Insights into Micellization of Octenylsuccinated Oat β-Glucan and

Jun 10, 2019 - (20) The above preparation of βC-OSβG-Ms was done in dark (absence of .... Second, the dispersion of various small signals may produc...
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Article Cite This: J. Agric. Food Chem. 2019, 67, 7416−7427

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Insights into Micellization of Octenylsuccinated Oat β‑Glucan and Uptake and Controlled Release of β‑Carotene by the Resultant Micelles Zhen Wu,†,‡ Chenyang Zhao,† Ruohua Li,† Fayin Ye,† Yun Zhou,† and Guohua Zhao*,†,§ †

College of Food Science, Southwest University, Chongqing 400715, PR China Chongqing Key Laboratory of Chinese Medicine & Health Science, Chongqing Academy of Chinese Materia Medica, Chongqing 400065, PR China § Chongqing Engineering Research Center of Regional Foods, Chongqing 400715 PR China

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‡

ABSTRACT: The core−shell structured micelles from octenylsuccinated oat β-glucan (OSβG) are able to solubilize βcarotene (βC). This study reveals molecular interactions governing the formation, stabilization, and βC uptake of OSβG micelles (OSβG-Ms) by means such as water contact angle, 1H nuclear magnetic resonance, dynamic light scattering, and confocal laser scanning microscopy. The results indicated that the micellization of OSβG molecules is triggered by hydrophobic interactions between octenylsuccinate (OSA) moieties, while OSβG-Ms are stabilized via both hydrophobic interactions and hydrogen bonds. For their uptake of βC, βC molecules are first adsorbed onto OSβG-Ms by interacting with OSA moieties scattered on micelle surface. By further interacting with OSA moieties located in micelle shell, βC molecules travel across the shell and finally are trapped in the hydrophobic core. In simulated gastrointestinal fluids, βC is controlled released from OSβGMs as an integrated consequence of its diffusion as well as the swelling and erosion of OSβG-Ms. As a result, this study first uncovered the mechanism underlying the uptake of βC by OSβG-Ms, which will certainly facilitate the effective loading of hydrophobic ingredients by OSβG-Ms. KEYWORDS: β-carotene, octenylsuccinated oat β-glucan, micelles, micellization process, loading process, controlled release

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INTRODUCTION

Natural and modified polysaccharides provide green, safe, and effective vehicles for encapsulation of lipophilic bioactive compounds in food and biomedical industries.10,11 Polysaccharides-based delivery systems (PBDSs) are appropriate for various industrial applications due to their outstanding merits such as bioavailability, nontoxicity, biocompatibility, biodegradability, and modifiability.12,13 Common PBDSs include amphiphilic nanoparticles (e.g., micelles, nanospheres, nanocapsules, and polymersomes), nanogels, microparticles, complexes, and emulsions.14−16 Among various PBDSs, modified polysaccharides-based micelles systems, formed by amphiphilic molecules with self-assembled properties in water, have various merits due to their unique structures.17,18 These advantages will give researchers a new opportunity to encapsulate hydrophobic nutraceuticals in their hydrophobic core and thus improve oral delivery and bioavailability. Chang et al.17 reported that self-aggregated micelles of octenylsuccinate (OSA) modified glucan had potential applications in food and biomedical industries because they could effectively encapsulate lipophilic bioactive food compounds or poorly soluble drugs and enhance their oral delivery. Previous findings from our group have successfully prepared OSA modified oat β-glucan (OSβG).18,19 Our later work found that βC could be solubilized by OSβG micelles (OSβG-Ms),

β-Carotene (βC) is a natural bioactive compound existing in numerous yellow−green vegetables (e.g., yellow pepper, carrot, spinach, and sweet potato) and fruits (e.g., mango, pink grapefruit and peach).1,2 βC is also a coloring agent and supplement that is ubiquitously added to food. More importantly, βC is an important source of vitamin A since it can be converted into vitamin A upon metabolism in the human body. βC has several functions such as radical scavenging activities,3 antioxidant activities,4 and anticarcinogenic activities.5 βC especially inhibits the growth of several cancer cell lines.6 Together, those studies indicate that βC plays a vital role in various metabolic and developmental processes. Therefore, dietary supplementation of βC is needed to overcome the dilemma of vitamin A deficiency. However, βC is difficult to dissolve in water because of its high hydrophobicity. Even in weakly lipophilic medium, only a small percentage of βC molecules exists in its monomolecular state.7 Besides, βC is easily oxidized in the existence of air, light, and high temperature. This insolubility in aqueous environments and susceptibility to degradation are the main drawbacks that hinder its application in numerous βC-fortified foods. 8 Therefore, researchers are now applying new techniques to fortify different food systems with encapsulated βC and other carotenoids in food industry.9 Nanoencapsulating lipophilic βC into suitable delivery systems is an effective method to protect βC from oxidation and thus improve its bioavailability. © 2019 American Chemical Society

Received: Revised: Accepted: Published: 7416

November 30, 2018 June 8, 2019 June 10, 2019 June 10, 2019 DOI: 10.1021/acs.jafc.8b06645 J. Agric. Food Chem. 2019, 67, 7416−7427

Article

Journal of Agricultural and Food Chemistry leading to formation of βC-loaded OSβG-Ms (βC-OSβGMs).20 However, the formation mechanism and molecular interactions of self-assembly and βC-loaded process is still unknown. The release mechanism of loaded βC is not clear. In light of the above studies, the objectives of the current work were thus to determine the hydrophilicity of oat β-glucan (OβG), OSβG-Ms, and βC-OSβG-Ms; to investigate the formation mechanism of self-assembly of amphiphilic OSβG polymers into micelles and βC-loaded process; and to evaluate in vitro controlled release behaviors of loaded βC by applying six mathematical models. The mechanism underlying βC release to improve the construction of gastrointestinal (GI) βC delivery systems was then analyzed.

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Powder X-ray diffractometry (PXRD) was performed to illustrate the crystal patterns of powdered samples. The test was carried out on an X-ray diffractometer (Bruker, Germany) using Cu Kα radiation. The X-ray tube was generated at 45 kV and 40 mA. All samples were measured in the 2θ angle range between 5° and 40° with a scan rate of 10° min−1 and a step size of 0.02. To analysis the βC-loading process and its mechanism, the diameter and zeta potential of βC-loading OSβG-Ms solution were investigated using a dynamic light scattering (DLS) detector (Malvern Instruments Ltd., UK) equipped with a He−Ne-laser (633 nm and scattering angle 90°). All measurements were performed at room temperature. The surface tension (SFT) measurement was obtained using a surface tension instrument (A101, USA KINO Industry CO. Ltd., NY, USA) by the Wilhelmy plate method at room temperature. Prior to each SFT test, the plate was cleaned up with distilled water and flamed to remove interferences. The SFT of pure water (72.0 mN m−1) was determined to calibrate the tensiometer and to guarantee the cleanliness of the glassware. The distribution of βC in OSβG-Ms aqueous solution was recorded by confocal laser scanning microscopy (CLSM) (LSM800, Zeiss, Germany) due to the fluorescent nature of βC. The excitation wavelength and emission wavelength for βC was fixed at 488 and 560 nm, respectively.22 All the samples were observed with a fixed micellization time interval. The morphology of unloaded micelles and βC-loaded micelles was observed using an atomic force microscopy (AFM) (Bruker Corporation, Camarillo, CA, USA). For specimen preparation, aliquots (2 μL) of each sample solution were pipetted onto a freshly cleaved mica surface, followed by natural air-drying at room temperature. The AFM experiment was done in an intermittent tapping mode. In Vitro Controlled Release Measurements. The kinetic release behavior of βC from βC-OSβG-Ms was assessed in simulated GI environments using a dialysis based method. Simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) were prepared according to the method reported by Wang et al.23 with minor modifications. Briefly, SGF (pH 2.0, 3.2 mg mL−1 pepsin solution) and SIF (pH 7.0, 10 mg mL−1 pancreatin solution) were mixed with equal volume of ethanol to prepare the release media, respectively. An aliquot (5 mL) of βC-OSβG-Ms solution was added in dialysis bag, and it was sealed tightly with clamps. The sealed bag was immersed into 30 mL of SGF for 120 min and then immediately moved to 30 mL of SIF for another 240 min at 37.5 °C under continuous magnetic stirring (100 rpm). During dialysis process, an aliquot (3 mL) of solution outside dialysis bag was sampled at preset time points and equal volume of corresponding fresh medium was supplemented. The dialysis test was performed in triplicate in dark (absence of light). The amount of released βC was determined by UV−vis method at 450 nm.24 The regression equation was fitted to y = 0.3447x − 0.0097 (R2 = 0.999). To analyze the mechanism that controlled the release kinetic process of βC from βC-OSβG-Ms, the following six models were applied. (1) Zero-order model:

MATERIALS AND METHODS

Materials. Oat β-glucan (OβG) was obtained from Zhangjiakou Yikang Biological Technology Co., Ltd. (Hebei, China), which had a β-glucan content of 80% and Mw of 1.68 × 105 g moL−1. 2-Octen-1ylsuccinic anhydride, all-trans β-carotene (98.0%, purity), deuterium oxide (D2O, 100% purity, 99.9 atom % D), sodium deuteroxide (99.5%, basicity 40%), deuterium chloride (DCl 20% in D2O), and deuterated dimethyl sulfoxide (d-DMSO, 100% purity, 99.96 atom % D) were purchased from Adamas Reagent Co. (Shanghai, China). KBr was purchased from Guangfu Reagent Co., Ltd. (Tianjin, China). Hydrochloric acid (HCl), sodium hydroxide (NaOH), and ethanol were purchased from Kelong Reagent Co., Ltd. (Chengdu, China). Pepsin (P-7000) and pancreatin (P-1500) were bought from Sigma− Aldrich, Inc. (St. Louis, MO, USA). The dialysis bag (molecular weight cutoff 12 kDa) was purchased from Union Carbide (USA). Preparation of OSβG, OSβG-Ms, and βC-OSβG-Ms. OSβG polymer and the self-aggregated micelles of OSβG were prepared according to our previous study.18 To obtain a satisfied βC-OSβGMs, ten milligrams βC was dissolved in 20 mL of 2.0 mg mL−1 OSβGMs solution, which was then quickly homogenized at 15 000 r min−1 for 1 min and 12 000 r min−1 for 1 min using a high-speed blender (IKA T18, Germany). After 24 h of stirring at 30 °C, βC was encapsulated by OSβG-Ms. Afterward, βC-OSβG-Ms were obtained by centrifuging the homogenate at 5000 g for 10 min. The final βCOSβG-Ms powder was collected by freeze-drying at −60 °C for 12 h.20 The above preparation of βC-OSβG-Ms was done in dark (absence of light). Instrumental Characterizations of OSβG, OSβG-Ms, and βCOSβG-Ms. Water contact angle (WCA) was measured by a Sessile drop method.21 The silicon surfaces were cleaned with acetone before each test. For specimen preparation, aliquots (50 μL) of each sample solution (2 mg mL−1) were dropped on a clean silicon wafer, followed by drying under room temperature. Newly prepared (t = 3 s) and after 60 s equilibrium, the microscopic measurement was observed with a WCA goniometer (Rame-Hart, USA). To record 1H nuclear magnetic resonance (NMR) spectra, all samples were deuterium-exchanged in D2O by continuous freezedrying three times. Each pretreated sample was first dissolved in D2O or d-DMSO (1 mL) at 30 °C for 24 h with continuous stirring. It was then transferred into an NMR glass tube. 1H NMR spectra were recorded with a Bruker Avance 600 spectrometer (Rheinstetten, Germany) at room temperature. The 1H NMR data were recognized by MestReNova software. To determine ultraviolet visible (UV−vis) absorption features of all samples, βC was dispersed in ethanol by successive stirring until complete solubilization, while OSβG-Ms and βC-OSβG-Ms were dispersed in pure water. UV−vis spectra were obtained using an UV2450 spectrometer (Shimadzu, Japan) with the scanning wavelength 300−650 nm. Fourier transform-infrared (FT-IR) spectra were acquired using a Spectrum 100 FT-IR detector (PerkinElmer, USA). All samples were ground with KBr to obtain thin pellets. The scanning wavelength was between 4000 and 400 cm−1 with 32 times at a resolution of 4 cm−1.

Q t = k0 × t + Q 0

(1)

where Qt and Q0 is the cumulative release rate of βC at interval t (min) and the initial amount of βC, respectively (the same below). k0 is the zero-order kinetic constant. (2) First-order model: Q t = Q 0 × e kt

(2)

where k is the first-order kinetic constant. (3) Higuchi model: Q t = kH × t 1/2

(3)

where kH is the Higuchi kinetic constant. This model showed that drug release process was consistent with the Fick’s law.25 (4) Hixson-Crowell model: 7417

DOI: 10.1021/acs.jafc.8b06645 J. Agric. Food Chem. 2019, 67, 7416−7427

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Figure 1. Water contact angle (WCA) of oat β-glucan (OβG), octenylsuccinated oat β-glucan micelles (OSβG-Ms), and β-carotene loaded OSβGMs (βC-OSβG-Ms). (A) Typical shape of a water droplet on the film surface of OβG, OSβG-Ms, and βC-OSβG-Ms. (B) Speculative model illustrating water wettability of the film surface of OβG, OSβG-Ms, and βC-OSβG-Ms. (C) Change in WCA for OβG, OSβG-Ms, and βC-OSβGMs. Different lower case letters indicate a significant difference between the values newly prepared and equilibrated sample within a specific sample (p < 0.05). Q 01/3 − Q t1/3 = kHC × t

The fitting was done by Origin 8.5 software (Systat Software Inc., USA). The obtained parameters such as adjusted coefficient of determination (R2adj) and root−mean−square error (RMSE) were used to evaluate the possible mechanism of βC release process. Statistical Analysis. All measurements were performed three times. Comparison of means was done by analysis of variance with least significant difference test (SPSS, version 15).

(4)

where kHC is the release constant.26 (5) Korsmeyer-Peppas model:

Q t = kKP × t n

(5)

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where kKP and n are the typical kinetic constant and exponent, respectively. In this model, n ≤ 0.45 indicates Fickian diffusion. Intermediate n values (0.45−0.89) represent anomalous (nonFickian) diffusion. n = 0.89 and n > 0.89 indicate Case II transport and super Case II transport, respectively.27,28 (6) Weibull model: Q t = 1 − exp[− ((t − tlag)b )/tscale]

RESULTS AND DISCUSSION Hydrophilicity of OβG, OSβG-Ms, and βC-OSβG-Ms. The hydrophilicity of the amphiphilic molecules showed an important effect on their micellar processes.30 In the current study, the hydrophilicity of OβG, OSβG-Ms, and βC-OSβGMs as well as their transformation during equilibration was evaluated in terms of WCA (Figure 1). Obviously, the native OβG is of hydrophilic nature and thus presents a relatively low WCA, approximately 53.3° (newly prepared WCA) and 39.4° (equilibrated) (Figure 1A). After the introduction of OSA moieties, the resultant modified OSβG-Ms displayed a higher

(6)

where tlag defines the lag time before the onset of the βC dissolution process. tscale defines the time scale of the βC dissolution process. The characteristic parameter b represents the shape of the release curve as exponential (b = 1), sigmoid (S-shaped, b > 1), or parabolic (b < 1).29 For the analysis of mathematical equations, the βC release data were plotted as Qt (%) as a function of the βC release time (min). 7418

DOI: 10.1021/acs.jafc.8b06645 J. Agric. Food Chem. 2019, 67, 7416−7427

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Figure 2. 1H NMR spectra of (A) octenylsuccinated oat β-glucan (OSβG) polymer at a concentration of 0.030 mg mL−1 (below CMC) and 25 °C, using D2O as deuterated solvents, (B) OSβG micelles (OSβG-Ms) using D2O as deuterated solvents, (C) pure β-carotene (βC), (D) βC-loaded OSβG-Ms (βC-OSβG-Ms) using d-DMSO as deuterated solvents, and (E) βC-OSβG-Ms using D2O as deuterated solvents. The gray solid line and blue-dashed line indicate the proton of octenylsuccinate and oat β-glucan (OβG) block, respectively. The dotted line indicates the proton of βC.

the WCA value of water onto the film formed on the plate depends on both chemical composition and morphology of the surfaces.31 In addition, the initial WCA solely depended on the surface hydrophilicity of a film substrate, while the changes of WCA during equilibration reflected the wettability of the film. The WCA of OβG decreases in a 60 s period due to the combined effects of surface diffusion and penetration of the

WCA of 72.3° (equilibrated), which implied the modification confers OβG with hydrophobicity and resulting in amphiphilic OSβG-Ms. However, unlike native OβG, the WCA of OSβGMs remained unchanged upon equilibration (Figure 1C). More interestingly, the initial WCA of βC-OSβG-Ms was comparable to that of OSβG-Ms, but it tended to decrease during the equilibration as it did by native OβG. In principle, 7419

DOI: 10.1021/acs.jafc.8b06645 J. Agric. Food Chem. 2019, 67, 7416−7427

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Figure 3. (A) UV−vis and (B) FT-IR spectra of (a) octenylsuccinated oat β-glucan micelles (OSβG-Ms), (b) pure β-carotene (βC), (c) βC-loaded OSβG-Ms (βC-OSβG-Ms), and (d) physical mixtures (PMs) of βC and OSβG-Ms (1:1, molar ratio).

droplet into the OβG film,32 which indicates the high wettability of OβG. On the basis of Wenzel’s equation, a micro/nano rougher surface tended to be more hydrophilic for the amphiphilic molecules.33 Much rough surface was formed caused by the self-aggregated OSβG-Ms (this phenomenon could also be found from AFM results, as will be shown later). However, the WCA of OSβG-Ms was higher than that of OβG and maintained steadily during equilibration. The speculative models in Figure 1B illustrated the surface formation of OβG, OSβG-Ms, and βC-OSβG-Ms. As depicted in Figure 1B, the possible reason might be that the OSA moieties of amphiphilic OSβG molecules were not completely oriented to the hydrophobic core, along with few part projecting onto the surface, and this lowered the hydrophilicity and wettability of the film surface of OSβG-Ms. Obviously, loading of βC could improve the wettability of OSβG-Ms surface. It might be adduced that after the loading process, the strong interaction between βC and hydrophobic segments of OSβG-Ms would be the primary driving force for octenyl residues migrating toward and completely orientating to the core of OSβG-Ms, resulting in a strong hydrophilic film surface. This was a coinciding process of oriented aggregation and encapsulation triggered by hydrophobic forces.34 Additionally, due to the availability of more hydrogen bonds that could interact with the drop of water, the hydrophilicity of shell surface of βC-OSβG-Ms could be improved. It was also confirmed that the loading of βC took place in the core of the micelles and not in the outer layers. Mechanism Underlying the Micellization of OSβG. 1H NMR studies have been widely applied to systematically explore the position and orientation of amphiphilic polymers during their micellar processes.35 The 1H chemical shifts were affected by the micellization behavior and self-assembled microstructure, which revealed the change in the distance between the polar group of OSβG molecules and the micellar conformation in OSβG-Ms.36 Moreover, the proton resonance variations were exceedingly useful to explain the micellar growth of OSβG molecules. The assignment of signals from protons of OSβG polymer was indicated in Figure 2A. The H1 of 4G3 residue of OβG moieties was masked by the strong signal of D2O at 4.70 ppm. The groups of overlapped signals at 4.46 ppm were attributed to the H1 of 3G4 and 4G4 OβG residues. The peaks in the 1H NMR spectra were recognized

based on previous literature.37,38 The OSβG molecules could self-assemble to form stable micelles when OSβG molecules concentration exceeded its critical micelle concentration (CMC).18 The 1H NMR spectra of OSβG polymer before and after micellar formation were shown in Figure 2A and B, respectively. The 1H NMR bands of OβG block started to broaden, and the signals corresponding to hydrophobic OSA block protons (h−j) in D2O solution decreased but did not disappear after micellization, which indicated rearrangement of OβG block and restricted mobility of OSA block. 39 Commonly, broad NMR resonances can be due to the following three reasons.40 First, the molecular motion is restrained by a rigid constricted microenvironment. Second, the dispersion of various small signals may produce an obvious large signal. Third, the conformation change of protons involved in chemical exchange may influence their signals. For OβG moieties, the first reason seemed more likely, implying that micellization increased the mobility of those protons belonging to same OβG moieties. Moreover, the 1H NMR signals of OSβG polymer shifted significantly toward high magnetic field, which was crucial to the formation of OSβG-Ms. Before micellization, the protons of OSA moieties were exposed to the bulk water and experienced a polar environment; with the formation of micelles, OSA chains were strongly bound to the hydrophobic core through hydrophobic effects. In other words, those protons moved from a polar media to an apolar media, and therefore, the proton signals shifted notably highfield.36 As shown in Figure 2A, the summary area ratio of peakOβG‑block to peakOSA‑block was 1:1.03. The ratio changed to about 1:0.37 (Figure 2B) when the OSA chains self-aggregated into hydrophobic core, which suggested the formation of micelles. Accordingly, it was assumed that the OSA moieties near the micellar core were largely masked and as a result showed the proton signals at lower chemical shift values (signals for a−j protons). Thus, 1H NMR analysis evidently revealed the core−shell structure of OSβG-Ms with hydrophobic domains in the core and hydrophilic domains in the shell in aqueous phase. The micellar structure was further elucidated by UV−vis and FT-IR spectra. As illustrated in Figure 3A (trace a), no absorption was observed for OSβG-Ms. As shown in Figure 3B (trace a), the typical absorption peaks of OSβG-Ms were 7420

DOI: 10.1021/acs.jafc.8b06645 J. Agric. Food Chem. 2019, 67, 7416−7427

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Journal of Agricultural and Food Chemistry shown at 3434 cm−1 (−OH stretching), 2926 cm−1 (−CH stretching), and 1640 cm−1 (−OH bending). Specifically, the FT-IR of OSβG-Ms was characterized by the presence of signals at 1727 cm−1 (C=O stretching vibration) and 1574 cm−1 (RCOO− stretching vibration), which proved that OSA modified reaction was successful.41 The broad peak at around 3434 cm−1 was due to the hydrogen-bonded hydroxyl. Therefore, the stabilization of OSβG-Ms was maintained by both hydrophobic effects of OSA moieties and hydrogen bond of OβG moieties. PXRD was applied to investigate the physical state of OSβG-Ms during self-assembly and later lyophillization. As shown in Figure 4a, OSβG-Ms were amorphous

the strong hydrophobic interactions resulting in the tight encapsulation. Compared with free OSβG molecules and OSβG-Ms, after βC loading the summary area ratio of peakOβG‑block to peakOSA‑block is 1:0.28 and that is due to continuous aggregation of the hydrophobic OSA chain in an orderly manner to form stable βC-loaded micelles. This is supported by the WCA observations. Table 1 shows the Table 1. Chemical Shift Values (δ) of Octenylsuccinated Oat β-Glucan Micelles (OSβG-Ms) and β-Carotene Loaded OSβG-Ms (βC-OSβG-Ms) at 25 °C chemical shift (ppm) OSβG-Ms protons a

H-1 H-2 H-(3−5)b H-6 H-a H-(b−d) H-e H-(f−g) H-(h−j)

δOSβG‑Ms

δβC‑OSβG‑Ms

Δδc

4.416 3.252 3.603−3.457 3.859 0.765 1.221−1.100 1.902 5.562−5.373 2.348−2.109

4.409 3.249 3.593−3.454 3.857 0.759 1.217−1.109 1.901 5.539−5.360 2.347−2.117

−0.007d −0.003 −0.007 −0.002 −0.006 −0.013 −0.001 −0.010 −0.009

a

Numbers or letters for protons in OSβG molecule follow the notation in Figure 2. bConsidered as a multiplet. cΔδ = δβC‑OSβG‑Ms − δOSβG‑Ms. The difference is calculated on the average point of each multiplet. d−, upfield shift.

chemical shift changes of OSβG-Ms and βC-OSβG-Ms based on 1H NMR investigations. According to the data, all the OSβG-Ms peaks exist in βC-loaded micelles spectrum, but due to the formation of βC-loaded micelles, their signals are shifted. The UV−vis study was conducted to investigate the impact of the loaded βC molecules on their aggregated structures in aqueous solution, which could assist us to understand the supramolecular structure changes of βC carriers in a microscopic perspective. Figure 3A (trace b) showed the UV−vis spectrum of βC. The structure of βC resulted in a strong absorption with peaks at 450 and 480 nm, which was similar to ref 43. In Figure 3A (trace c), the two typical absorption peaks of βC-OSβG-Ms were observed at 505 and 550 nm, which were characteristic of βC. In contrast, βC incorporated into OSβG-Ms underwent a red shift compared to βC in ethanol. The UV−vis spectrum of physical mixtures (PMs) (Figure 3A, trace d) was different from that of βC; specifically, the first maximum absorbance was found to have shifted slightly from 450 to 435 nm, which might be explained by the following assembling behaviors. Generally, the blue shift of UV−vis absorption peak of βC molecule is caused by the “H-type” aggregates and the red shift is due to the “J-type” aggregates.7 The formation of “H-type” or “J-type” aggregates is due to the capacity of βC to form hydrogen bonding between conjugated chains.44 The existence of free hydroxyl groups at both sides of βC molecule is crucial for the production of the “H-type” aggregate. The spectral features described above give evidence for the existence of domains of regularly J-aggregated type molecules after the loading of βC. This can be explained that the strong hydrophobic interactions of βC and OSβG-Ms prevent the formation of more hydrogen bonds. Moreover, the inner cavity of OSβG-Ms has a mass of carboxyl groups of OSA moieties, which is an electron-withdrawing group to βC; this makes hydrogen bonds unstable and lack of the driving

Figure 4. Powder X-ray diffractometry (PXRD) of (a) octenylsuccinated oat β-glucan micelles (OSβG-Ms), (b) pure β-carotene (βC), (c) βC-loaded OSβG-Ms (βC-OSβG-Ms), (d) physical mixtures (PMs) of βC and OSβG-Ms (1:1, molar ratio), and (e) superposition of measured diffractograms.

because there was no typical signals of crystalline portions, which suggested that crystal was not formed at any micellar process or during lyophillization as crystalline form might impact its delivery and bioavailability. Mechanism Relating to the Loading of βC by OSβGMs. Next, we will discuss the formation process of βC-loaded micelles. A typical 1H NMR spectrum of βC (Figure 2C) obtained is very similar to the reported spectra.42 As shown in Figure 2C, the methyl groups 16/16′ and 17/17′ of βC molecule show a characteristic proton peak at 1.02 ppm, methyl groups 18/18′ a proton peak at 1.69 ppm, and methyl groups 19/19′ and 20/20′ a proton peak at 2.26 ppm.42 1H NMR spectrum of βC-OSβG-Ms (Figure 2D) contains multiple proton resonance peaks of βC in the region between 1.00 and 2.35 ppm. The spectrum also contains characteristic βC proton peaks (e.g., characteristic methyl groups 14/14′ around 6.16 ppm) (Figure 2D). However, for D2O as a deuterated solvent, these βC proton peaks completely disappear (Figure 2E). This is due plausibly to suppressed molecular motion of the aggregated hydrophobic chains and 7421

DOI: 10.1021/acs.jafc.8b06645 J. Agric. Food Chem. 2019, 67, 7416−7427

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Figure 5. (A) Particle size, (B) zeta potential, and (C) surface tension (SFT) of β-carotene (βC) loaded octenylsuccinated oat β-glucan micelles (βC-OSβG-Ms) prepared by direct stirred dissolution method as a function of micellization time. Confocal laser scanning microscopy (CLSM) images of βC-loading OSβG-Ms solution stirred for (D) 3 and (E) 24 h. The white arrows denote red-colored βC-loaded system. (F) Schematic of morphological illustration of βC loading by octenylsuccinated oat β-glucan micelles (OSβG-Ms) driven by unoriented octenylsuccinate moieties in aqueous solution. Graphical depictions show that the free and unoriented octenylsuccinate moieties pull βC molecules into the hydrophobic inner core of OSβG-Ms. OβG, oat β-glucan; OSA, octenylsuccinate.

force to form the “H-type” aggregates. Similarly, Cao-Hoang, Fougère, and Waché45 reported that βC could form micrometric-uncharged J-aggregates in Tween-80 micelles. Ribeiro et al.46 found that when βC molecule was incorporated into polymer carrier systems in the absence of any oily core material, this system possessed the protective ability for the βC-enriched nanodispersion with high stabilization. Additionally, different molecular aggregates of βC might impact its stability and bioactivity.45 These results indicated that OSβGMs as a solubilizer improved the aqueous solubility of βC and formed the micellization of βC, which might penetrate into OSβG-Ms cavities by hydrophobic interactions. After βC loading, the hydrophilicity of βC greatly improved. The results agreed well with those WCA observations. Such change might facilitate the application of OSβG-Ms in food and biomedical industries. To further probe the possible macromolecular structure and interactions between βC and OSβG-Ms, FT-IR studies were also used to analyze the molecular packing in βC-loaded

micelles. For βC (Figure 3B, trace b), the FT-IR spectrum was characterized by principal peaks at 3029 cm−1 (for C=C stretching), 2924 and 2866 cm −1 (for aliphatic CH 2 asymmetric and symmetric stretching), 1628 cm−1 (for aromatic ring stretching), 1562 cm−1 (for C=C bending), and 966 cm−1 (for trans-conjugated alkene CH out of plane deformation mode).47 As Figure 3B (trace c) shows, such characteristic peaks of βC almost disappeared in βC-OSβGMs, which suggested that βC was entrapped into the micelles. Evidently, no significant change was observed in the absorption band between 3200 and 3500 cm−1, which suggested no obvious variation of hydrogen bonds after loading of βC. Furthermore, the hydrophobic chains led to the steric hindrance, which prevented the formation of intra- and intermolecular hydrogen bonds. This further supported the “J-type” aggregates of loaded βC stated above. Therefore, the formation of βC-OSβG-Ms was driven by hydrophobic effects, following the assistance of hydrogen bonding between carbohydrate backbone shells. 7422

DOI: 10.1021/acs.jafc.8b06645 J. Agric. Food Chem. 2019, 67, 7416−7427

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Figure 6. Atomic force microscope (AFM) images of (a) octenylsuccinated oat β-glucan micelles (OSβG-Ms) and (b) β-carotene loaded OSβGMs (βC-OSβG-Ms).

of βC-OSβG-Ms. The macromolecular interaction of βC with OSβG-Ms decreased crystalline state and aggregated size of βC efficiently. This will be more beneficial for the delivery and uptake than pure βC, probably because the micellar form provides significantly more bioavailability for βC than its crystal form,49 which we will verify in the discussion section on βC release. Additionally, the diffractogram of PMs (Figure 4d) showed weak intensity of the peaks at 14.66°, 15.36°, 18.50°, and 24.44° at 2θ scale, which revealed that the βC signals were not completely obscured when blended with OSβG-Ms. The crystalline structure of βC was still present in PMs. To further understand the loading growth of βC-OSβG-Ms, DLS and SFT experiments were carried out to follow the slow βC loading process. As shown in Figure 5A, increasing size was observed over stirring time. Further increase of stirring time beyond 10 h did not significantly increase particle size, and after 13 h the particle size started to remain stable. The zeta potential was widely used to reveal the nanoparticle delivery systems stabilities controlled by the sum of repulsive and attractive forces.50 Figure 5B exhibited the effect of stirring time on zeta potential of βC-loaded micelles. The zeta potential of the system decreased rapidly within the first 10 h because of a large number of βC as well as free OSA moieties binding into the hydrophobic inner core, leading to formation of the stable micelles. Similarly, the SFT generally decreased with the increase of stirring time until it reached equilibrium (Figure 5C). Because the adsorption of βC by exterior surface OSA moieties and βC diffusion into hydrophobic core was a slow process, almost all unoriented OSA moieties adsorption sites of OSβG-Ms were occupied and translated to the hydrophobic inner core; hence, the surface layer of βC-loaded OSβG-Ms aqueous solution provided completely oriented molecular arrangement, which resulted in an abrupt decrease

Generally, the methylene (−CH2−) chains inside micelles core are almost disordered.48 After the loading of βC, their intensities decreased, which indicated the highly ordered state of hydrocarbon chain aggregates of OSβG-Ms. Besides this, the characteristic absorption peaks of stretching vibration of −COOH and C=O were not found in the spectrum of βCloaded micelles, as compared with that in the spectrum of OSβG-Ms. The reason probably is that the formation of βCloaded micelles accompanied by completely oriented OSA moieties leads to the diminished signals of −COOH and C=O as mentioned above. Additionally, compared to βC-OSβG-Ms, the characteristic peaks of βC were detected in PMs (Figure 3B, trace d). These findings revealed that βC molecule was slowly entrapped into hydrophobic cores during the βC loading process. As a result, FT-IR spectroscopy of βC-OSβGMs only presented the characteristic bands of OβG moieties, which formed the stable OβG shell and shielded the bands of the hydrophobic core contained by OSA moieties and loaded βC. In all, these results demonstrated that hydrophobic interaction between βC and OSA moieties was the main driving forces for accelerating βC-OSβG-Ms formation. PXRD was applied to investigate crystalline nature of βC after being entrapped into OSβG-Ms because this would impact its release from the micellar formulations. The diffractogram of βC (Figure 4b) proved a crystalline pattern with prominent sharp peaks at 2θ equal to 11.76°, 14.58°, 15.40°, 16.58°, 18.80°, 21.80°, and 24.50°. This was in good agreement with the reported literature.34 In contrast, βCOSβG-Ms were characterized by the complete absence of any diffraction peak (Figure 4c) corresponding to βC, which indicated βC entrapment within the micelles and aqueous βCloaded nanomicellar formulation in an amorphous state. The amorphous form βC may have crucial impact on the formation 7423

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Journal of Agricultural and Food Chemistry of surface tension. Typically, the incorporation of lipophilic compounds into micellar hydrophobic core can be interpreted in two ways: (i) the dissociation and arrangement of micelles leading to a more stable aggregation processes (demicellization−transfer−micellization); (ii) adsorption by free hydrophobic segment and translation to micellar hydrophobic core driven by strong hydrophobic effects. For the growth of βC-loaded OSβG-Ms, the second possibility seems more likely, implying that adding βC molecules are attracted by the unoriented OSA moieties in OSβG-Ms aqueous solution (as stated in WCA and NMR sections) and driven by strong hydrophobic effects. This result is also confirmed by CLSM image of βC-loading OSβG-Ms solution stirred for 3 and 24 h (Figure 5D,E). After stirring for 3 h, a small part of βC was adsorbed and incorporated into the hydrophobic core (Figure 5D). Then the majority of βC was translated into the core following a long period of stirring (Figure 5E). The above results suggested that βC was gradually incorporated inside core of OSβG-Ms. As illustrated in Figure 5F, the βC incorporation happens via available OSA chains, which pull βC molecule into the hydrophobic inner core of OSβG-Ms. Consequently, the micelles may become swollen. A combination of DLS, SFT, and CLSM offers a particularly useful way for exploring the micellar process of βC loading and can help in understanding its mechanism. To obtain a clear understanding on micellar morphology, AFM was applied to investigate the conformations of unloaded micelles and βC-loaded OSβG-Ms in aqueous solution. The three-dimensional and two-dimensional AFM images (Figure 6) showed a dramatic difference in size and monodispersity before and after βC-loading. As presented in Figure 6, the average micelle sizes of unloaded micelles and βC-OSβG-Ms were obtained as about 140 and 598 nm, respectively. βCOSβG-Ms showed a homogeneous and smooth spherical morphology (Figure 6b), while OSβG-Ms exhibited several sharp and cone shapes (Figure 6a). AFM proved that βCloaded micelles were in a more swollen state, and more hydrophobic chains joined the micelles to keep the micellar stability by increased hydrophobic interactions between βC and OSA chain. Similarly, the enlarged micelle size was observed after loading βC by chitosan-graf t-poly(lactide) micelles.4 It could be explained by the fact that with the introduction of βC, the OSA blocks forming the hydrophobic core were more stretched and the interaction between OβG main chains forming the hydrophilic corona was reduced.4 It was of interest to compare these findings with the morphology of curcumin-loaded OSβG-Ms. Our previous studies found the decreased size of OSβG-Ms after curcumin incorporation.19 This converse phenomenon might be due to the chemical nature of lipophilic ingredients (e.g., polarity and hydrophobicity/hydrophilicity) as well as their intra- and intermolecular interactions.51 The hydrogen bonds and hydrophobic interactions might both be the main forces to entrap curcumin into micelles. Additionally, it was likely to strengthen OSβG−OSβG and OSβG−curcumin interactions via intermolecular π−π stacking interactions of phenyl moieties and hydrogen bonds, resulting in more stable and compact curcumin-loaded micelles. Mechanism Governing the in Vitro Release of βC from OSβG-Ms. Modeling is an effective way to elucidate the mechanism of micellar carrier systems in controlled releasing active ingredients.52 The controlled release profile of βC encapsulated in OSβG-Ms was depicted in Figure 7. Around

Figure 7. In vitro controlled release profile and fitting curves obtained using the different mathematical models for β-carotene (βC) loaded octenylsuccinated oat β-glucan micelles (βC-OSβG-Ms) in the simulated gastric fluid (SGF, 0−120 min) and simulated intestinal fluid (SIF, 120−360 min) at 37.5 ± 0.1 °C.

32.24% of initial loaded βC was released in the SGF within the first 120 min and only 18.82% diffused into SIF during later 240 min dialysis. Interestingly, an initial rapid and burst βC release (12.49%) within the initial 20 min was observed in SGF and followed with a plateau (20−80 min), and then the accumulative βC release increased gradually, while the βC release percentage grew quickly during the initial SIF stage (120−180 min). Afterward, the release rate slowed down and remained stable. There were at least two possible explanations. First, the protonation of the carboxyl segments at pH 2.0 might drastically change micelles structure, leading to the shrinkage of hydrophilic shells of OSβG-Ms and triggering βC release.18 Second, at pH 7.0, the micelle size might increase because of the deprotonation of the carboxyl segments and the increasing hydrophilicity of OβG segments, resulting in the swelling of the micelles.19 However, the swollen micelles exhibited a fair stability, which was reflected by the slow increase of cumulative βC release rate in later SIF stage. Generally, the maximal accumulated release rate used for all classical mathematical models was 60%.29 To overcome the limitation of single mathematical modeling and to achieve a satisfactory prediction, the six models were performed to interpret the βC release from the core−shell OSβG-Ms in different perspectives (Figure 7). The fitting results were listed in Table 2. The fitting results showed that the βC release data fit very poorly to the Hixson-Crowell model, which indicated that this model could not be used to describe the βC release kinetic. Valuable information on the mechanism of the βC release was obtained from the Korsmeyer-Peppas and Weibull models. As a result, a n value of 0.5451 was obtained for Krosmeyer-Peppas model, which located between 0.45 and 0.89 and revealed that βC release from OSβG-Ms was governed mainly by anomalous (non-Fickian) diffusion.27 Moreover, the βC release data fit well with the Weibull model, as it presented a highest R2adj (0.9792) and lowest RMSE (2.180) among all models. In this model, b value, characterized the diffusion curve, indicated the controlled release mechanism of βC transport within the core−shell OSβG-Ms. The result showed that b value was greater than 1.0, which suggested that βC release from OSβG-Ms in simulated GI environment was controlled by a complex mechanism including non-Fickian diffusion, swelling (induced by relaxation of OSA and OβG moieties), and erosion. The βC release process was not a reverse version of its loading into the micelles. The diffusion of 7424

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molecules and their uptake mechanism of βC may help us to control the related processes and generate novel food materials via the interaction between food components. In light of the present results, the temperature dependence of the micellization of OSβG molecules and their uptake mechanism of βC should be addressed in further studies due to the fact that thermal treatment is necessary for a lot of food products.

Table 2. Release Constants and Correlation Coefficient of Different Mathematic Equations Applied to β-Carotene (βC) Release from βC-Loaded Octenylsuccinated Oat βGlucan Micelles (βC-OSβG-Ms)a model

R2adj

RMSE

model parameters

zero-order first-order Higuchi Hixson-Crowell Korsmeyer-Peppas Weibull

0.8422 0.7075 0.9283 0.7503 0.8968 0.9792

6.522 8.881 4.562 8.206 4.619 2.180

k0 = 0.1395 k = 0.0034 kH = 2.931 kHC = 0.0039 kKP = 2.299 n = 0.5451 b = 12.75

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +86 23 68 25 03 74. Fax: +86 68 25 19 47.

a 2

R adj represents the adjusted correlation coefficient obtained from the release curves according to different mathematic models. RMSE is root−mean−square error. The parameters of k0, k, kH, kHC, kKP, and b represent the release rate constants of the different models. The parameter n is the release exponent applied to the Korsmeyer-Peppas model.

ORCID

Zhen Wu: 0000-0002-1047-1856 Guohua Zhao: 0000-0002-2870-0549 Notes

The authors declare no competing financial interest.

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loaded βC from hydrophobic core to surface was a slow process, which might be the key step of βC release process. In addition, it was known that erosion always occurred along with the swelling action, especially in an acidic environment.53 For anticancer chemicals, their accelerated release in an acidic environment was preferred due to the fact that most tumor tissues presented acidic microenvironments.54 The burst release of loaded βC from βC-OSβG-Ms is helpful to attain an effective concentration, and the continuous release could provide an effective dose for a long time and thus improve the uptake efficiency of βC.2 Notably, even at the end of the test, βC was not completely released in this study. This incomplete release would be due to the stable hydrophobic interaction between βC and OSA moieties of OSβG-Ms. A previous investigation reported that the release rate of lutein from the polymeric micelles was higher at acidic condition compared to that at neutral condition due to the pH-triggered de-crosslinking and micelle disassembly.55 The environment dependence of release properties of core−shell OSβG-Ms warranted their future applications in oral delivering βC or other lipophilic bioactive food compounds. Although the release of vitamin D3 from chitosan derivative-based micelles was characterized with an initial burst stage connected to a sustained stage, its mechanism was well described by Super Case II (Higuchi model).2 Previous studies indicated that βC-loaded nanoparticles were rarely released in SGF but almost completely released in SIF, which facilitated βC absorption in the small intestine and protected it from being destroyed during oral delivery.47 Recently, researchers have devoted to improve the solubility, stability, and efficacy of carotenoids through different strategies such as incorporation of carotenoids into foods by using micelles. Sáiz-Abajo et al.9 proved that micelles could protect βC against degradation during the most common industrial treatments. Moreover, Xu et al.56 reported that mixed micelles stabilized lycopene by preventing isomerization and degradation in cell culture media. Therefore, to select optimal biocompatible and biodegradable polymers is a critical task in constructing polymeric core−shell micelles with expected characters. In summary, the mechanism relating to the micellization formation of OSβG molecules in aqueous solution and their loading of βC as well as the controlled release were deeply analyzed. The clarification of the micellization of OSβG

ACKNOWLEDGMENTS We acknowledge support from the National Natural Science Foundation of China (Grant Nos. 31771932 and 31371737) and Chongqing Science and Technology Bureau (Grant No. cstc2019jcyj-msxm1936).

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ABBREVIATIONS USED βC, β-carotene; PBDSs, polysaccharide-based delivery systems; OβG, oat β-glucan; OSA, octenylsuccinate; OSβG, octenylsuccinated oat β-glucan; OSβG-Ms, octenylsuccinated oat βglucan micelles; βC-OSβG-Ms, βC-loaded octenylsuccinated oat β-glucan micelles; PMs, physical mixtures; WCA, water contact angle; 1H NMR, 1H-nuclear magnetic resonance; UV− vis, ultraviolet visible; FT-IR, Fourier transform-infrared; PXRD, powder X-ray diffractometry; DLS, dynamic light scattering; SFT, surface tension; CLSM, confocal laser scanning microscopy; AFM, atomic force microscopy; GI, gastrointestinal; SGF, simulated gastric fluid; SIF, simulated intestinal fluid; CMC, critical micelle concentration.

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