABSTRACT: Due to their biocompatibility and biodegradability in vivo, natural polysaccharides

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are effective nanocarriers for delivery of active ingredients or drugs. Moreover, bioactive

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polysaccharides, such as tea, Ganoderma lucidum, and Momordica charantia polysaccharides (TP,

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GLP, and MCP), have antibacterial, antioxidant, antitumor, and antiviral properties. In this study,

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tea, Ganoderma lucidum, and Momordica charantia polysaccharide nanoparticles (TP-NPs,

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GLP-NPs, and MCP-NPs) were prepared via the nanoprecipitation approach. When the ethanol to

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water ratio was 10:1, the diameter of the spherical polysaccharide nanoparticles was the smallest,

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and the mean particle size of the TP-NPs, GLP-NPs, and MCP-NPs was 99±15, 95±7, and 141±9

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nm, respectively. When exposed to heat, increased ionic strength and pH levels, the nanoparticles

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exhibited superior stability and higher activity than the corresponding polysaccharides. In

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physiological conditions (pH 7.4), the nanoparticles underwent different protein adsorption

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capacities in the following order: MCP-NPs> TP-NPs> GLP-NPs. Moreover, the 2,

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2-Diphenyl-1-picrylhydrazyl (DPPH), hydroxyl radical, and superoxide anion radical scavenging

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rates of the nanoparticles were increased by 9%–25%, as compared to the corresponding

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polysaccharides. Compared to the bioactive polysaccharides, the nanoparticles enhanced

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antimicrobial efficacy markedly and exhibited long-acting antibacterial activity.

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KEYWORDS bioactive polysaccharides, nanoprecipitation, free radicals scavenge, protein

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adsorption, cytotoxicity


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INTRODUCTION

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Bioactive polysaccharides are natural polymers that exist widely in plants, fungi, animals,

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and microorganisms. They engage in a broad spectrum of physiological activities, including

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antioxidation, antimicrobial, antitumor, antiviral, and lipid-lowering.1-4 Moreover, bioactive

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polysaccharides are biocompatible and biodegradable in vivo and are nontoxic in a wide range of

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doses. Thus, there is growing interest among researchers regarding the benefits of bioactive

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polysaccharides for human health and other areas.5,6 However, bioactive polysaccharides belong to

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Class III of the Biopharmaceutics Classification System, which means that the oral absorption of

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polysaccharides is poor and erratic. Furthermore, due to their hydrophilic and uncharged nature,

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neutral polysaccharides demonstrate low bioavailability and clearance by the reticuloendothelial

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system, which indicates their restricted bioactivity in vivo.7

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To improve the bioavailability and bioactivity of functional ingredients, various nanoparticle

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delivery systems have been developed, such as nanomicelle, nanovesicle, nanoliposome, and

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nanogels.8-11 Because bioactive polysaccharides provide a wide range of benefits for various

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biological processes,12,13 nanocarriers loaded with bioactive polysaccharides have become a

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prevalent research topic. For example, Kong et al. prepared silica–chitosan nanoparticles for

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encapsulation of Antrodia camphorata polysaccharides and demonstrated their increased

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antitumor effects.14 Sun et al. found that the ophiopogon polysaccharide liposome could activate

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macrophages, and its efficacy was significantly better than Ophiopogon polysaccharide.15 Liu, et

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al. reported that Ganoderma lucidum polysaccharides (GLP) encapsulated in liposomes induce

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more powerful antigen-specific immune responses than GLP alone.16 Wang et al. found that the

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histidine modified Auricularia auricular polysaccharide nanomicelles with a diameter of 157.2



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nm were more easily ingested by cells than Auricularia auricular polysaccharide.17 Qiu et al.

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prepared bioactive polysaccharide-loaded maltodextrin nanoparticles with sizes of 80–120 nm,

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and they found that the stability of polysaccharide-loaded maltodextrin nanoparticles was

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improved at high salt concentrations.18

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Due to the small size and high surface/volume ratio, polysaccharide-loaded nanoparticles

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display specific characteristics that differ from those of the corresponding bulk material, such as

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better dispersibility, higher stability, and higher penetration rates through biological barriers.

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However, to the best of our knowledge, there are no studies that report the preparation of bioactive

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polysaccharide nanoparticles using only bioactive polysaccharides. We hypothesized that

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polysaccharide nanoparticles may improve bioactivities of polysaccharides. To these this

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hypothesis, we fabricated three bioactive polysaccharide nanoparticles using tea polysaccharide

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(TP), Ganoderma lucidum polysaccharide (GLP), and Momordica charantia polysaccharide (MCP)

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via the nanoprecipitation method. We first investigated the morphological characteristics and size

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distribution of the bioactive polysaccharide nanoparticles: tea polysaccharide nanoparticles

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(TP-NPs), Ganoderma lucidum polysaccharide nanoparticles (GLP-NPs), and Momordica

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charantia polysaccharide nanoparticles (MCP-NPs). Moreover, we explored the stability,

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cytotoxicity, and protein adsorption of the polysaccharide nanoparticles. We further explored

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antioxidant and antimicrobial activities of polysaccharide nanoparticles in vitro by comparison

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with the polysaccharides.

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MATERIALS AND METHODS

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Materials. TP, GLP, and MCP were provided by Ci Yuan Biotechnology Co., Ltd. (Shanxi,

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China). 2, 2-Diphenyl-1-picrylhydrazyl (DPPH) and hydrogen peroxide (H2O2) were purchased


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from Sigma Chemical Co. (St. Louis, MO, United States). Mouse embryonic fibroblast (MEF)

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was provided by the American Type Culture Collection (ATCC). 3-(4, 5-dimethylthiazol-2-yl)-2,

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5-diphenyltetrazolium bromide (MTT), and Dulbecco’s Modified Eagle’s Medium (DMEM) were

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obtained from Sigma Chemical Co. (St. Louis, MO, United States). Gram-negative bacteria

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Escherichia coli (E. coli, ATCC 25922) and Salmonella typhus (S. typhus, ATCC 6897), and

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Gram-positive bacteria Staphylococcus aureus (S. aureus, ATCC 25923) and bacillus subtilis (B.

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subtilis, ATCC 60511) were purchased from Nanjing Bianzhen Biological Technology Co., Ltd.

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Luria-Bertani (LB) broth powder was supplied by Thermo Fisher Scientific Inc. (Beijing, China).

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All other reagents used were of analytical grade.

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Preparation of bioactive polysaccharide nanoparticles. Three types of bioactive

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polysaccharides (TP, GLP, and MCP) were used to prepare nanoparticles using the

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nanoprecipitation method of Qiu et al.19 with some modifications. In brief, polysaccharide solution

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(2%, w/v) was prepared by mixing 2 g of polysaccharide powder in 100 mL of deionized water,

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stirring the solution for 1 h at room temperature (25 °C), and then centrifuging the solution at

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3,500 g for 5 min to remove any insoluble components. The polysaccharide solution was adjusted

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to different pH levels using a 0.5 M NaOH solution (pH 5.0 for GLP; pH 6.0 for TP and MCP).

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Afterwards, a fixed quantity of 95% ethanol (30, 40, 50, 100, and 200 mL) was added drop-wise

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into 10 mL of polysaccharide solution at room temperature, which was continually stirred using a

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magnetic stirrer. Furthermore, the solution was kept under continuous mechanical stirring for

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another 2 h. Afterward, the suspension was centrifuged (10,000 g for 15 min), and the precipitates

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were rinsed three times with 95% ethanol to remove excess water. Subsequently, the dried TP-NPs,

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GLP-NPs, and MCP-NPs were obtained by conducting the lyophilization process (-80 °C for 72 h)



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and kept in a plastic bag until further use.

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Size, ζ-potential, and polydispersity index. The size distributions, average size, and

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polydispersibility index (PDI) of polysaccharide nanoparticles were determined by the dynamic

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light scattering (DLS) technique using a Zetasizer Nano ZS90 (Malvern Instruments, U.K.). The

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electrical characteristic (ζ-potential) was determined by particle electrophoresis using the same

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instrument. The nanoparticle dispersions were diluted (0.1%) with ultrapure water to avoid

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multiple scattering effects and were placed into the measurement chamber. Then, the dispersions

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were equilibrated at 25±1 °C prior to analysis.

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Transmission electron microscopy (TEM). The morphologies of the TP-NPs, GLP-NPs,

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and MCP-NPs were analyzed with a Hitachi 7700 TEM (Tokyo, Japan) at an acceleration voltage

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of 80 kV. A small droplet of nanoparticle suspension with a concentration of 0.1% (w/v) was

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deposited onto a carbon-coated copper grid (300 meshes) and then lyophilized for more than 6 h to

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obtain dry samples for further observation.

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Fourier transform infrared spectroscopy (FTIR). The chemical structures of the TP and

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TP-NPs were measured using FTIR (Tensor 27, Jasco Inc., Easton, MD, USA). The background

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obtained from the scan of KBr was automatically subtracted from the sample spectra. The

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measured spectral region was between 4,000 and 400 cm−1.

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Temperature, ionic strength, and pH stability. The turbidity, average size, and PDI of the

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TP-NPs (1 mg/mL) at different temperatures, ionic strength (NaCl) levels, and pH levels were

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determined using a spectrophotometry (Shimadzu-2600, Kyoto, Japan) and a Zetasizer Nano ZS90

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(Malvern Instruments, U.K.). The particle suspensions were divided into fifteen groups: four

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groups were incubated at temperatures of 4, 25, 37, and 65 °C for 2 h and then returned to room


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temperature. Another six groups were dispersed in NaCl solutions (0, 100, 200, 300, 400, and 500

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mM) at 0.1% (w/v) for 3 h at room temperature. The other groups were adjusted to the desired pH

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values (2, 5, 7.4, and 9) using 0.5 M HCl/NaOH solution.

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In vitro cytotoxicity. The in vitro cytotoxicity of the TP-NPs, GLP-NPs, and MCP-NPs were

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assessed using an MTT assay according to the method used by Carmichael et al.20 with some

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modifications. MEF cells in DMEM media (8,000 cells/well) were respectively seeded into each

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well of a 96-well tissue culture plate (Costar, Corning, NY, USA) and cultured until they reached

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confluence (24–36 h). One hundred microliter aliquots of TP-NP dispersions at concentrations of 0,

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25, 50, 100, 150, and 200 µg/mL (the concentrations of the GLP-NPs and MCP-NPs were 0; 250;

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500; 1,000; 1,500; and 2,000 µg/mL) were added to the cell culture wells, respectively. The plate

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was incubated for 24 h (at 37 °C) in a 5% CO2 humidified atmosphere. Next, 100 µL of MTT

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solution (5 mg/mL) were added to each well for further incubation for 4 h at 37 °C. The total

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number of cells was determined by the absorbance at 570 nm. Cell viability was expressed as a

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percentage of the control absorbance, defined as 100%.

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Protein adsorption assay. To study the protein adsorption of the TP-NPs, GLP-NPs, and

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MCP-NPs, bovine serum albumin (BSA) was chosen as a model protein. The polysaccharide

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nanoparticles (0.15 mg/mL) were coincubated with BSA (0.25 mg/mL) in a phosphate buffer

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solution (pH 7.4) at 37 °C. At different time points, 1 mL aliquots of each sample were centrifuged

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(10,000 g, 20 min) to precipitate protein-adsorbed aggregates. Afterward, a BSA standard curve

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was established using a BCA Protein Assay Kit. Meanwhile, at the same condition, the

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concentration of the protein that had not been adsorbed was measured using a microplate reader.

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The ratios of adsorbed protein at different time points were then calculated.



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Free radical scavenging activity assays. The DPPH free radical scavenging activity was

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determined using the standard DPPH assay method with slight modifications. In brief, 2 mL of

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DPPH solution (0.2 mM in 95% ethanol) mixed with 2 mL of various concentrations of bioactive

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polysaccharide or nanoparticle solutions (10-300 µg/mL for TP and TP-NPs; 200-2000 µg/mL for

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GLP and GLP-NPs; 30-2000 µg/mL for MCP and MCP-NPs), respectively. The reaction mixture

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was shaken well and then incubated in the dark for 30 min at room temperature. Afterward, the

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absorbance of the resulting solution was read at 517 nm against a blank. DPPH radical scavenging

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activity was calculated using equation (1):

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DPPH scavenging rate (%) = [1-(A1- A2)/A0] ×100% (1)

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A0 is the absorbance of the control (DPPH solution with no sample); A1 is the absorbance of the

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test sample (DPPH solution with sample or positive control); and A2 is the absorbance of the

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blank (sample with no DPPH).

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Hydroxyl radicals (•OH) were generated by the Fenton reaction. Briefly, the reaction system

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contained 1.0 mL FeSO4 (9.0 mM), 1.0 mL H2O2 (8.8 mM), 1.0 mL salicylic acid (9.0 mM), and

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1.0 mL of various concentrations of the bioactive polysaccharides or nanoparticles (10-300 µg/mL

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for TP and TP-NPs; 200-2000 µg/mL for GLP and GLP-NPs; 30-2000 µg/mL for MCP and

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MCP-NPs). The mixed solution was incubated at 37 °C for 1 h, and then the absorbance of the

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mixture was recorded at 510 nm against a blank. The •OH scavenging activity was calculated by

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equation (2):

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Scavenging rate (%) = [1-(A1-A2)/A0] × 100% (2)

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A0 is the absorbance of the control (reaction solution with no sample); A1 is the absorbance of the

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test sample (reaction solution with sample or positive control); and A2 is the absorbance without


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salicylic acid.

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The superoxide radical scavenging activity was determined using the same method as that

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used for the free radical scavenging activity. In brief, the system that generates superoxide radicals

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was based on the autoxidation of the pyrogallol reaction. Five milliliters of 50.0 mM Tris-HCl

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buffer (pH 8.1) was mixed with 4.0 mL bioactive polysaccharides or nanoparticles at different

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concentrations (10-300 µg/mL for TP and TP-NPs; 200-2000 µg/mL for GLP and GLP-NPs;

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30-2000 µg/mL for MCP and MCP-NPs), respectively. After the mixture was incubated for 20 min

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at 25 °C, 1.0 mL of 3.0 mM pyrogallol was added to the mixture, and the mixture was incubated

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for 5 min (at 25 °C). Then 1.0 mL HCl (10.0 mM) was added to terminate the reaction, and the

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absorbance was monitored at 320 nm. The superoxide radical scavenging activity was calculated

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by equation (3):

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Scavenging rate (%) = [1-(A1-A2)/A0] × 100% (3)

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A0 represents the absorbance of the control (reaction solution with no sample); A2 represents the

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absorbance of the blank (sample with no pyrogallol); and A1 represents the absorbance of the test

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sample (reaction solution with sample or positive control).

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Additionally, ascorbic acid (Vc) was used as positive control in DPPH, •OH, and superoxide radical scavenging assays.

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Determination of antibacterial activity. The inhibitory effects of bioactive polysaccharides

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(GLP and MCP) and nanoparticles (GLP-NPs and MCP-NPs) on the growth rate of four types of

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bacteria (S. aureus, Salmonella, E. coli, and B. subtilis) were measured using the broth

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microdilution test recommended by the Clinical and Laboratory Standard Institute.21 Serial

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doubling dilutions of the GLP and GLP-NP suspensions at concentrations of 0; 250; 500; 1,000;



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1,500; 2,000; and 3,000 µg/mL and the MCP and MCP-NP suspensions at concentrations of 0; 250;

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500; 1,000; 1,500; 2,000; 3,000; and 4,000 µg/mL in fresh broth were placed in a test tube,

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receptively. Bacteria suspensions were inoculated to achieve approximately 1 × 106 CFU/mL of

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bacterial concentration in each tube. Different bacteria suspensions were grown in LB broth

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supplemented with different concentrations of bioactive polysaccharides or nanoparticles at 37 °C

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for 24 h. The optical density (OD) of each tube was monitored by a UV–vis spectrometer at 600

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nm (Shimadzu-2600, Kyoto, Japan).

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Statistical analysis. Each measurement was conducted in at least triplicate samples, and the

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results were reported as the mean ± standard deviation. The data were analyzed using SPSS V.17

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software (SPSS Inc., Chicago, IL). Duncan's multiple range test was also applied to compare the

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difference of means from the ANOVA, using a significance level of 5% (p < 0.05).

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RESULTS AND DISCUSSION

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Morphology and size of bioactive polysaccharide nanoparticles. The particle size and

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ζ-potential of the TP-NPs prepared at different ethanol to water ratios (3:1, 4:1, 5:1, 10:1, and 20:1)

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are shown in Figure 1. The ratios of ethanol to water displayed a significant impact on the particle

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size distribution of the obtained nanoparticles. As the ratio increased from 3:1 to 10:1, the mean

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particle size of the TP-NPs significantly decreased from 248±11 nm to 99±15 nm, and the PDI

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value also decreased from 0.45 to 0.33. The results suggested that small and well-distributed

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TP-NPs could be fabricated at higher ethanol to water ratios. Similarly, Tan et al. obtained starch

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acetate nanospheres using nanoprecipitation and found that the average particle size can be

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reduced by increasing the volume of anti-solvent.22 Furthermore, the ζ-potential of the TP-NPs

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ranged from -24.5 mV to -22 mV.


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The TEM images of the TP-NPs also show a similar trend (Figure 2). As the ratio of ethanol

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to water increased from 3:1 to 20:1, the size decreased, and the particles were gradually changed

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from an irregular to a more spherical shape. At low volume ratios (3:1 and 4:1), the nanoparticles

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coalesced rapidly and formed large aggregates with loose structures in the presence of excess

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water; therefore, the formed nanoparticles were mostly non-spherical in shape and demonstrated

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an inhomogeneous size distribution (Figure 2A, B). When a high volume of ethanol (5:1, 10:1,

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and 20:1) was added to the polysaccharide solution, a high degree of supersaturation probably

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resulted in a spatially uniform distribution of nuclei and a low crystal growth rate. The decrease of

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the amount of coalescence leads to a decrease in particle size.23,24

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It should be pointed out that the TP solution did not show any change when illuminated by a

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laser beam (Figure 3). After ethanol was added drop-wise into the solution, Tyndall light

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scattering was observed along the beam path, which indicated the presence of nano-sized

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particles. Moreover, the beam intensity of the TP-NPs suspension gradually increased as the

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ethanol to water ratio increased.

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Furthermore, the mean size and morphologies of the GLP-NPs and MCP-NPs at different

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ethanol to water ratios are shown in Figures S1, S2, S3, and S4, respectively. When the ethanol to

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water ratio was high (5:1, 10:1, and 20:1), the PDI values of both the nanoparticles were smaller

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than 0.50 (ranged from 0.39 to 0.26), indicating that the nanoparticles were monodispersed

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without obvious aggregation. At an ethanol to water ratio of 10:1, the GLP-NPs and MCP-NPs

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were spherical, with sizes of 95±7 and 141±9 nm, respectively. Moreover, according to the TEM

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results, we speculated that the ethanol to water ratios improved the morphologies of the

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nanoparticles, while the polysaccharide types did not play a role. These results showed that the



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ethanol to water ratio of 10:1 was optimal to prepare the TP-NPs, GLP-NPs, and MCP-NPs, and

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thus the obtained polysaccharide nanoparticles were used for further investigation of their

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stability, antioxidant, and antimicrobial activity.

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Fourier transform infrared spectroscopy (FTIR). The spectra of the TP and TP-NPs are

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shown in Figure 4. The absorption spectra of the TP and TP-NPs exhibited a broad peak at

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3,600–3,200 cm–1 corresponding to intra- or inter-molecular hydrogen bonds with O-H stretching

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vibration and a weak band at around 2,940 cm-1, which is characteristic of weak C-H asymmetric

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vibration.25 There were characteristic peaks for the asymmetric (1,700–1,600 cm−1) and

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symmetric (1,400–1,300 cm−1) stretching of carboxylate anions groups, indicating that carboxyl

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groups existed in the samples.26,27 The stretching vibrations of S=O had an absorption peak of

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about 1,300–1,200 cm−1, which was evidence of sulfuric radicals and suggested that all the

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TP-NPs had sulfated groups.28 The spectra also indicated that TP-NPs had similar adsorption

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peaks, but the intensities of the peaks were different among them. Compared with TP, the

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characteristic bands at 3,600–3,200 cm−1 in the TP-NPs shifted to a shorter wavelength, which

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indicated that the hydrogen bonds between molecular chains in the TP-NPs became stronger.

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Particle stability. Bioactive polysaccharide nanoparticles may be used in foods and

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beverages with different external environments depending on the nature of the product. Moreover,

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they may be exposed to considerable variations in temperature, ionic strength, or pH level as they

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pass through the human gastrointestinal tract after ingestion. Consequently, it is important to

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understand the influence of temperature, ionic strength, and pH level on the size, charge, and

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stability of polysaccharide nanoparticles.

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Figure 5 shows the effect of different temperature treatments (4, 25, 37, and 65 °C) on the


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mean size, PDI, and turbidity of the TP-NPs. As temperature increased from 4 to 65 °C, no

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significant changes were observed for particle size, PDI, and turbidity, indicating that the TP-NPs

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were stable and homogeneous. Presumably, the excellent thermal stability of the TP-NPs was

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probably due to the steric repulsion between the nanoparticles.

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The effects of salt content (0–500 mM NaCl) on the size distribution, mean size, PDI, and

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turbidity of the TP-NPs are shown in Figure 6. Compared with the control, the size distribution,

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the values of mean size, PDI, and turbidity of the TP-NP suspensions did not change markedly

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with the addition of 100–300 mM NaCl. These results suggested that the TP-NPs exhibited

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excellent salt stability. When the salt concentration was over 300 mM, there was an increase in

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particle size and turbidity. This could be because the inter-particle repulsion was reduced by

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sufficiently high ionic strength through the electro-screening effect, which allowed for potential

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contact to occur between nanoparticles, and formed agglomerations, thereby increasing the size of

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the TP-NPs.

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Variations in the mean size, ζ-potential, PDI, and turbidity of the TP-NPs over a wide pH

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range (pH 2.0–9.0) are shown in Figure 7. The size distribution of the TP-NPs remained relatively

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stable under neutral and alkaline conditions, but it increased significantly under acidic conditions.

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The TP-NPs had a small size of ~ 95 nm in neutral and alkaline conditions, but the size increased

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remarkably to about 220 nm in acidic conditions. Likewise, the PDI and turbidity of the TP-NPs

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showed the same trend regarding size. Jahanshahi and Babaei reported that the ζ-potential of

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protein nanoparticles was around -30 mV, which should be large enough to ensure good stability

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through electrostatic repulsion.29 Because the ζ-potential of the TP-NPs was nearly zero or slightly

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negative at lower pH values (2.0), the particles gathered to form large aggregates. These results



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indicated that the stability of the TP-NPs in neutral and alkaline conditions was better than in

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acidic conditions. Additionally, the PDI values of the TP-NPs were about 0.29–0.36, and TP-NPs

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appeared relatively stable across the various pH levels (Figure 7C). Tang et al. suggested that good

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stability of camptothecin nanosuspension provides its ability to target blood fluid, which helps

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improve bioactivity of camptothecin.30

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Cell

viability

of

polysaccharide

nanoparticles.

The

cytotoxicity

of

bioactive

277

polysaccharides and the nanoparticles (TP-NPs, GLP-NPs, and MCP-NPs) against MEF cells

278

were estimated by MTT assay. After 24 h of cell exposure in the medium containing various

279

concentrations of samples, the cell viabilities were still higher than 80% (Figure 8), suggesting

280

that the bioactive polysaccharides and nanoparticles exhibited no toxicity. Polysaccharide

281

extracted from tea materials can be classified as very low toxicity polymers or as unclassified with

282

oral administration.31,32 Our results suggested that the TP-NPs, GLP-NPs, and MCP-NPs could be

283

safely used as functional food materials.

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Protein adsorption assay of polysaccharide nanoparticles. We evaluated the interaction of

285

the TP-NPs, GLP-NPs, and MCP-NPs with proteins, using BSA as a model plasma protein. Figure

286

9 shows that in physiological conditions (pH 7.4), the nanoparticles underwent different protein

287

adsorption capacities in the following order: MCP-NPs> TP-NPs> GLP-NPs. This could be due to

288

the surface charge and size of particles. Compared to the other two nanoparticles, the GLP-NPs

289

showed reduced nonspecific protein adsorption, thereby indicating prolonged blood circulation.

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Functionality and opsonization of the nanoparticles begins immediately after they are introduced

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into plasma. Therefore, bioactive polysaccharide nanoparticles, especially MCP-NPs, could be

292

used as a functional ingredient or nanocarrier with improved stability in blood.


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Antioxidant activity assays of polysaccharide nanoparticles

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DPPH radical scavenging activity. Polysaccharides that can donate hydrogen have been

295

proved to reduce the stable DPPH radical to yellow diphenylpicrylhydrazine.12,33 Total DPPH

296

scavenging effects of bioactive polysaccharides and nanoparticles at varying concentrations were

297

measured (Figure S5). Evidently, of all the test samples, the DPPH radical scavenging activity was

298

displayed in the most concentration-dependent manner. The higher the concentrations of the

299

samples were, the stronger the radical scavenging activity was. Furthermore, all kinds of

300

polysaccharide nanoparticles presented stronger scavenging activities than their corresponding

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bioactive polysaccharides at every concentration point. The scavenging rates of the TP-NPs,

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GLP-NPs, and MCP-NPs at the highest test concentrations (300; 2,000; and 2,000 µg/mL)

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increased by 12.5%, 10.1%, and 11.25%, compared with the TP, GLP, and MCP, respectively

304

(Figure S5). As shown in Table 1, the 50% inhibitory concentration (IC50) values of TPS, GLP,

305

and MCP were 71.70, 870.42, and 746.82 µg/mL, respectively. In contrast, the IC50 values of the

306

TP-NPs, GLP-NPs, and MCP-NPs were 46.49, 687.91, and 485.47 µg/mL. These results indicated

307

that these polysaccharide nanoparticles had a noticeable effect on scavenging free radicals.

308

Previously, Schaffazick et al.34 and Yen et al.35 reported a similar effect, in which the nanoparticle

309

system increased the antioxidant ability of melatonin anti-lipid peroxidation. Similarly, previous

310

studies have suggested that the nanoparticles of natural bioactive components (kaempferol and

311

resveratrol) demonstrate improved antioxidant activity in vitro.36,37

312

The NaCl and thermal treatments showed a decreased antioxidant activity capacity for most

313

of the bioactive polysaccharides (Figure S5 and Table 1). The antioxidant activities of the

314

bioactive polysaccharides especially decreased after heating treatment. However, the TP-NPs,



315

GLP-NPs, and MCP-NPs were stable in heating solutions and had higher DPPH radical

316

scavenging activities than the corresponding bioactive polysaccharides did. The ionic strength

317

treatment also inhibited the antioxidant activities of the three bioactive polysaccharides, but when

318

they formed nanoparticles, the polysaccharide nanoparticles exhibited stronger antioxidant activity.

319

At the highest tested concentration, compared with TP, GLP, and MCP in the presence of heating

320

and NaCl treatment, the scavenging rates of the TP-NPs, GLP-NPs, and MCP-NPs were increased

321

by 19.8% and 24.7%; 13.7% and 14.4%; and 17.8% and 14.5%, respectively. These results

322

indicated that nanoparticles had stronger stability against ionic strength and thermal treatment.

323

Hydroxyl radical scavenging activity. The •OH possesses extremely high reactivity and

324

can induce severe damage to functioning biomolecules in living cells and functional ingredients in

325

food systems.38 Though not as strong as that of Vc, all samples exhibited •OH scavenging effects

326

within the test concentrations (Figure S6). Previous studies of the antioxidant activity of

327

polysaccharides have suggested that the OH group, carboxyl group, sulfate group, and

328

monosaccharide constituent may affect the antioxidant activity of polysaccharides.39-42 As shown

329

in Figure S6, polysaccharide nanoparticles possessed higher •OH scavenging activities than the

330

corresponding bioactive polysaccharides. The scavenging rate of the TP-NPs at a concentration of

331

300 µg/mL increased by 9.6%, and the scavenging rates of the GLP-NPs and MCP-NPs at a

332

concentration of 2,000 µg/mL increased by 12.4% and 15.2%, respectively. Under NaCl and

333

thermal treatment, the •OH scavenging activity of the bioactive polysaccharides was decreased

334

obviously, while the polysaccharide nanoparticles only slightly decreased.

335

After heating treatment, the IC50 values of the TP-NPs, GLP-NPs, and MCP-NPs (Table 2)

336

for scavenging •OH were calculated to be 51.26, 820.29, and 579.67 µg/mL, which were smaller


Page 16 of 43

Page 17 of 43


337

than those of bioactive polysaccharides (the IC50 values of the TP, GLP, and MCP were 193.75,

338

1209.3, and 1061.54 µg/mL). At the highest concentration, the •OH scavenging rates of the

339

TP-NPs, GLP-NPs, and MCP-NPs were increased by 14.8% and 20.5%; 20.7% and 15.4%; and

340

21.8% and 15.1%, respectively (Figure S6). These results revealed that the polysaccharide

341

nanoparticles exhibited stronger antioxidant activity than the bioactive polysaccharides.

342

Superoxide anion radical scavenging activity. In organisms, superoxide anion radical

343

indirectly initiates lipid peroxidation and can form strong reactive oxidative species, and thus the

344

presence of superoxide anion radicals is harmful for biomolecules.43-45 Therefore, the superoxide

345

anion radical scavenging activities of bioactive polysaccharides and nanoparticles at different

346

concentrations were evaluated, and the results are shown in Figure S7 and Table 3. All samples

347

exhibited scavenging activities for superoxide anion radicals. Zhao et al. speculated that

348

polysaccharides had electron-withdrawing groups (like keto or aldehyde) that could facilitate the

349

liberation of hydrogen from the O-H bond and thus stabilize the superoxide anion.28 The

350

polysaccharide nanoparticles also had higher scavenging activities for superoxide anion radicals

351

than the corresponding bioactive polysaccharides. After NaCl and heating treatment, compared

352

with the bioactive polysaccharides, the scavenging rates of the TP-NPs, GLP-NPs, and MCP-NPs

353

at the highest concentration were increased by 14.9% and 13.7%; 24.1% and 21.2%; and 16.6%

354

and 18.3%, respectively. Raveendran et al. reported that mauran polysaccharide/chitosan

355

nanoparticles could inhibit the oxidative damage caused under natural conditions, and they also

356

presented a prospective incipient polysaccharide nanoparticle made of extremophilic bacterial

357

origin for defending oxidative stress in vitro.46

358

Antibacterial activity in vitro. A normal intestinal microbial environment is important for



359

reducing the risk of gut diseases triggered by pathogenic microorganisms.47,48 Therefore, the

360

effects of the GLP-NPs and MCP-NPs on the growth of the Gram-positive bacteria S. aureus and

361

B. subtilis and the Gram-negative bacteria E. coli and Salmonella after an incubation period of 24

362

h were investigated. For comparison, the antibacterial activities of GLP and MCP were also

363

determined. The OD profiles of the bacteria as a function of concentrations are presented in Figure

364

10.

365

For all of the bacteria types, the OD values of all the samples reduced as the concentration

366

increased, indicating that all the tested samples exhibited antimicrobial activity. Li et al. reported

367

that Cyclocarya paliurus polysaccharides at a concentration of 1 mg/mL exhibited considerable

368

inhibitory activities against S. aureus and B. subtilis.49 As shown in Figure 10, GLP and MCP

369

exhibited higher antibacterial activity against the Gram-positive bacteria than against the

370

Gram-negative bacteria. E. coli had a more negatively charged cell surface than S. aureus and B.

371

subtilis.50 The interaction between bacterial cells and sub-micrometric materials depends on the

372

van der Waals and electrostatic interactions. Furthermore, for the same kind of bacteria, the OD

373

values of the GLP-NPs and MCP-NPs were lower than those of GLP and MCP, respectively.

374

Quantitatively, when the concentration of samples was 3,000 µg/mL, the OD values of the

375

GLP-NPs and MCP-NPs against S. aureus were 0.073 and 0.105, respectively, while the OD

376

values of GLP and MCP were 0.142 and 0.162, respectively. The polysaccharide nanoparticles

377

acted as excellent antibacterial agents against both Gram-positive and Gram-negative bacteria

378

when compared to polysaccharides. This was most likely due to the smaller size and higher zeta

379

potential of the polysaccharide nanoparticles. Likewise, Nguyen et al. reported that the

380

chitosan/sodium tripolyphosphate nanoparticles with smaller size and more positive ζ-potential


Page 18 of 43

Page 19 of 43


381

showed higher antibacterial activity against Streptococcus pneumoniae.51

382

Additionally, antibacterial activity as a function of the incubation time of bioactive

383

polysaccharide and nanoparticles against bacteria is shown in Figure S8. Antimicrobial activity of

384

the test samples against bacteria displayed a significant increasing trend (p < 0.05) as the

385

incubation time increased from 2 to 24 h. However, when the incubation time exceeded 24 h,

386

especially for 48 h, the OD values of GLP and MCP against bacteria increased markedly, while the

387

OD values of the GLP-NPs and MCP-NPs were almost constant, which indicated that the

388

GLP-NPs and MCP-NPs could effectively limit the increase in viable cell numbers, and exhibited

389

long-acting antibacterial activity. This could be because that the nanoparticles adhered to the

390

bacterial surface, irreversibly disrupted the membrane structure of the bacteria, and subsequently

391

penetrated cells and effectively inhibited protein activity, which ultimately led to bacteria

392

apoptosis.52,53

393

In summary, we successfully prepared the TP-NPs, GLP-NPs, and MCP-NPs using

394

nanoprecipitation. The polysaccharide types did not affect the morphology of the corresponding

395

nanoparticles. The high ethanol to water ratios produced monodispersed spherical nanoparticles.

396

The diameter of polysaccharide nanoparticles decreased as the ethanol/polysaccharide ratio

397

increased from 3:1 to 10:1. The nanoparticles exhibited better antioxidant activity and radical

398

scavenging activity than the bulk polysaccharides. Furthermore, the functional performances of

399

the TP-NPs, GLP-NPs, and MCP-NPs were more stable than those of the bioactive

400

polysaccharides when exposed to heat and increased ionic strength. In comparison to native

401

polysaccharides, GLP-NPs and MCP-NPs exhibited greater antimicrobial activity and prolonged

402

the antibacterial effect of the Gram-positive bacteria, S. aureus and B. subtilis, and the



403

Gram-negative bacteria, E. coli and Salmonella. The results suggest that the functional

404

nanoparticles prepared in this study could be applied as powerful antioxidant and antibacterial

405

materials in the food, cosmetics, and biopharmaceutical industries.

406

Supporting Information

407

DLS and TEM analysis of GLP-NPs (Figure S1 and Figure S2) and MCP-NPs (Figure S3

408

and Figure S4); the DPPH, •OH, and superoxide anion radical scavenging effects of TP-NPs,

409

GLP-NPs, and MCP-NPs at different concentrations after NaCl (100 mM) and 65 °C treatment

410

(Figure S5, Figure S6, and Figure S7); the antibacterial activity of GLP and GLP-NPs at the

411

concentration of 1.25 mg/mL, and MCP and MCP-NPs at the concentration of 2.0 mg/mL (Figure

412

S8).

413

Notes

414 415

The authors declare no competing financial interest. ACKNOWLEDGMENTS

416

This work was supported by the Special Funds for Taishan Scholars Project of Shandong

417

Province (No. ts201712058) and the Natural Science Foundation of Shandong Province of China

418

(ZR2017MC044).


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419

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569

FIGURE CAPTION

570

Figure 1 Size distribution (A: Intensity; B: Number) and mean size, PDI, and ζ-positional (C) of

571

TP-NPs prepared at different ethanol to water ratio (3:1, 4:1, 5:1, 10:1, and 20:1) at pH 6.0.

572

Figure 2 TEM images of TP-NPs prepared at different ethanol to water ratio (A: 3:1, B: 4:1, C: 5:1,

573

D: 10:1, and E: 20:1) at pH 6.0, respectively. Scale bar is 200 nm.

574

Figure 3 Photo showing the aspect of TP solution (a), and TP-NP suspensions (0.5%) prepared at

575

different ethanol to water ratio (b: 3:1, c: 4:1, d: 5:1, e: 10:1, and f: 20:1) and the Tyndall effect by

576

a light beam incident from the left side.

577

Figure 4 FTIR images of TP-NPs prepared at different ethanol to water ratio at pH 6.0.

578

Figure 5 Effect of different temperature (4, 25, 37, and 65 °C) on size distribution (A: Intensity; B:

579

Number), mean size, PDI, and turbidity of TP-NPs prepared with ethanol to water ratio of 10:1 at

580

pH 6.0. Data are presented as the average of triplicate measurements with standard deviation (n =

581

3).

582

Figure 6 Effect of various ionic strength (0-500 mM, NaCl) on size distribution (A: Intensity; B:

583


584


585

3).

586

Figure 7 Effect of various pH-levels (2, 5, 7.4, and 9) on size distribution (A: Intensity; B:

587

Number), mean size, PDI, ζ-potential, and turbidity of TP-NPs prepared with ethanol to water

588

ratio of 10:1 at pH 6.0. Data are presented as the average of triplicate measurements with standard

589

deviation (n = 3).

590

Figure 8 Cytotoxicity of MEF treated with TP-NPs (A), GLP-NPs (B), and MCP-NPs (C) at


Page 28 of 43

Page 29 of 43


591

different concentrations.

592

Figure 9 BSA adsorptions of TP-NPs, GLP-NPs, and MCP-NPs after incubating at 37 °C (pH 7.4)

593

for various time intervals.

594

Figure 10 Antibacterial activity of native polysaccharides and polysaccharide nanoparticles against

595

various microorganisms in LB broth after 24 h incubation at 37 °C. The red arrow represents the

596

concentration of samples from low to high.

597

A: GLP and GLP-NPs at the concentration of 0, 250, 500, 1000, 1500, 2000, and 3000 µg/mL; B:

598

MCP and MCP-NPs at the concentration of 0, 250, 500, 1000, 1500, 2000 3000, and 4000 µg/mL.



Page 30 of 43

599

Table 1 IC50 values of DPPH radical scavenging activity of bioactive polysaccharides and

600

nanoparticles after NaCl (100 mM) and 65 °C treatment. Sample

IC50 (µg/mL) Control

NaCl

Temperature

TP

71.70±3.28c

80.85±3.01b

123.71±9.81a

TP-NPs

46.49±3.12b

46.95±2.74b

56.26±3.25a

GLP

870.42±19.32c

961.11±23.19b

1180.43±20.15a

GLP-NPs

687.91±20.73b

681.19±22.31b

860.67±19.41a

MCP

746.82±22.80c

921.35±20.88b

1215.38±22.57a

MCP-NPs

485.47±18.33c

525.16±19.43b

643.48±17.20a

601

Values mean ± SD indicates the replicates of three experiments. Different letters in the same row (a-c) indicate

602

significant differences (upper case) (P < 0.05).


Page 31 of 43


603

Table 2 IC50 values of hydroxyl radicals scavenging activity of bioactive polysaccharides and

604



NaCl

Temperature

TP

68.49±2.78c

87.61±3.08b

193.75±6.87a

TP-NPs

39.54±2.23c

46.72±2.01b

51.26±2.16a

GLP

854.95±18.92c

961.11±19.90b

1209.33±25.77a

GLP-NPs

481.58±20.71b

427.91±21.59c

820.29±23.81a

MCP

526.55±21.29c

773.28±21.78b

1061.54±22.40a

MCP-NPs

273.50±14.27c

300.61±17.83b

579.67±16.97a

605


606




Page 32 of 43

607

Table 3 IC50 values of superoxide radical scavenging activity of bioactive polysaccharides and

608



NaCl

Temperature

TP

100.53±4.34c

189.75±3.13b

327.31±5.72a

TP-NPs

78.20±3.31b

68.91±3.39c

109.86±4.75a

GLP

771.27±20.89c

949.12±30.01b

1362.37±29.77a

GLP-NPs

426.18±19.72b

415.29±21.59b

760.67±28.06a

MCP

452.86±19.94b

765.22±20.82b

1320.21±29.79a

MCP-NPs

246.74±19.89b

279.61±16.52b

543.49±30.64a

609


610



Page 33 of 43


25

A

TP-NPs 1:3 TP-NPs 1:4 TP-NPs 1:5 TP-NPs 1:10 TP-NPs 1:20

20

15

B

TP-NPs 1:3 TP-NPs 1:4 TP-NPs 1:5 TP-NPs 1:10 TP-NPs 1:20

20

N um ber ( % )

Intensity （ % ）

25

10

5

15

10

5

0

0

10

100

1000

3000

10

100

1000

3000

Size (nm)

Size (nm)

300

C

0.5

Mean size (nm) PDI ζ -positional (mV) 0.4

250

0 -5 -10

200 0.3

-15

150 0.2

-20

100 0.1

50

-25

0.0 -30

0 3

4

5

10

20

The ratio of ethanol to water

611

Figure 1 Size distribution (A: Intensity; B: Number) and mean size, PDI, and ζ-positional (C) of

612

TP-NPs prepared at different ethanol to water ratio (3:1, 4:1, 5:1, 10:1, and 20:1) at pH 6.0.



A

Page 34 of 43

C

B

200 nm

200 nm

200 nm

E

D

200 nm

200 nm

613

Figure 2 TEM images of TP-NPs prepared at different ethanol to water ratio (A: 3:1, B: 4:1, C: 5:1,

614

D: 10:1, and E: 20:1) at pH 6.0, respectively. Scale bar is 200 nm.


Page 35 of 43


a

b

c

d

e

f

615

Figure 3 Photo showing the aspect of TP solution (a), and TP-NP suspensions (0.5%) prepared at

616

different ethanol to water ratio (b: 3:1, c: 4:1, d: 5:1, e: 10:1, and f: 20:1) and the Tyndall effect by

617

a light beam incident from the left side.



TP-NPs 1:4 TP-NPs 1:10 TP

Intensity (%)

TP-NPs 1:3 TP-NPs 1:5 TP-NPs 1:20

Page 36 of 43

4000

3500

3000

2500

2000

1500

1000

500

-1

Wavenumber (cm )

618

Figure 4 FTIR images of TP-NPs prepared at different ethanol to water ratio at pH 6.0.


Page 37 of 43


25

A

4 °C 25 °C 37 °C 65 °C

20

B

4 °C 25 °C 37 °C 65 °C

20

Number ( % )

Intensity ( % )

25

15

10

15

10

5

5

0

0 10

100

1000

10

3000

100

350 300

1000

3000

Size (nm)

Size (nm)

C

Mean size (nm) PDI Turbidity

0.6

0.6

0.5

0.5

0.4

0.4

0.3

0.3

0.2

0.2

0.1

0.1

250 200 150 100 50 0 4

25

37

65

Temperature (°C) 619

Figure 5 Effect of different temperature (4, 25, 37, and 65 °C) on size distribution (A: Intensity; B:

620


621


622

3)



30

A

0 mM NaCl 100 mM NaCl 200 mM NaCl 300 mM NaCl 400 mM NaCl 500 mM NaCl

Intensity ( % )

15

B

25

N u m b e r (% )

20

10

5

Page 38 of 43

0 mM NaCl 100 mM NaCl 200 mM NaCl 300 mM NaCl 400 mM NaCl 500 mM NaCl

20 15 10 5

0

0 10

100

3000

1000

10

100

Size (nm)

300 250

1000

3000

Size (nm)

0.6

Mean size (nm) PDI Turbidity

C

0.5

0.4 0.5

200 0.3 150

0.4 0.2

100 0.3 50 0

0.2 0

100

200

300

400

0.1

0.0

500

NaCl (mM) 623

Figure 6 Effect of various ionic strength (0-500 mM, NaCl) on size distribution (A: Intensity; B:

624


625


626

3).


Page 39 of 43


A

18 16

Intensity ( % )

40

pH = 2.0 pH = 5.0 pH = 7.4 pH = 9.0

14

B

35

N u m b e r (% )

20

12 10 8 6

pH= 2.0 pH= 5.0 pH= 7.4 pH= 9.0

30 25 20 15 10

4

5

2 0

0

10

100

1000

3000

10

100

10

300

0

250

-10

1000

3000

Size (nm)

Size (nm)

C

Mean size (nm) Turbidity

PDI ζ - potential

0.7 0.6

0.6 0.5

200 0.5 0.4

-20

150 0.4 0.3

-30 100 -40

0.3 0.2

50 -50 -55

0.2 0.1

0 2

5

7.4

9

pH 627

Figure 7 Effect of various pH-levels (2, 5, 7.4, and 9) on size distribution (A: Intensity; B:

628

Number), mean size, PDI, ζ-potential, and turbidity of TP-NPs prepared with ethanol to water

629

ratio of 10:1 at pH 6.0. Data are presented as the average of triplicate measurements with standard

630

deviation (n = 3).



120

A

TPS

TP-NPs

Cell viability ( % )

100 80 60 40 20 0 25

0

50

100

200

150

Concentration (µg/mL)

120

B

GLP GLP-NPs

Cell viability ( % )

100 80 60 40 20 0 0

250

500

1000

1500

2000


120

C

MCP MCP-NPs

Cell viability (% )

100 80 60 40 20 0 0

250

500

1000

1500

2000

Concentration (µg/mL) 631

Figure 8 Cytotoxicity of MEF treated with TP-NPs (A), GLP-NPs (B), and MCP-NPs (C) at

632

different concentrations.


Page 40 of 43


Protein adsorption ratio ( % )

Page 41 of 43

35

TPS-NPs GLP-NPs MCP-NPs

30 25 20 15 10 5 0

5

10

15

20

25

30

35

40

Time (h) 633

Figure 9 BSA adsorptions of TP-NPs, GLP-NPs, and MCP-NPs after incubating at 37 °C (pH 7.4)

634

for various time intervals.



1.0

A

0.8

OD 600

0.6

0.4

0.2

0.0

GLP

GLP-NPs

GLP GLP-NPs GLP

S. aureus Bacillus subtilis

1.0

GLP-NPs

E. coil

B

OD 600

0.8

0.6

0.4

0.2

0.0

MCP MCP-NPs MCP MCP-NPs MCP MCP-NPs

S. aureus

E. coil

Salmonella

635

Figure 10 Antibacterial activity of native polysaccharides and polysaccharide nanoparticles against

636

various microorganisms in LB broth after 24 h incubation at 37 °C. The red arrow represents the

637

concentration of samples from low to high.

638

A: GLP and GLP-NPs at the concentration of 0, 250, 500, 1000, 1500, 2000, and 3000 µg/mL; B:

639

MCP and MCP-NPs at the concentration of 0, 250, 500, 1000, 1500, 2000 3000, and 4000 µg/mL.


Page 42 of 43

Page 43 of 43


Graphic Abstract Antimicrobial MCP S. aureus MCP-NPs MCP E.coli MCP-NPs MCP Salmonella MCP-NPs

1.0

Light beam

0.8

OD600

Tyndall effect

0.6 0.4 0.2

Add dropwise

0.0

0

5

10 15 20 25 30 35 40 45 50

Time (h)

Stirring

TEM image

Ethanol Polysaccharide Bioactive

polysaccharide

Antioxidant

100

Scavenging ability (%)

640

80 60 40 Vc TP TP/NaCl TP/65 °C

20 0

0

50

100

150

TP-NPs TP-NPs/NaCl TP-NPs/65 °C

200

250



300

Preparation of bioactive polysaccharide nanoparticles with enhanced

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