Resveratrol-loaded chitosan–γ-poly(glutamic acid) nanoparticles: Optimization, solubility, UV stability, and cellular antioxidant activity

Journal Pre-proof Resveratrol-loaded chitosan–␥-poly(glutamic acid) nanoparticles: optimization, solubility, UV stability, and cellular antioxidant activity Joo Hee Chung, Ji-Soo Lee, Hyeon Gyu Lee

PII:

S0927-7765(19)30846-X

DOI:

https://doi.org/10.1016/j.colsurfb.2019.110702

Reference:

COLSUB 110702

To appear in:

Colloids and Surfaces B: Biointerfaces

Received Date:

29 June 2019

Revised Date:

4 November 2019

Accepted Date:

1 December 2019

Please cite this article as: Chung JH, Lee J-Soo, Lee HG, Resveratrol-loaded chitosan–␥-poly(glutamic acid) nanoparticles: optimization, solubility, UV stability, and cellular antioxidant activity, Colloids and Surfaces B: Biointerfaces (2019), doi: https://doi.org/10.1016/j.colsurfb.2019.110702

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(To be submitted to the Colloids and Surfaces B: Biointerfaces)

Resveratrol-loaded chitosan–γ-poly(glutamic acid) nanoparticles: optimization, solubility, UV stability, and cellular antioxidant activity

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Joo Hee Chung1, Ji-Soo Lee1, and Hyeon Gyu Lee*

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Department of Food and Nutrition, Hanyang University, Republic of Korea

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*Corresponding author.

Tel.: +82-2-2220-1202; Fax: +82-2-2281-8285;

Co-first author.

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1

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E-mail: [email protected]

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Graphical abstract

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The solubility and stability of resveratrol could be more effectively improved by

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controlling size of resveratrol-loaded nanoparticle. Highlights

Resveratrol (RSV) was nanoencapsulated using chitosan and γ-poly

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

(glutamic acid).

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The solubility and stability of RSV was significantly increased by nanoencapsulation.

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As nanoparticle size increased, the stability increased but the solubility decreased. 2

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The particle size was controlled by chitosan and γ-poly (glutamic acid) concentration.

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The solubility and stability of RSV could be controlled by particle size.

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ABSTRACT

The objective of this study was to investigate the effects of the particle size of resveratrol (RSV)-loaded nanoparticles (NPs) on their solubility and stability and to optimize their preparation conditions for their solubility and stability. RSV-loaded NPs

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were prepared using chitosan and γ-poly(glutamic acid) (γ-PGA). Although the

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solubility and stability of RSV have been significantly increased using chitosan/γ-PGA nanoencapsulation, as the NP size decreased, the solubility increased, but the stability

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decreased. In order to understand the interrelationship of particle size, solubility, and stability, the target values of RSV solubility and ultraviolet (UV) stability for the

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aforementioned optimization were determined at two levels: solubility > 153 μg/mL, UV stability > 12% (S153U12) and solubility > 150 μg/mL, UV stability > 18%

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(S150U18). The S150U18-NPs (258 nm) showed a significantly higher UV stability and tyrosinase inhibition activity against UVA than S153U12-NPs (87 nm) (p < 0.01).

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Although insignificant, the S153U12-NPs exhibited higher solubility than the S150U18NPs. In addition, the cellular antioxidant activity was significantly higher in the S153U12-NPs than the S150U18-NPs (p < 0.05). These results demonstrated that the solubility and stability of RSV-loaded NPs may be influenced by their particle size, which could be controlled by the chitosan and γ-PGA concentrations.

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Keywords: resveratrol; solubility; stability; particle size; response surface methodology;

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nanoencapsulation

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1. Introduction

Resveratrol (3,5,4′-trihydroxy-stilbene; RSV), a phytoalexin found in grape skins and peanuts [1] has been reported to have cardioprotective, antioxidant, anti-obesity, anti-inflammatory, and anti-carcinogenic effects [2]. Despite these health benefits of RSV, its utilization as a nutraceutical ingredient in the food industry is limited owing to

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its poor water-solubility, low bioavailability, and rapid degradation [2, 3]. As RSV had poor water-solubility (0.02–0.03 mg/mL) [3, 4], it is difficult to incorporate relatively high amounts of RSV into beverages and aqueous food products. In addition, RSV is sensitive to chemical degradation and is easily degraded by ultraviolet (UV) light, high

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temperatures, alkaline pH, or enzymes. The chemical degradation of RSV often

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involves trans-to-cis isomerization [3]. The cis-isomer exhibits lower radical scavenging and anti-inflammatory activities than the trans-isomer [5]. Moreover, the

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poor solubility and stability of RSV result in its low intestinal absorption and short biological half-life after oral administration, thus resulting in low bioavailability [1, 3].

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To overcome these drawbacks of RSV, several encapsulation strategies have been adopted, including the use of polymer particles, emulsions, liposomes,

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cyclodextrin complexation, and solid lipid nanoparticles (NPs) [3, 6]. Encapsulation is a technique used to incorporate active ingredients into a type of matrix. This can

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improve the stability of RSV by providing a physical barrier against light, heat, and oxygen [3]. In particular, polymer particles can effectively protect RSV from environmental factors that could cause its chemical degradation as they exhibit high stability in hydrated, dehydrated (dry), and rehydrated states [6]. Nano-sized particles may increase the solubility and passive cellular absorption owing to their subcellular size and provide a greater surface area per mass unit, thus improving bioactivity [7]. 5

Therefore, biopolymer-based nanoencapsulation is suitable for increasing the application of bioactive ingredients having poor water solubility and stability (such as RSV) in the food industry [8]. Typically, delivery systems comprising smaller particles facilitate higher dissolution rate of the core material, thus enhancing its solubility. However, larger particles can be expected to expose a limited surface area to the oxidative

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environment, thus improving stability [9]. Therefore, it appears reasonable to hypothesize that the characteristics required for improving the solubility and stability of NPs may differ. However, few studies on the interrelationship between particle size, solubility, and stability have been reported. Our previous study [10] also showed that

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the solubility, stability, and cellular uptake of RSV could be improved by using

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nanoencapsulation with chitosan and γ-poly(glutamic acid) (γ-PGA). However, as the solubility and stability of RSV-loaded NPs having a diameter of 100–150 nm were

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compared with those of non-nanoencapsulated RSV, no information was provided on how the solubility and stability vary with particle size. Therefore, it is necessary to

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investigate the effects of the NP size on the solubility and stability and to determine the NP preparation conditions that improve not only the solubility but also the stability

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in the case of poorly soluble and unstable bioactive ingredients such as RSV. Chitosan, which is obtained via N-deacetylation of chitin, is commonly used as

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the wall material of biopolymeric NPs because of its cationic, biodegradable, and nontoxic properties. Owing to the positive surface charge of chitosan, chitosan-based NPs have muco-adhesive properties and can transiently open tight junctions between epithelial cells, thus enhancing the intestinal permeation of entrapped core materials [11]. γ-PGA, which is found in natto, a traditional Japanese fermented food, is an anionic, edible, and non-toxic polypeptide. As γ-PGA is a water-soluble and 6

biodegradable polyanion that can be safely consumed [12], it has been used as the wall material of NPs and microcapsules in food and medicine [13-16]. It contains repetitive glutamic acid units connected by an amide bond between the α-amino and γ-carboxylic acid groups [12]. Chitosan/γ-PGA NPs are prepared via ionic gelation between the positively charged amino groups of chitosan and the negatively charged carboxyl groups of γ-PGA [13, 14]. In previous studies, chitosan/γ-PGA NPs were

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found to protect insulin against the gastrointestinal environment and increase the absorption of insulin by the intestinal epithelium through the paracellular pathway [16, 17]. Chitosan/γ-PGA NPs have also been used as carriers for DNA and fibroblast growth factors, thus facilitating their controlled release and enhanced skin penetration

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[18]. Furthermore, our previous study [10] also suggested that chitosan/γ-PGA

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nanoencapsulation could improve the solubility and stability of RSV. Therefore, the objectives of this study were to investigate the effects of the

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particle size of RSV-loaded chitosan/γ-PGA NPs on their solubility and stability. For this purpose, the preparation conditions of RSV-loaded NPs were optimized for the

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solubility as well as stability of RSV by using response surface methodology (RSM). The solubility, stability, cellular antioxidant activity, and tyrosinase-activity inhibition of

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the optimized RSV-loaded NPs at two levels were investigated.

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2. Materials and methods

2.1. Materials

γ-PGA (molecular weight (MW) 50 kDa) and chitosan (MW 1,000–3,000 kDa) were purchased from BioLeaders Corp. (Daejeon, Korea) and Kitto Life Co. (Seoul, Korea), respectively. Mushroom tyrosinase, 2′,7′-dichlorofluorescein diacetate (DCFH7

DA), 2,2′-azobis (2-amidinopropane) dihydrochloride (ABAP), and RSV having a purity of ≥99% were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Ltyrosine was purchased from Junsei Chemical Co. (Tokyo, Japan). Human hepatoma (HepG2) cell lines were purchased from the Korean Cell Line Bank (Seoul, Korea). Minimum essential media, non-essential amino acids, Hank’s balanced

salt

solution

(HBSS),

fetal

bovine

serum,

and

0.25%

trypsin-

Island, NY, USA).

2.2. Preparation of RSV-loaded chitosan/γ-PGA NPs

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ethylenediamine tetraacetic acid were purchased from Gibco Invitrogen Co. (Grand

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Chitosan/γ-PGA NPs were prepared via ionic gelation [15]. RSV was dissolved

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in ethanol at a concentration of 2.0 mg/mL, and 0.5 mL of the RSV solution was premixed with 5 mL of an aqueous chitosan solution. Then, 0.5 mL of the γ-PGA

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solution was added dropwise into the premixed RSV/chitosan solution using a pipette tip under magnetic stirring (1,000 rpm) at 25 °C. The final obtained concentration of

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RSV in the suspension was 166.67 μg/mL.

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2.3. Physical properties of NPs

The mean particle diameter and polydispersity index of the RSV-loaded

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chitosan/γ-PGA NPs were measured using a dynamic light scattering (DLS) method on a Zetasizer Nano ZS (Malvern Instruments Ltd., Worcestershire, UK) at 25±1 °C. Samples were measured in triplicate at the least for each treatment. A transmission electron microscope (TEM; JEM 2100F, JEOL, Tokyo, Japan) was used to analyze the morphology of the NPs [13]. For the TEM samples, the nanosuspension was dropped onto a 200-mesh carbon-coated copper grid and dried 8

at 37°C. The dried samples were stained with a 2%-phosphotungstic acid solution for 30 min. The samples were subsequently dried at 37°C for 24 h before making the TEM observation.

2.4. Determination of entrapment efficiency The entrapment efficiency (EE) of the RSV-loaded chitosan/γ-PGA NPs was

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calculated based on the measurement of the free RSV content in the supernatant after ultracentrifugation (Optima TL Ultracentrifuge, Beckman, Fullerton, CA, USA) at 30,000 × g for 30 min. The EE was determined using Eq. 1 [19].

Total content of RSV – free content of RSV in supernatant EE (%) =

X 100

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Total content of RSV

(1)

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The RSV content was measured using high-performance liquid chromatography (HPLC; Waters 486 Tunable Absorbance Detector, Waters Corporation, Milford, MA,

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USA). The HPLC system comprised a Waters 515 pump and Waters 486 tunable absorbance detector. A Puresil C18 column (4.6 × 150 mm, 5 μm) (Waters, Milford,

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MA, USA) was used, and the mobile phase consisted of 0.5% (v/v) acetic acid in methanol–water (50:50 v/v). The flow rate was 1.0 mL/min, and the injection volume

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was 20 μL. Detection was performed at 306 nm [20].

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2.5. Experimental design and data analysis A two-factor, five-level central composite design was used to study the effects of

two independent variables X1 (chitosan concentration) and X2 (γ-PGA concentration) on three response variables Y1–Y3 (particle size, solubility, and UV stability). The design comprised 10 experimental points that included fractional 22 factorial points, four star points, and two center points. The actual values of each independent variable 9

were coded at five levels. The ranges of the independent variables were based on the results of preliminary experiments (Table 1). The proposed model for each response of Y is described in Eq. 2:

(2) where b0, bi, bii, and bij are the regression coefficients for the constant, linear, quadratic,

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and cross-product, respectively. Xi and Xj represent independent variables. The Statistical Analysis System program 9.4 (SAS Institute Inc., Cary, NC, USA) was used to analyze the experimental data. Response surfaces and contour plots were

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generated using the resulting regression models.

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2.6. Solubility

The effect of chitosan/γ-PGA nanoencapsulation on the solubility of RSV was

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investigated. For the measurement of the RSV solubility, both non-nanoencapsulated and nanoencapsulated RSV suspensions were filtered through a mixed-cellulose-

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ester membrane filter comprising 0.45-μm pores (Jet Biofil, Guangzhou, China) to eliminate the remaining insoluble RSV. The filtered solutions containing RSV were

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measured using HPLC. The final concentrations of RSV in the non-nanoencapsulated and nanoencapsulated RSV suspensions were fixed at 167.7 μg/mL. In order to

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eliminate the interference of ethanol in the RSV solubility, the ethanol concentrations in both suspensions were fixed at 8.3% (the same ethanol concentration that was used for the RSV-loaded NP suspension).

2.7. UV light stability 10

The chemical stability of the RSV against isomerization was assessed using a UVA lamp (UVT series, Dongseo Science Co., Seongnam, Gyeonggi, Korea). A 1-mL aliquot of each sample was pipetted into a 2-mL Eppendorf tube and placed 15 cm below the UV-light source (365 nm) at 25°C for 3 h. The remaining RSV content was analyzed using HPLC. The effect of nanoencapsulation on the degradation under UV light was assessed based on the retention percentage of trans-RSV [21]. The final

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concentration of the RSV in the suspension was fixed at 167.7 μg/mL, at which both the non-nanoencapsulated and nanoencapsulated RSV suspensions were in a liquid state without precipitation, so that the samples could be prepared under the same

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conditions.

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2.8. Inhibition of tyrosinase activity

Tyrosinase activity was performed using the modified method of Tomita et al. [22].

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Tyrosinase inhibition was assayed with L-tyrosine as the substrate, and the formation of dopachrome was monitored. Subsequently, 440 μL of 0.1-M phosphate buffer (pH

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6.5), 40 μL of mushroom tyrosinase (1500 U/mL), and 40 μL of the sample were briefly mixed and incubated for 10 min at 37°C. Then, 80 μL of 1.5-mM L-tyrosine was added

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to this mixture to initiate the enzymatic reaction. The mixture was incubated at 37°C for 10 min and placed on ice to stop the reaction. After the mixture was centrifuged at

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9,200 × g for 4 min, 0.5 mL of the supernatant was transferred to an Eppendorf tube. The amount of dopachrome produced was then measured spectrophotometrically at 490 nm. The percent inhibition of tyrosinase activity was calculated as follows: Inhibition of tyrosinase activity (%) = [(C−S)/C] × 100 where C is the control absorbance, and S is the sample absorbance.

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(5)

2.9. Cellular antioxidant activity assay The cellular antioxidant activity (CAA) was evaluated using the method presented by Wolfe and Liu [23]. HepG2 cells (6 × 104 cells/well) were seeded on a 96-well plate with 100 µL of medium/well. After incubation for 24 h, the medium was removed from the well plate, and the wells were washed using 100 μL of phosphatebuffered saline (PBS). The cells were then treated for 1 h with 50 µL of the sample and 50 µL of 25-μM DCFH-DA dissolved in medium. After washing the plate with PBS, the

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plate was refilled with 600-μM ABAP dissolved in 100 μL of HBSS. ABAP, which is an exogenous source of peroxyl radicals, oxidizes DCFH to fluorescent DCF. In this assay, the cells treated with samples that have antioxidant activity are expected to exhibit a

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lower fluorescence than cells that are not treated with antioxidants. The fluorescence

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of each well was measured every 5 min for 60 min at 37°C on a Synergy HT Multimicroplate Reader (BioTek Instruments, Winooski, VT, USA). The emission and

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excitation wavelengths were 538 and 485 nm, respectively. Each plate comprised control and blank wells; the control wells were treated with DCFH-DA and ABAP, while

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the blank wells were treated with DCFH-DA and HBSS without ABAP. The CAA values were calculated using the following formula: CAA units = 100 − (∫SA /∫CA) × 100

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(6)

where ∫SA and ∫CA are the integrated areas under the fluorescence versus time curves

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for the sample and control curves, respectively.

2.10. Statistical analysis All the experiments were performed at least three times, and the obtained results were expressed as the mean±standard deviation (SD). Statistical analysis was performed using the Statistical Package for the Social Sciences (SPSS, Version 21.0, 12

SPSS Inc., Chicago, IL, USA). Significant differences were observed among the mean values in an independent samples t-test and a one-way analysis of variance (ANOVA), and these were followed by a Duncan's multiple-range test with a confidence level of 95%.

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3. Results and discussion

3.1. Effect of NP preparation conditions on particle size, solubility, and UV stability

RSM was used to evaluate the effects of the NP preparation conditions, including

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the concentrations of chitosan (X1) and γ-PGA (X2), on the particle size (Y1), solubility

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(Y2), and UV light stability (Y3). As shown in Table 1, the particle sizes, determined from the 10 experimental runs generated by the central composite design, ranged from

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50.59 to 607.22 nm. The solubility of the NPs was in the range of 146.62–153.73 μg/mL, and the percentage of the remaining trans-RSV after the UVA-light exposure

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for 3 h was 11.88–25.80%.

The mathematical relationships of the response variables (Yn) with the

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concentrations of wall materials (Xn) obtained from the regression analysis of Eq. 2 are given as follows:

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Particle size (nm, Y1)

= 473 + 237X1 − 3,996X2* − 1,948X1X2** + 172X12*** + 10,642X22**

(7)

Solubility (µg/mL, Y2) = 124.88*** + 10.82X1** + 149.52X2*** − 12.16X1X2 − 3.44X12*** − 219.60X22*** (8) UV stability (%, Y3) = 39.86** − 12.40X1 − 132.28X2 + 40.42X1X2 + 2.85X12* + 92.22X22 13

(9)

where ***, **, and * indicate the significance at p < 0.01, p < 0.05, and p < 0.1, respectively. The ANOVA of the regression model demonstrated that the models were significant and adequate (Y1: p < 0.01, R2 = 0.9919; Y2: p < 0.01, R2 = 0.9852; Y3: p < 0.01, R2 = 0.9652). Therefore, this model could be used to predict the effects of the NP preparation conditions on the particle size, solubility, and UV-light stability of NPs. The predicted values of particle size, solubility, and UV stability obtained using Eqs. 7, 8, and 9 were used to generate three-dimensional response-surface plots (Fig.

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1). The size of the NPs increased as the chitosan concentration increased and γ-PGA concentration decreased. This could be interpreted in terms of the chitosan/γ-PGA ratio, which was the main factor influencing the particle size. Tang et al. [14] and Jeon

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et al. [10] also observed that the NP size increased as the chitosan/γ-PGA ratios

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increased. At higher concentrations of γ-PGA, the electrostatic interaction between γPGA and chitosan was found to be stronger, thus resulting in a more compact NP

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structure [24, 25].

The solubility of nanoencapsulated RSV was greater than 146.62 μg/mL,

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irrespective of the chitosan and γ-PGA concentrations (Table 1), while the solubility of non-nanoencapsulated RSV was only 66.28±5.75 μg/mL (data not shown). These

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results suggest that the water dispersibility of nanoencapsulated RSV can be improved by hydrophilic materials such as chitosan and γ-PGA on the surface of the NPs. In

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particular, hydrogen bonding between chitosan and RSV appears to increase the solubility of the RSV by increasing solvent–solute interactions. Similar results were reported by Kim et al. [26], who demonstrated that chitosan NPs improved the solubility of retinol, and by Davidov-Pardo et al. [27], who found that the formation of complexes of RSV and sodium caseinate increased the RSV solubility. The majority of studies on this subject indicate that the trans-isomer of RSV has 14

higher bioactivity (e.g., tyrosinase inhibition activity and radical scavenging activity) than the cis-isomer [5]. Furthermore, trans-RSV is a photosensitive compound and can easily be isomerized to cis-RSV via UV irradiation, thus limiting its application [28, 29]. Therefore, the assessment of the UV-light stability of RSV is necessary. The stability of all nanoencapsulated RSV in this study was greater than 11.88% (Table 1), while the stability of the non-nanoencapsulated RSV was only 9.25±0.70% (data not

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shown). These results indicate that the nanoencapsulated RSV was more stable than the non-nanoencapsulated RSV as the trans-RSV degraded more slowly and less cisRSV was formed; this was because the chitosan/γ-PGA nanoencapsulation provided the RSV with an effective protective barrier against UV-light-induced degradation [9].

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Careful observation of Fig. 1 reveals that, as the γ-PGA concentration increased

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and the chitosan concentration decreased, the NP size and the UV stability decreased significantly while the solubility increased significantly. That is, as the particle size

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increased, the stability increased but the solubility decreased. In addition, as shown in Table 1, the NPs in Run No. 8 that were prepared with the highest chitosan

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concentration had the largest particle size (Y1), lowest solubility (Y2), and highest UVlight stability (Y3). These results were obtained because a lower surface area was

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exposed to the UVA light in the larger NPs than in the smaller NPs, thus reducing the degradation of the RSV in the larger NPs. However, in terms of solubility, smaller NPs

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having a larger surface area in contact with the dissolution solution could be more advantageous. These results suggest that the size of the NPs can influence their solubility and UV stability [9, 26], and that the tendencies of the solubility and UV stability are inversely related to each other. Larger particles can entrap core materials more stably and smaller particles can increase the water-dispersibility. Therefore, the preparation conditions of NPs should be determined while taking into consideration 15

not only their solubility but also their stability.

3.2. Optimization of chitosan and γ-PGA concentrations to enhance solubility and UV stability of RSV Because the chitosan and γ-PGA concentrations for increasing RSV solubility and UV stability showed the opposite trend, their target values for optimization were

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determined by a compromise between RSV solubility and UV stability. The target solubility and UV stability values of the RSV for the optimization were determined at two levels: solubility > 153 μg/mL, UV light stability > 12% (S153U12) and solubility > 150 μg/mL, UV light stability > 18% (S150U18). To obtain the optimized chitosan and

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γ-PGA concentrations for S153U12 and S150U12, the contour plots generated using

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Eqs. 8 and 9 were superimposed and regions that satisfy all the constraints were selected. The S153U12-NPs were obtained when the chitosan and γ-PGA

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concentrations were 1.325 and 0.3 µg/mL, respectively (chitosan/γ-PGA = 4.41), and the S150U18-NPs were obtained when the chitosan and γ-PGA concentrations were

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1.975 and 0.27 µg/mL, respectively (chitosan/γ-PGA = 7.31). The experimental values of the S153U12-NPs and S150U18-NPs, which include

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the particle size (Y1), solubility (Y2), and UV stability (Y3), were in a close agreement with the predicted values (Table 2); that is, the differences between the experimental

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and predicted values were within the SDs. The particle size and UV stability of S150U18-NPs were significantly higher than those of S153U12-NPs (p < 0.01). The S153U12-NPs also showed higher solubility than the S150U18-NPs; however, this had no statistical significance. These results could be attributed to the difference in the solubility of S153U12-NPs and S150U18-NPs being only 2.9 μg/mL, which was also within the SDs. These results suggested that the solubility and stability of RSV-loaded 16

NPs may be influenced by the particle size, which could be controlled by the chitosan and γ-PGA concentrations. In addition, the NP-preparation conditions can be optimized to enhance the solubility or stability according to the purpose of the NPs.

3.3. Morphology of NPs The morphology of the RSV-loaded chitosan/γ-PGA NPs observed via TEM is shown in Fig. 2. The NPs appeared to be round or oval with relatively smooth surfaces.

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The shapes of the chitosan/γ-PGA NPs were similar to those observed in a previous study [14]. The sizes of the S153U12-NPs (Fig. 2A) and S150U18-NPs (Fig. 2B) ranged from 50 to 150 nm and from 250 to 350 nm, respectively, which is similar to

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the sizes measured in the DLS analysis.

3.4. Entrapment efficiency

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The EEs of the S153U12-NPs and S150U18-NPs were 33.0% and 37.1%, respectively (Fig. 3). These values did not differ significantly from each other; however,

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the S150U18-NPs showed a higher EE than the S153U12-NPs. This result could be attributed to the chitosan content of the NPs. Similar results were reported by Sanna

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et al. [30], who reported that the EE of RSV-loaded chitosan/poly (D,L-lactic-co-glycolic acid) microcapsules increased as the chitosan concentration increased. These results

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could have been obtained because the RSV entrapment efficiency may have been related to the hydrogen bonding between the hydroxyl or amine groups of chitosan and the phenolic hydroxyl groups of RSV [6].

3.5. Inhibition of tyrosinase activity Many studies have demonstrated that RSV and its derivatives inhibit tyrosinase 17

activity [31, 32]. Tyrosinase causes the browning reaction (which may cause undesirable changes in the nutritive value and color of foods) and is also responsible for melanin biosynthesis [33]. Thus, effective tyrosinase inhibitors have been studied for their potential application in preventing food quality degradation and melaninrelated health problems in humans [34]. The tyrosinase inhibition activity of RSV was observed under UVA irradiation to

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determine the protective effect of nanoencapsulation (Fig. 4). While the tyrosinase inhibition activity of the NPs decreased slowly, that of the free RSV decreased sharply until 2 h after UV irradiation and degraded gradually thereafter. The protective effect of the NPs was most pronounced at the 2-h exposure time, when the tyrosinase

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inhibition activities of the free RSV, S153U12-NPs, and S150U18-NPs were 10.5%,

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49.7%, and 57.6%, respectively. This result displayed a similar tendency to that of the UV stability determined using HPLC. The decrease in tyrosinase inhibition activity with

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increasing exposure to UV irradiation may be attributed to the reduction of trans-RSV (owing to its conversion to cis-RSV). The S150U18-NPs had higher inhibitory activity

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than the S153U12-NPs, thus indicating that the S150U18-NPs had a stronger protective effect against UVA irradiation, which is consistent with the previous

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experiment.

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3.6. Cellular antioxidant activity assay The CAA assay, which is a cell-based method for determining antioxidant activity,

takes into consideration factors such as the cellular uptake and distribution of bioactive compounds [35]. Therefore, this assay is appropriate for evaluating antioxidant behavior in biological systems. The antioxidant activity of RSV before and after nanoencapsulation was measured in the CAA assay. 18

As shown in Fig. 5, the CAA value of the nanoencapsulated RSV was significantly higher than that of the non-nanoencapsulated RSV. The highest CAA value was observed in the S153U12-NPs, which had higher RSV solubility and were smaller than the S150U18-NPs. These results could be because the decrease in particle size led to an increase in contact surface area between the entrapped RSV and the environment, solubility, and cellular uptake, resulting in improved CAA. These results were similar to the results obtained by Yu et al. [36], who reported that

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encapsulation of curcuminoids in modified ϵ-polylysine increased the CAA value of curcuminoids by facilitating higher solubility, more specific interaction with the cell, and lower degradation. Therefore, it can be concluded that nanoencapsulation with a

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natural polymer such as chitosan and γ-PGA could improve the solubility and stability

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of the entrapped core material. Furthermore, the solubility and stability of NPs could be more effectively improved by controlling the NP size, which was influenced by the

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NP preparation conditions.

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Declaration of interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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Acknowledgements

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This work was supported by the Technological Innovation R&D Program (S2230695) funded by the Small and Medium Business Administration (SMBA, Korea).

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Figure captions

Fig. 1. Three-dimensional response-surface plots displaying the effects of the chitosan and γ-poly(glutamic acid) concentrations on the particle size (A), solubility (B), and UV stability (C) of RSV-loaded NPs. Fig. 2. TEM micrograph of RSV-loaded chitosan/γ-poly(glutamic acid) NPs: (A) S153U12-NPs, (B) S150U18-NPs.

error bars represent mean±SD (n = 3). Fig.

4.

Tyrosinase

inhibitory

activities

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Fig. 3. Entrapment efficiency of RSV-loaded chitosan/γ-poly(glutamic acid) NPs. The

nanoencapsulated

and

non-

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nanoencapsulated RSV under UV irradiation. The error bars represent

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mean±SD (n = 3). Different letters indicate significant differences between samples (p < 0.05).

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Fig. 5. Cellular antioxidant activity of nanoencapsulated and non-nanoencapsulated RSV. The error bars represent mean±SD (n = 3). Different letters indicate

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significant differences between samples (p < 0.05).

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Table 1. Experimental values of difference activities determined from 10 experimental runs generated by central composite design Independent variables1) (Xn) Run

Response variables (Yn)

Coded

Process

Particle size

Solubility

UV-light

variables

variables

(nm)

(μg/mL)

stability2) (%)

X1

X1

Y1

Y2

Y3

X2

X2

-1

-1

1.0

0.20

107±11

2

1

-1

2.0

0.20

447±10

3

-1

1

1.0

0.30

62±2

4

1

1

2.0

0.30

207±11

5

0

0

1.5

0.25

169±5

153±8

16.0±2.8

6

0

0

1.5

0.25

159±11

153±8

16.2±3.5

7

-2

0

0.5

0.25

51±2

152±11

11.9±2.3

8

2

0

2.5

0.25

607±36

147±8

25.8±6.9

9

0

-2

1.5

0.15

432±36

148±6

19.5±4.4

10

0

95±6

152±7

14.5±2.1

2

0.35

16.7±7.4

149±5

19.0±6.6

154±7

11.9±1.8

151±8

18.2±6.4

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1.5

151±9

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number

X1= Chitosan concentration (mg/mL), X2= γ-PGA concentration (mg/mL)

2)

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1)

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Percentage of remaining trans-RSV after direct exposure to UVA light for 3 h

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Table 2. Comparison of predicted and experimental values for particle size, solubility, and UV stability Response variables

Nanoparticles

Predicted

Experimental

S153U12-NPs

72

87±15*

S150U18-NPs

269

258±22*

S153U12-NPs

153

153±2

S150U18-NPs

151

150±2

S153U12-NPs

13.1

16.75±1.2*

Particle size (nm)

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Solubility (μg/mL)

S150U18-NPs

-p

UV stability (%)

19.1

23.23±0.4*

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in the Student’s t-test at P < 0.01.

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One asterisk indicates a significant difference between S153U12- and S150U12-NPs

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Fig. 1

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(B)

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Fig. 2

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Fig. 5

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