Self-Assembly of Protein Nanoparticles from Rice Bran Waste and

Research Article pubs.acs.org/journal/ascecg

Self-Assembly of Protein Nanoparticles from Rice Bran Waste and Their Use as Delivery System for Curcumin Hailong Peng,†,‡,§ Zhaodi Gan,† Hua Xiong,*,† Mei Luo,† Ningxiang Yu,† Tao Wen,§ Ronghui Wang,§ and Yanbin Li*,§ †

State Key Laboratory of Food Science and Technology, Nanchang University, No. 235 Nanjing East Road, Nanchang 330047, China School of Resources, Environmental, and Chemical Engineering, Nanchang University, No. 999 Xuefu Avenue, Nanchang 330031, China § Department of Biological and Agricultural Engineering, University of Arkansas, 230 Engineering Hall, Fayetteville, Arkansas 72701, United States ‡

ABSTRACT: Rice bran waste, a massive byproduct from rice milling, is rich in proteins, lipids, vitamins, and trace minerals. However, rice bran is an underutilized sustainable resource that is ultimately discarded as agricultural waste. Herein, rice bran albumin (RBA) was extracted from rice bran waste using water, and its properties of amino acid compositions, molecule weight, secondary structure, denaturation temperature, and ζ-potential were characterized. The results indicated that RBA possessed negative charges and amphiphilic molecules with high value and good digestibility. RBA was then proposed to prepare nanoparticles with chitosan (CS) (RBA−CS nanoparticles) by a self-assembly method. The RBA−CS nanoparticles exhibited spherical shape with core (RBA)−shell (CS) structures, and the average size was about 384 nm. The solubility of curcumin (CUR) was greatly improved after loading into the RBA−CS nanoparticles, and higher entrapment efficiency (EE, 93.56%) was obtained. CUR released from the RBA−CS nanoparticles was governed by two distinct stages (burst release and sustained release) and dependent on the pH value of the release media. Diffusion, swelling, and erosion mechanisms might coexist for the full release processes for the RBA− CS nanoparticles. The RBA−CS nanoparticles have properties of delayed degradability in gastric conditions and complete digestibility in the small intestine with good biodegradability. Additionally, the CUR loaded RBA−CS nanoparticles exhibited higher cytotoxicity than free CUR for Caco-2 cells. Thus, a sustainable and higher value byproduct (RBA−CS nanoparticles) was obtained from rice bran waste, which has great potential application for hydrophobic active agent delivery. KEYWORDS: Rice bran waste, Rice bran albumin, Curcumin, Self-assembly, Nanoparticle

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INTRODUCTION

bran albumin (RBA) exhibits high solubility in water with the beneficial properties of less allergenic properties, nontoxic, lowcost, biodegradability, and biocompatibility.6 Meanwhile, RBA has pharmacological actions of antiaging, antioxidative, anticancer, and antidepressant activities.7 Nowadays, RBA researches mostly focus on the extraction and physical− chemical properties, but there is, as yet, no report on the applications of RBA. If natural and functional RBA can be applied to produce high value byproducts, a sustainable protein platform is then achieved to broaden practical rice bran waste applications. Proteins and polysaccharides are polymers ubiquitous in nature, and they can form complexes by covalent or hydrogen binding or by hydrophobic interactions.8 Recently, protein− polysaccharide complexes have been widely applied in the food and pharmaceutical industry due to their functional properties

Rice (Oryza sativa) is consumed as a staple food globally with an estimated production of 645 million tons per year.1 Rice grains consist of bran, embryo, endosperm, and hull. Rice bran constitutes about 10% of the rough rice and is an underutilized byproduct of the rice milling industry.2 Rice bran is considered to have high nutritional values, including 15%−22% lipids, 34.1%−52.3% carbohydrates, 7%−11.4% fiber, and 10%−16% protein.3 Until now, however, rice bran is underexploited and generally discarded as agricultural waste, which leads to resource waste and environmental pollution. Thus, renewable applications of rice bran waste and production of high value and green byproducts are needed. As an abundant and cheap agricultural byproduct, rice bran protein has many desirable nutritional factors, such as hypoallergenic, antioxidant, anticancer, and significant quantities of essential amino acids, which can be a useful supplement of proteins in milk and soy infant formulations.4 Rice bran protein compositions include 37% albumins, 36% globulins, 22% glutelins, and 5% prolamins.5 Among these proteins, rice © 2017 American Chemical Society

Received: March 21, 2017 Revised: June 1, 2017 Published: June 29, 2017 6605

DOI: 10.1021/acssuschemeng.7b00851 ACS Sustainable Chem. Eng. 2017, 5, 6605−6614

Research Article

ACS Sustainable Chemistry & Engineering

Medical Technology Co Ltd. (Hangzhou, Zhejiang). Fetal bovine serum (FBS), nonessential amino acids, penicillin, and streptomycin were all purchased from Fisher Scientific (Shanghai, China). Extraction and Characterizations of RBA. RBA was extracted using distilled water (1:10, w/v) with stirring at room temperature for 3 h and centrifuged at 4000g for 10 min to obtain the albumin fraction (supernatant). The supernatant was adjusted to about pH 4.0 with HCl solution (0.5 M) and then allowed to precipitate for 2 h at 4 °C. The precipitate was washed with distilled water three times, dialyzed (Mw: 8000−14000) for 3 days at 4 °C, and then freeze-dried for 24 h. After that, the precipitated protein (RBA) was obtained and the purity was determined by Kjeldahl methods. The RBA characterizations of amino acid compositions, molecular weight, denaturation temperature, ζ-potential, and secondary structure were all analyzed according to Zhao’s method29 to evaluate the potential of forming nanoparticles. Preparation of CUR Loaded RBA−CS Nanoparticles. CUR ethanol solution (5 mL, 8 mg mL−1) was dispersed directly into RBA aqueous solution (15 mL, 8 mg mL−1) under magnetic agitation for 30 min. CS was dissolved in aqueous acetic acid solution (5 mg mL−1) and stirred overnight to dissolve completely. Then, the CS solution (8 mL) was added dropwisely into the RBA solution and mixed by stirring for 30 min. The pH of the RBA−CS solution was adjusted to 4.0 with HCl (0.1 N) or NaOH (0.1 N), and the solution was stirred for 3 h at room temperature. The CUR loaded RBA−CS nanoparticles were obtained after heating at 90 °C for 60 min in the sealed vial. Characterizations of CUR Loaded RBA−CS Nanoparticles. The morphology of the nanoparticles was evaluated using scanning electron microscopy (SEM, Quanta 200F, FEI) and atomic force microscopy (AFM, Agilent 5500). The particle size and distribution of nanoparticles were examined using a nanoparticle size analyzer (NICOMP380/ZLS, PSS). ζ-Potentials of nanoparticles were determined using a Zetasizer (DTS1060, Malvern Instruments). Selfassembly behavior of nanoparticles was evaluated using fluorescence spectroscopy (F-4500, Hitachi). In Vitro Digestibility of CUR Loaded RBA−CS Nanoparticles. In vitro digestibility of nanoparticles were carried out according to the previous report with minor modification.30 Pepsin (1 mg mL−1, pH 2.0) and pancreatin (10 mg mL−1, pH 6.8) solutions were prepared as stock solutions. The pH values of nanoparticle samples (4 mL) were preadjusted to pH 2.0 using HCl (1.0 N), followed with 100 μL of the pepsin stock solution to obtain simulated gastric digestion. The nanoparticles in simulated gastric solutions were incubated at 37 °C for 2 h with mild stirring and pH was raised to 7.0 with NaOH (1.0 N) to stop the proteolytic reaction. After pepsin digestion, the pancreatin stock solution (100 μL) was added into the nanoparticles samples, incubated for 4 h at 37 °C, and then boiled for 3 min to deactivate proteases. For evaluation of nanoparticles digestibility, 50 μL of nanoparticles solution after pepsin and pancreatin digestion was withdrawn and analyzed by SDS-PAGE, size distribution, and SEM. Entrapment Efficiency (EE) and Drug Loading (DL). The CUR loaded RBA−CS nanoparticle solutions were ultracentrifugated (125000g) for 45 min, and the supernatant was taken out and diluted suitably with ethanol. The unloaded CUR in the supernatant was determined using a UV spectrophotometer (Lambda 18, PerkinElmer) at 428 nm. The EE and DL were determined by the following equations.

of hydration and structuration and to their surface properties. Among these properties, structuration has received considerable attention because such complexes have the ability to selfassemble and then form nanoparticles that are eco-friendly, cost-effective, and convenient.9 Chitosan (CS) is a natural polysaccharide composed of randomly distributed β-(1−4)-linked D-glucosamine (deacetylated unit) and N-acetyl-D-glucosamine (acetylated unit).10,11 Due to its advantages of nontoxicity, sustainability, and biodegradability, some protein−CS coacervation complexes have been researched, such as α-lactalbumin−CS and βlactoglobulin−CS,12 soy globulin−CS,13 pea protein isolate− CS,14 soybean protein isolate−CS,15 xanthan−CS,16 and gum arabic−CS.17 However, the complex coacervation between RBA and CS has not been reported yet. Meanwhile, to the best of our knowledge, the application of RBA−CS complexes to form nanoparticles by a self-assembly method has been rarely reported. Curcumin (CUR) is the main phenolic pigment extracted from the dietary spice turmeric, and it is commonly used as a spice, food preservative, and flavor agent. Clinical trials indicated that CUR possesses diverse pharmacologic effects, including anti-inflammatory, antioxidant, antitumor, anti-HIV, antiproliferative, and antiangiogenic activities.18 CUR also inhibits lipid peroxidation and scavenges superoxide anion, nitric oxide, and hydroxyl radicals.19 However, the application of CUR in the pharmacologic field is limited owing to its disadvantages of poor solubility, low bioavailability, rapid metabolism, and rapid systemic elimination.20 To overcome these drawbacks, several delivery systems have been used to encapsulate CUR, such as liposomes,21−23 nanoparticles,24−26 and hydrogels.27,28 However, there still remain some noticeable problems for these CUR delivery systems. One is that the synthetic polymer is usually used as wall-materials, which may still be toxic for humans and the environment. Another is that organic solvent was often involved during the preparation processes, leading to residues in these delivery systems. For the purpose of sustainable development and environmental protection, a nontoxic material based delivery system and eco-friendly encapsulation technique should be developed for CUR. Herein, in this study, the natural and nontoxic protein RBA was extracted from rice bran waste and its properties amino acid composition, molecule weight, secondary structure, denaturation temperature, and ζ-potential were investigated. RBA is proposed to develop nanoparticles with CS (RBA−CS nanoparticles) by a self-assembly technique under eco-friendly conditions, and then it is used to encapsulate CUR. In addition, the physical properties of RBA−CS nanoparticles, including loading efficiency, particle size, surface morphology, ζ-potential, digestion ability, drug release profile, and cytotoxicity, were all investigated.

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EXPERIMENTAL PROCEDURES

Materials. Rice bran was obtained from Tian Yu Oil Co., Ltd. (Jiangxi, China). Chitosan (CS, deacetylation degree ≥95%) was purchased from Sigma-Aldrich (Germany). Curcumin (CUR, AR: 99.00%) was purchased from Xiya Reagent (Chengdu, Sichuan). Pepsin (enzyme activity ≥ 250 units/mg) and pancreatin (enzyme activity ≥ 250 units/mg) were purchased from Sigma-Aldrich (Germany). Caco-2 cell line was purchased from the China Center for Type Culture Collection (Wuhan, China). Dulbecco’s modified Eagle’s medium (DMEM) and 0.25% trypsin with ethylenediaminetetraacetic acid (EDTA, 0.02%) were obtained from Gino Biological

EE% = [(Total CUR − FreeCUR )/Total CUR ] × 100%

(1)

DL% = [(Total CUR − FreeCUR )/Total mass] × 100%

(2)

In Vitro Release and Mechanisms of RBA−CS Nanoparticles. CUR loaded RBA−CS nanoparticles (5 mL) were placed in dialysis bags (14 kDa) and were suspended in 50 mL of simulated gastric fluid (SGF), phosphate buffer saline (PBS), and simulated intestinal fluid (SIF), respectively, and then incubated at 37 ± 0.1 °C with stirring. At specified time intervals, 5 mL of the release medium was taken and the same volume of fresh media was added to maintain a constant volume. The amount of CUR release from the RBA−CS nanoparticles was estimated via a UV method at 428 nm and was calculated by the following equation. 6606


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ACS Sustainable Chemistry & Engineering n−1

Cumulative Release% =

Ve ∑1

Ci + V0Cn M

× 100%

Scheme 1. Schematic Representation Formation of RBA−CS Nanoparticles

(3)

where Ve is the removed volume of release medium (5 mL), V0 is the total volume of release medium (50 mL), Ci is the CUR concentration at i time, Cn is the CUR concentration at n time, and M is the content of the CUR in the nanoparticles. In order to gain insight into the release mechanism of CUR, the release sample data was analyzed according to the zero-order, firstorder, Higuchi, and Hixson−Crowell models, and the release exponent n of the Peppas model was also calculated by Ritger and Peppas’s method.

zero‐order model:

M t /M∞ = kt

(4)

first‐order model:

ln(1 − M t /M∞) = − kt

(5)

Higuchi model:

M t /M∞ = kt 1/2

Hixson−Crowell model:

Peppas model:

(1 − M t /M∞)1/3 = − kt

ln M t /M∞ = n ln t + ln k

(6) (7)

potential of self-assembly ability to form nanoparticles, the RBA properties of amino acid compositions, molecular weight, secondary structure, denaturation temperature, and ζ-potential were all evaluated. The essential amino acid compositions and scores of RBA are presented in Table 1. The results indicated that RBA contained

(8)

where Mt/M∞ is the fractional active agent released at time t, k is a constant incorporating the properties, and n gives an indication of the release mechanism. The correlation coefficient (r2) is the linear relationship between CUR release and time. In Vitro Cytotoxicity. Caco-2 cells were grown and routinely maintained at 37 °C in DMEM supplemented with 25 mM D-glucose containing FBS (10%), nonessential amino acids (1%), L-glutamine (1%), and penicillin (100 U mL−1)−streptomycin (100 μg mL−1) in an atmosphere of CO2 (5%) and 90% relative humidity. Culture medium was changed after washing with PBS every 2 days, and the nonadherent cells were removed by washing with PBS and medium changes. The cells were passaged every 4 days when the cell density reached 80−90% confluence at a 1/3 split ration by treating with a solution of 0.25% trypsin and 0.02% EDTA for 3 min after washing with PBS (pH = 7.4). For detection the cell viability, cells were seeded in a 96-well tissue culture plate with 200 μL of culture medium and were exposed to various concentrations of free CUR and CUR loaded RBA−CS nanoparticles (0, 0.4, 0.8, 3.0, 9.0, and 15.0 μg mL−1). After 24 h of incubation, MTS solution (40 μL) was then added and reacted for another 4 h. The absorbance was recorded immediately at 490 nm using a Microplate Reader (Model 680, Bio-Rad, CA), and cell viability was calculated using the following equation. Reported values are means of three replicates.

Table 1. Amino Acid Composition of RBAa and FAO/WHO Suggested Essential Amino Acid Requirements (g/100g of protein) FAO/WHO(1985)

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RBA

Child

Adult

Thr Val Ile Leu Lys His Met+Cys Phe+Tyr

4.10 6.31 3.92 7.79 6.09 4.18 2.86 8.41

3.40 3.50 2.80 6.60 5.80 1.90 2.50 6.30

0.90 1.30 1.30 1.90 1.60 1.60 1.70 1.90

a

The values of amino acid composition reported represent the average of three determinations.

Cell viability (%) = (Aborbance of test cells /Absorbance of control) × 100

Essential Amino acids

(9)

all the essential amino acids with higher scores of Phe and Tyr (8.41), Leu (7.79), and Val (6.31). The lowest proteins were Met and Crs, and the score was only about 2.86. Furthermore, the scores of all the essential amino acids were used to compare with those of the FAO/WHO (1973)31 and the results shown in Table 1, which indicated that the essential amino acids scores of RBA were all higher than that of FAO/WHO. Thus, RBA was shown to have a higher content of functional proteins for health. SDS-PAGE was performed to explore the molecular weight and subunit of RBA. Three major subunit bands (i.e., 53−55 kDa, 35−37 kDa, and 22 kDa) and a dense band (10−17 kDa) were clearly observed (Figure 1A). The subunit bands were almost exclusively distributed in the small molecular weight range (lower than 17 kDa), indicating that RBA was composed mainly of small molecules, which can be easily digested and absorbed in the body. The secondary structure of RBA was investigated using the FT-IR spectrum (Figure 1B). The peak of amide I was located at 1648.85 cm−1, corresponding to the stretching vibration of

RESULTS AND DISCUSSION Preparation Processes of CUR Loaded RBA−CS Nanoparticles. Scheme 1 illustrates the preparation processes of CUR loaded RBA−CS nanoparticles. For decreasing environmental pollution and improving the value of rice bran waste, the soluble rice bran protein RBA was extracted from rice bran waste using water and then introduced to prepared nanoparticles with CS using a self-assembly method. The RBA−CS nanoparticles formed a core−shell structure, and CUR was then loaded into the nanoparticle cores for improving the solubility. Therefore, rice bran waste was reused and produced a higher value byproduct (RBA−CS nanoparticles). Meanwhile, the preparation processes of RBA−CS nanoparticles were environmentally green, and no organic solvents or harsh conditions were involved. Extraction and Characterizations of RBA. RBA was extracted using water as a green extraction methodology, and the purity 85.58% was obtained. To further confirm the 6607


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Figure 1. Characterizations of RBA: (A) SDS-PAGE; (B) FT-IR (bottom) and curve fitting (top); (C) DSC; and (D) ζ-potential.

Figure 2. (A) Optical images and Dh of RBA−CS nanoparticles under different conditions of (B) pH, (C) temperature, and (D) MR of RBA:CS before and after 4 weeks storage.

form aggregations by self-assembly.33 Therefore, the higher βsheet content in RBA helped it to form nanoparticles. The denaturation temperature of RBA was determined by differential scanning calorimetry (DSC), and the result is shown in Figure 1C. The DSC result showed that only one endothermic peak (denaturation temperature) was observed at 85.35 °C for RBA, which indicated that the extracted RBA has higher purity and good thermal stability. The ζ-potential of RBA was measured, and the isoelectric point (pI) was about 3.8

CO (Figure 1B, bottom), which was representative of the secondary structure of the protein because the amide I band consisted of α-helix, β-sheet, β-turn, and random coil forms.32 The amide I of the RBA fitting results in Figure 1B (top) showed that the peaks of the α-helix, β-sheet, β-turn, and random coil structures were 1658.7 cm−1; 1617.8, 1630.8, and 1686.1 cm−1; 1672.8 cm−1; and 1644.6 cm−1, respectively, and the corresponding content was 22.66%, 38.60%, 15.05%, and 23.69%. The β-sheet structure of the protein was available to 6608


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Figure 3. (A) EE of RBA−CS nanoparticles under different MRs of RBA:CS:CUR; and (B) optical image of CUR in water and RBA−CS nanoparticle solutions.

Figure 4. (A) SEM, (C) AFM, and (E) size distribution of RBA−CS nanoparticles; and (B) SEM, (D) AFM, and (F) size distribution of CUR loaded RBA−CS nanoparticles.

time with a self-assembly method and then was used to encapsulate CUR to improve its solubility. Optimization of RBA−CS Nanoparticles Preparation. The RBA−CS nanoparticles were prepared by a self-assembly method, and the effects of pH, temperature, and mass ratio (MR) of RBA:CS were investigated. The optical image, particles size, and polymer disperse index (PDI) of the

(Figure 1D). RBA and CS have opposite potentials and higher ζ-potential products when the pH value was between 4.0 and 7.0 (Figure 1D), indicating that the RBA−CS complexes have strong electrostatic attraction in that pH range. Based on these above-mentioned RBA properties, the sustainable material of RBA was then proposed to prepare nanoparticles with CS (RBA−CS nanoparticles) for the first 6609


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Figure 5. (A) ζ-potential of RBA−CS mixture and RBA−CS nanoparticles at pH from 3 to 12; (B) I1/I3 ratio of pyrene fluorescence in RBA−CS nanoparticle solutions at pH from pH 2 to 10; and (C) Forming mechanisms for RBA−CS nanoparticles.

3:1 were considered the optimum conditions to develop the RBA−CS nanoparticles at 90 °C for 60 min. Encapsulation and Stability of CUR Loaded RBA−CS Nanoparticles. Recently, more and more nanotechnologybased delivery systems were used to load active molecules.34,35 Meanwhile, CUR has also been encapsulated into different nanotechnology-based delivery systems, such as liposomes,21−23 nanoparticles,24−26 and hydrogels.27,28 The highest EE of CUR in these delivery systems was up to 90.00%. Under the optimum preparation conditions of the RBA−CS nanoparticles, different CUR amounts were encapsulated into the RBA−CS nanoparticles and the optimum MRs of RBA:CS:CUR were investigated. The highest EE (93.56%) and LC (26.20%) were obtained with the MR of RBA:CS:CUR at 9:3:3 (Figure 3A). It was clear that the EE of CUR within the RBA− CS nanoparticles was higher than that of these previous delivery systems.21−28 The higher EE of the RBA−CS nanoparticles might be attributed to the higher MR of RBA, which favored the formation of inner cores with more hydrophobic space. The solubility of CUR in water and the RBA−CS nanoparticles shown in Figure 3B suggested that the solubility of CUR was greatly improved without any precipitation after loading into the RBA−CS nanoparticles. For investigation into the stability of the CUR loaded RBA−CS nanoparticles, the samples were stored at room temperature for 4 weeks. The EE and LC remained at 85.23% and 23.11% without precipitation (Figure 3B), which suggested that the CUR loaded RBA−CS nanoparticles have good stability. Morphology and Size of CUR Loaded RBA−CS Nanoparticles. The morphology of RBA−CS nanoparticles was observed by SEM and AFM. Smooth sphere morphology was observed for CUR loaded RBA−CS nanoparticles with some aggregation (Figure 4A,B). The average height of the RBA−CS nanoparticles was 26.36 and 35.90 nm before and after loading CUR (Figure 4C,D), respectively. The average size and PDI of the RBA−CS nanoparticles was 384 nm and 0.23 (Figure 4E), respectively. After loading CUR, the average size of the RBA−CS nanoparticles increased to 778 nm (Figure 4F). The reason that the average size increased might be due, in

nanoparticle solutions before and after 4 weeks storage were assessed to determine the optimum conditions for nanoparticle formation. Figure 2A showed the optical images of the RBA−CS nanoparticle solutions at different pH values from 4.0 to 7.0. The results indicated that the RBA−CS nanoparticle solutions exhibited a stable dispersion system from pH 4.0 to 5.75, and precipitation was observed when the pH value was higher than 5.75. After 4 weeks storage, the RBA−CS nanoparticle solutions still remained as a stable system without precipitation from pH 4.0 to 5.00. However, the particle size and PDI changes in pH 4.0 solutions were lower than those in pH 5.0 solutions (Figure 2B), suggesting that the nanoparticles were more stable in pH 4.0 solutions than in other pH solutions. Therefore, pH 4.0 was considered the optimum solution condition for developing the RBA−CS nanoparticles. Due to the denaturation temperature of RBA at 85.35 °C and heating treatment that can promote the self-assembly of protein, the effects of heating treatment at 80 and 90 °C at different times (10, 20, 30, and 60 min) were evaluated. The heat-treated RBA−CS nanoparticles still existed in a stable system without precipitation after 4 weeks storage (Figure 2A), and the particle size changes were lowest (from 372 to 483 nm) with an ideal PDI value (0.41) with heating at 90 °C for 60 min (Figure 2C). Therefore, 90 °C and 60 min were chosen as the ideal heating conditions for developing the RBA−CS nanoparticles. Under the optimum pH, heating treatment, and time conditions, the influence of the MR of RBA:CS on the size and PDI was also investigated. Figure 2A and Figure 2D show the optical images and particle size change produced by the MR from 5:1 to 1:5 after 4 weeks storage, respectively. The precipitations could be observed clearly for the pure RBA solution after heating treatment without adding CS. However, the RBA−CS solutions exhibited a stable dispersion system after 4 weeks storage (Figure 2A). The size change (from 384 to 389 nm) was the smallest for the RBA−CS nanoparticles at MR 3:1 (Figure 2D). However, the PDI value increased to 0.52, which may be due to some additional assembly between nanoparticles after 4 weeks storage. Therefore, pH 4.0 and MR 6610


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Figure 6. In vitro digestibility of CUR loaded RBA−CS nanoparticles: (A) SDS-PAGE (Lane 1: RBA; Lane 2: RBA−CS mixture; Lane 3: RBA−CS nanoparticles; Lines 4/6 and Lanes 5/7: RBA−CS nanoparticles and RBA−CS mixture digested in simulated gastric and simulated intestinal solutions, respectively); Size distribution of CUR loaded RBA−CS nanoparticles digested in (B) simulated gastric and (C) simulated intestinal solutions; (D) SEM of CUR loaded RBA−CS nanoparticles, (E) digestion in simulated gastric solutions for 2 h, and (F) in simulated intestinal solutions for 4 h.

After heating treatment, the hydrophobic interaction, disulfide bonds, and hydrogen bonds among RBA molecules appeared and formed the hydrophobic core. The CS chains electrostatically combined with the RBA were also trapped in the core, and the remaining unattached CS chains formed the shell on the core surface. Thus, the RBA−CS nanoparticles exhibit stable dispersion systems with core (RBA)−shell (CS chain) structure and can be used to load the hydrophobic drugs into the core to improve their solubility. Digestibility of CUR Loaded RBA−CS Nanoparticles. The digestibility of protein-based nanoparticles plays an important role for the oral delivery of drugs and nutrients. Thus, the digestibility of CUR loaded RBA−CS nanoparticles was investigated in simulated gastric and intestinal conditions using SDS-PAGE and SEM (Figure 6). It was observed that the pure RBA, simple mixture of RBA−CS, and RBA−CS nanoparticles have the same composites before digestibility. After 2 h digestion in the simulated gastric conditions with pepsin, the distinct bands of 35−40 kDa and 55 kDa were still observed for the RBA−CS nanoparticles. However, such bands were missing for the simple mixture of RBA−CS. The reduced proteolytic hydrolysis of the RBA−CS nanoparticles was attributed to the fact that CS shell could protect the RBA core from degradation by pepsin. After the following 4 h simulated intestinal digestion with pancreatin, the bands of 35− 40 kDa and 55 kDa of the RBA−CS nanoparticles were also absent, and the proteolysis of the RBA core was complete (Figure 6A). This may be due to the fact that the solubility of the CS shell was much lower in the simulated intestinal solutions, and the digestive enzymes can easily enter into the core and be rapidly degraded. The SEM and average diameter were also used to further confirm the nanoparticles digestibility. The results of Figure 7B showed that the average diameter of

part, to the formation of looser and larger hydrophobic cores after CUR loading. ζ-Potential and Pyrene Fluorescence of RBA−CS Nanoparticle. Figure 5A shows the ζ-potential of the simple RBA−CS mixture and RBA−CS nanoparticle solutions. The MR of 3:1 RBA−CS nanoparticles showed an amphoteric ζpotential curve between the curves of RBA and CS, as well as the MR of 3:1 simple mixture solutions. However, the ζpotential curve of the MR of 1:1.3 RBA−CS nanoparticles was closer to the CS ζ-potential curve. These results agreed with the previous report,36 which suggested that the surfaces of the nanoparticles were full of CS chains and RBA molecules were enriched in the nanoparticles core. For further confirmation that RBA−CS nanoparticles form a core−shell structure, pyrene was used as a fluorescence probe to characterize the nanoparticles hydrophobicity. Pyrene is very sensitive to the polarity of the microenvironment, and the intensity ratio of the first peak (I1, 372 nm) and the third peak (I3, 383 nm) in its fluorescence spectrum is always used to describe this subtle change.37 Pyrene molecules preferably locate inside or close to the hydrophobic region once the core− shell nanoparticle forms, resulting in a decreasing of the intensity ratio of the first to third band (I1/I3) in its fluorescence emission spectrum. The I1/I3 ratios of the RBA−CS nanoparticle solutions were between 1.01 and 1.33 (Figure 5B), which was much lower than the ratio of 1.9 in water, suggesting that the RBA−CS nanoparticles have core− shell structure and hydrophobic areas which exist inside the nanoparticles core.38 Figure 5C illustrates the RBA−CS nanoparticle complexforming mechanisms. The RBA and CS possess anionic patches and cationic groups at pH 4.0 solutions, respectively, and the RBA−CS complexes are formed by electrostatic attraction. 6611


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Figure 7. (A) Accumulated release; and (B) Higuchi kinetics of CUR release from RBA−CS nanoparticles in SIF, PBS, and SGF solutions, respectively; (C) Accumulated release of free CUR solutions and CUR loaded RBA−CS nanoparticles in SGF (0−2 h) and SIF (2−6 h) solutions.

Table 2. Correlation Coefficients (r2) and Release Exponent (n) of Different Models in SIF, PBS, and SGF Solutions for CUR Loaded RBA−CS Nanoparticles

nanoparticles decreased from 778 to 368.2 nm after 2 h simulated gastric digestion while still maintaining sphere morphology (Figure 6E). However, the nanoparticles shrank considerably after 4 h simulated intestinal digestion (Figure 6F), with average diameter of about 54.6 nm (Figure 6C). These results suggested that the RBA−CS nanoparticles exhibit good biodegradability in the digestive environment with the properties of delayed degradation in gastric conditions and complete digestibility in the small intestine, which gives the RBA−CS nanoparticles promising features for development of small intestine targeted delivery systems. In Vitro Release and Mechanisms of CUR Loaded RBA−CS Nanoparticles. The amount of accumulative release of CUR from the RBA−CS nanoparticles in SGF, PBS, and SIF solutions was investigated according to the previous method with some modification,39 and the results are shown in Figure 7A. The CUR release amount was about 91.86%, 77.01%, and 36.06% in SIF, PBS, and SGF solutions after 10 h, respectively, and the CUR release amount was approximately 94.60%, 81.12%, and 47.96% after 24 h. As the control, free CUR solutions showed a fast release rate with approximately 97.34% release amount after 6 h (Figure 7C). These results also suggested that the release rate of the RBA−CS nanoparticles was dependent on pH value of media conditions, with higher release rate in SIF solutions. The swelling of the CS shell on the core surface increased, with CS solubility increasing in SGF solution, leading to CS shell layers becoming increasingly thick and then hindering the SGF solution from entering into the core. Conversely, the solubility of CS was lower in SIF solutions and the shell collapsed and disintegrated, allowing SIF easily to enter into the core, causing rapid degradation with a higher CUR release rate. For investigation of the probable CUR release mechanisms from the RBA−CS nanoparticles, the release data were fitted by zero-order, first-order, Higuchi, Hixson−Crowell, and Peppas models. As shown in Table 2, the Higuchi model was the most appropriate model to fit the release kinetics of CUR from

SIF solution

PBS solution

SGF solution

Model

Stage 1

Stage 2 Stage 1 Stage 2 Stage 1 Stage 2

Zero-order (r2) First-order (r2) Higuchi (r2) Hixson−Crowell (r2) Peppas (n)

0.98 0.98 1.00 0.99

0.33 0.42 0.86 0.39

0.99 0.97 0.99 0.99

0.65 0.7 0.94 0.68

1.00 0.99 0.99 1.00

0.62 0.67 0.85 0.65

0.85

0.03

0.89

0.05

1.00

0.28

nanoparticles because of the highest r2 value. Thus, the release data were presented as the fractional release and plotted against the square root of time by using the Higuchi model (Figure 7B). These plots revealed that there were two stages for the CUR release from the RBA−CS nanoparticles. The first release stage was initially rapid (burst release), and the second release stage was slow (sustained release). The burst release may help to rapidly reach the effective concentration of CUR in plasma, whereas the sustained release would maintain the effective concentration of CUR in plasma for an extended time period. Thus, the CUR bioavailability might be improved after loading into the RBA−CS nanoparticles. The n value of the Peppas model is applied to further investigate the detailed release mechanisms for the RBA−CS nanoparticles, such as n < 0.43 for Case I Fickian diffusion, n > 0.85 for Case II transport (swelling and erosion), and 0.43 < n < 0.85 for anomalous behavior or non-Fickian transport.40 The n values for CUR release from the RBA−CS nanoparticles are listed in Table 2. The n values of the RBA−CS nanoparticles for stage 1 in the SIF and SGF solutions are all higher than 0.85, suggesting that swelling and erosion are the main release mechanisms at stage 1. However, the n value was lower than 0.43, suggesting Fickian diffusion as the main release mechanism for stage 2. These results indicated that the diffusion, swelling, and erosion release mechanisms coexisted 6612



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for the CUR release from the RBA−CS nanoparticles during the release processes. In Vitro Cytotoxicity. The cytotoxicity effects of unloaded RBA−CS nanoparticles, free CUR, and CUR loaded RBA−CS nanoparticles against Caco-2 cells were investigated. The results (Figure 8) showed that the unloaded RBA−CS nanoparticles

Research Article

AUTHOR INFORMATION

Corresponding Authors

*Tel: +86-791-86634810. E-mail address: huaxiong100@126. com (H. Xiong). *Tel: +01-479-575-2881. E-mail address: [email protected] (Y. B. Li). ORCID

Hailong Peng: 0000-0003-3150-4618 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was supported by the Planning Subject of “the Twelfth Five-Year-Plan” National Science and Technology for the Rural development of China (2013AA102203-05), the National Natural Science Foundation of China (31660482 and 21667018), and the China Scholarship Council. The authors would like to thank Lisa Kelso for her help on the review of this manuscript.

Figure 8. Cell cytotoxicity of free CUR, unloaded NPs, and CUR loaded NPs against Caco-2 cells at various concentrations. (NPs: RBA−CS nanoparticles).

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REFERENCES

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did not exhibit any effect on cell viability, indicating no toxicity of unloaded RBA−CS nanoparticles for Caco-2 cells. However, the cell viability of Caco-2 cells decreased from 92.12% to 29.56% and from 92.45% to 39.06% for CUR loaded RBA−CS nanoparticles and free CUR with concentration increasing, respectively. It was clearly observed that the cytotoxicity of CUR loaded RBA−CS nanoparticles for Caco-2 cells was significantly higher than that of free CUR, which is probably due to the significantly higher stability of CUR, the improved solubility of CUR, and subsequently the enhanced cellular uptake after loading into RBA−CS nanoparticles.

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CONCLUSIONS In this study, RBA was extracted from rice bran waste using water as a green extraction methodology, and its properties of amino acid compositions, molecule weight, secondary structure, denaturation temperature, and ζ-potential were all characterized. The results showed that RBA has the advantages of being nontoxic, having higher nutritional value, being easily digested, and exhibiting self-assembly capability with negative charges. Therefore, RBA was explored as a nontoxic and sustainable biomaterial for preparation of nanoparticles with CS by an ecofriendly method. The RBA−CS nanoparticles exhibited sphere morphology with core−shell structure. The CUR solubility was greatly improved after loading into the RBA−CS nanoparticles with higher EE (93.56%). The RBA−CS nanoparticles have good biodegradability with delayed degradability in gastric conditions and complete digestibility in the small intestine. The release of CUR from the RBA−CS nanoparticles was controlled by two release stages of burst release and sustained release. The CUR loaded RBA−CS nanoparticles showed a greater cytotoxicity than free CUR for Caco-2 cells. In summary, the renewable and green RBA−CS nanoparticles obtained from rice bran waste have great potential applications for hydrophobic active agent delivery. 6613


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