Liposome-chitosan hydrogel bead delivery system for the encapsulation of linseed oil and quercetin: Preparation and in vitro characterization studies

LWT - Food Science and Technology 117 (2020) 108615

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Liposome-chitosan hydrogel bead delivery system for the encapsulation of linseed oil and quercetin: Preparation and in vitro characterization studies

T

Juan Huanga,c,e, Qiang Wangd, Lanling Chub, Qiang Xiae,∗ a

School of Biology and Food Engineering, Changshu Institute of Technology, Changshu, China Faculty of Food Science and Engineering, School of Light Industry and Food Engineering, Nanjing Forestry University, Nanjing, China c College of Biosystems Engineering and Food Science, Zhejiang University, Hangzhou, China d Collaborative Innovation Center of Tissue Repair Material of Sichuan Province, College of Life Sciences, China West Normal University, Nanchong, China e School of Biological Science and Medical Engineering, State Key Laboratory of Bioelectronics, Southeast University, Nanjing, China b

A R T I C LE I N FO

A B S T R A C T

Keywords: ω-3 polyunsaturated fatty acids Quercetin Liposome Chitosan Ionic crosslinking

This study concentrates on developing linseed oil and quercetin co-loaded liposomes-chitosan hydrogel beads to enhance their stability and solubility. Scanning electron microscope (SEM) analysis revealed that the constructed hydrogel beads had three-dimensional network structure and liposomes were entrapped evenly within polymer network. X-ray diffraction (XRD) study illustrated that quercetin was in an amorphous or dissolved form in hydrogel beads. Thermodynamic properties of chitosan and soybean lecithin with phosphatidyl cholines of 60% (PC60) changed after ion crosslinking. After loaded into hydrogel beads, the release of quercetin and ɑ-linolenic acid became slow and can be explained by Higuchi model which indicated that bioactives were released from the skeleton of the hydrogel beads by the mechanism of Fick diffusion. The in vitro simulated digestion study showed that hydrogel beads could improve the stability of liposomes in gastrointestinal tract and inhibit the rapid release of fatty acids. The accelerated oxidation and photostability studies showed that the chemical stability of functional bioactives could be improved by loading liposomes into hydrogel beads. Hence, chitosan hydrogel bead is an attractive candidate for the encapsulation of functional bioactives for use in food industry.

1. Introduction Linseed oil and quercetin are lipophilic nutrients, which have many health-promoting effects, including promoting brain function and development, improvement of cardiovascular health, coping with inflammatory disorders and reducing risk for cancer (Kajla, Sharma, & Sood, 2015; Wang et al., 2016). Due to their poor chemical stability, low solubility in aqueous media and extremely poor oral bioavailability, their application in hydrophilic food systems is limited (Li et al., 2016; Piornos, Burgos-Díaz, Morales, Rubilar, & Acevedo, 2017). Various carrier systems have been used to encapsulate these compounds to enhance their stability, solubility, and hence, bioavailability, including emulsions, nanostructured lipid carriers, solid lipid nanoparticles, liposome and micelles (Nakajima, Wang, Chaudhry, Park, & Juneja, 2015). Liposome are concentric vesicles composed of phospholipid bilayer, and their structure are similar to cell membranes. Compared with other carrier systems (emulsions, nanostructured lipid carriers, solid lipid

nanoparticles et al.), liposomes are made up of biocompatibility materials which leads to low toxicity (Shin, Kim, & Park, 2015). Besides, liposomes could encapsulate of multi-components, improve the solubility of lipophilic nutrients, extend the circulation lifetime and hence increase the bioavailability of the core active ingredients (Maurer, Fenske, & Cullis, 2001; Torchilin, 2007). Hence, liposome acts as an alternative delivery system to others. Liposomes have been extensively studied for the delivery of unstable active ingredients such as vitamins, flavors, antioxidants and antimicrobials (Fathi, Mozafari, & Mohebbi, 2012). However, the application of liposomes in functional foods is limited due to their poor storage stability, easy oxidation of phospholipids, low encapsulation efficiency and short release time (Kamaly, Xiao, Valencia, Radovic-Moreno, & Farokhzad, 2012). The filled hydrogel bead delivery system is particularly useful to overcome these issues. Hydrogel beads are swellable polymer networks which own the hydrophilic functionalities on the polymer gel backbone. This propriety gives the hydrogel beads the ability to imbibe a substantial amount of fluids (Ahmed, 2015). Besides, the crosslinks

∗ Corresponding author. School of Biological Science and Medical Engineering, State Key Laboratory of Bioelectronics, Southeast University, Nanjing, 210096, China. E-mail address: [email protected] (Q. Xia).

https://doi.org/10.1016/j.lwt.2019.108615 Received 3 July 2019; Received in revised form 25 August 2019; Accepted 10 September 2019 Available online 11 September 2019 0023-6438/ © 2019 Elsevier Ltd. All rights reserved.

LWT - Food Science and Technology 117 (2020) 108615

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obtain liposomes with incorporated linseed oil and quercetin.

between polymer chains can render hydrogel beads resistant to dissolution (Ahmed, 2015). Using filled hydrogel beads as food-grade colloidal delivery systems for the encapsulation of various lipophilic bioactives is a promising formulation approach to improve their water dispersibility, chemical stability, and bioavailability characteristics (Kosaraju, Weerakkody, & Augustin, 2009; Liang, Line, Remondetto, & Subirade, 2010). The combination of liposome and hydrogel can avoid their disadvantages and integrate advantages. In addition, more designable release kinetics of lipophilic bioactives could be achieved by liposomes in hydrogel beads compared with that by hydrogels or liposomes alone. Chitosan is non-toxic, biodegradable and biocompatible natural polysaccharide (Illum, 1998), which is generally regarded as safe (GRAS) by FDA. Chitosan gel is especially suitable for oral applications due to its properties of mucoadhesivity (Bhattarai, Gunn, & Zhang, 2010), bacteriostaticity (Jumaa, Furkert, & Muller, 2002) and improvement in transport across biological barriers (Mooren, Berthold, Domschke, & Kreuter, 1998). Chitosan hydrogels have been mainly studied for wound-healing (Obara et al., 2003), cartilage tissue engineering (Montembault et al., 2006), pharmaceutical delivery applications as well as oral delivery of food bioactives. Inspired by these knowledges, the aim of this study was to fabricate hydrogel beads containing liposomes with incorporated linseed oil and quercetin. Such liposome/hydrogel beads are completely made up of natural polymers which can meet the demand of label friendliness for customers. The characterization of physicochemical properties, accelerated oxidation stability and in vitro digestion were then presented. Finally, an investigation about the release of quercetin and ɑ-linolenic acid encapsulated in liposomes and hydrogel beads was described and discussed. Liposome filled hydrogel beads will provide valuable information for the incorporation of nutritional bioactives into functional food products.

2.3. Chitosan bead preparation Chitosan beads were fabricated by a modified injection-gelation method according to Yuan, Jacquier, & O'Riordan (2018). Chitosan hydrochloride was added to the above liposomes to obtain a final concentration of 2.5% w/w. The above mixture was stirred until all the chitosan hydrochloride dissolved and then placed in ultrasound power (80 W, 5 min) to eliminate air bubbles. The chitosan solutions were then injected into a 1.5% (w/w) sodium tripolyphosphate solution drop by drop using a syringe with a diameter of 0.6 mm. The tip of the syringe is 15 cm from the surface of the sodium tripolyphosphate solution. The chitosan beads were then incubated in the phosphate solution for 30 min at room temperature to ensure sufficient crosslinking to occur. Subsequently, the chitosan beads were washed with double distilled water to remove any excess phosphate ions. At this point, the wet hydrogel beads have been prepared. Finally, the wet hydrogel beads were dried in a vacuum drying oven until constant weight. 2.4. Determination of particle size

2. Materials and methods

The particle sizes of liposome and hydrogel beads were measured by dynamic light scatting (DLS) technology and Imagine J software, respectively. DLS technology was applied to measure the particle size of liposome with a particle size analyzer (Malvern Zetasizer ZS90, Malvern Instruments, UK). The measurement was conducted with a scattering angle of 90° at 25 °C. The samples were diluted with distilled water prior to measurements. 20 hydrogel beads were randomly selected and distributed on a scaled paper uniformly. Photograph of the paper was taken and the particle sizes of hydrogel beads were measured by Imagine J software. Finally, the average particle size of hydrogel beads was calculated.

2.1. Materials

2.5. Morphological analysis

Cold-pressed linseed oil with a ɑ-linolenic acid content of 57% was produced by Ta Foods Co. Ltd (Canada). Quercetin (99%) were purchased from Shaanxi Kingsci Biotechnology Co. Ltd (China). PC60 was provided by Beijing Yuan Hua Mei Lecithin Sci-Tech Co. Ltd (Beijing, China). Chitosan hydrochloride with a degree of deacetylation of 91.5% and a molecular weight of 20 000 was brought from Zhengzhou Gebbers Food Additive Company Ltd (Zhengzhou, China). Sodium tripolyphosphate was purchased from Shandong Qinye Chemical Co. Ltd (Jinan, Shandong). All the ingredients were food grade. Cumene hydroperoxide (85%) was supplied by Adamas Reagent Co. Ltd (Shanghai, China). 1,1,3,3-Tetraethoxypropane (97%) was obtained from Shanghai Macklin Biochemical Co. Ltd (Shanghai, China). 2Thiobarbituric acid (≥98.5%) was brought from Sinopharm Chemical Reagent Co. Ltd (Shanghai, China). Pepsin (Sigma-Aldrich, USA), lipase (Ruibio, Germany) and cholic acid salt (Amresco, USA) were used. All other chemical reagents were of analytical grade with no further purification.

A scanning electron microscope (SEM, Ultra Plus, Zeiss, Germany) was applied to study the structural feature of hydrogel bead with modified method of Torres, Murray, and Sarkar (2017). The hydrogel bead was washed with hexane removing all oil excipients. After removal of the oil, the deliberately fractured particles were fixed onto a chrome coated steel plate with a double-sided carbon conductive adhesive tape. The SEM images were then obtained by an In-len secondary electron detector (GEMINI). 2.6. Quercetin content Quercetin in liposome and hydrogel beads was extracted by ethanol. The extracted solutions were filtered by a 0.45 μm nylon syringe filter and measured at 374 nm by an UV spectrophotometer (755B, JINGHUA, Shanghai, China). Before extraction, hydrogel beads need to be crushed with a mortar. 2.7. ɑ-linolenic acid content

2.2. Liposome preparation ɑ-linolenic acid in liposomes and hydrogel beads was extracted by n-hexyl alcohol. The extracted solutions were filtered by a 0.45 μm nylon syringe filter and measured at 300 nm by an UV spectrophotometer (Chen et al., 2017). Before extraction, hydrogel beads need to be crushed with a mortar.

Linseed oil and quercetin loaded liposomes were prepared using the ethanol injection method described previously with some modifications (Balanč, Trifković; Ðorđević, Marković, Pjanović, Nedović, & Bugarski, 2016). In brief, 1 g of linseed oil, 200 mg of quercetin, 4 g of PC 60 and small quantity of ethanol were stirred at 50 °C until all the crystals disappeared. Then the ethanol solution was incorporated into 94.8 g of distilled water drop by drop at the same temperature with agitation (500 rpm). After that, the mixture was stirred at 50 °C for 1 h with speed of 500 rpm. Finally, the solution was cooled at room temperature to

2.8. XRD The crystal state of quercetin in hydrogel beads was analyzed by Xray powder diffraction (D8 Discover, Bruker Corporation, Germany). 2

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0.3 M CaCl2 solution prepared by simulated intestinal digestive solution were added. After being adjusted to pH 7.0, 5 mL of pancreatin solution (800 U/mL) fabricated with simulated intestinal digestive solution was added into the above mixture. At this point, the hydrolysis of linseed oil started and a pH-stat titration method was used to control the pH and keep it at constant pH 7.0 by titrating 0.01 M NaOH solution into the mixture for 2 h at 37 °C. The volumes of NaOH solution consumed were recorded during the incubation period at different time intervals.

Diffraction patterns of quercetin, physical mixture and hydrogel beads were recorded over a range of 2θ angles from 5° to 60° with speed of 0.15 s/step and a scan step size of 0.02°. Physical mixture was composed of PC60, quercetin, linseed oil and chitosan. Samples were irradiated by Cu-Ka radiation at a 30 mA current, and a voltage of 40 kV. 2.9. DSC analyses The thermodynamic property of hydrogel beads was evaluated by differential scanning calorimetry (DSC) analyses. The DSC curves were determined with a PerkinElmer DSC calorimeter (DSC8000, Waltham, MA, USA). The samples (about 10 mg) were hermetically sealed in an aluminum pan with an empty pan sealed as the reference. The sample was heated from 20 to 400 °C at a rate of 10 °C/min under nitrogen.

2.12. Accelerated oxidation study of linseed oil The oxidation stabilities of linseed oil in liposome and hydrogel beads were compared by accelerated oxidation experiment. Samples were putted in transparent bottles and maintained in the oven without light under 15 days. The oxidation stability of linseed oil was investigated by measuring peroxides as primary oxidation products and thiobarbituric acid reactive substances as second oxidation products. Before measurement, dry hydrogel beads need to be powdered in a mortar. Peroxide values (PV) were measured by the method with slight modifications (Huang, Wang, Li, Xia, & Xia, 2018a). Approximately 0.5 mL of liposome and equal amount of dry hydrogel beads were incorporated into 1.6 mL of solvent composed of isooctane-2-propanol (3:1, v/v). Ultrasound was used to extract peroxide. The above mixture was vigorously vortexed three times and centrifugated for 10 min at 633 × g. 0.2 mL of upper solvent was added into 2.8 mL of methanol–1butanol mixed solvent (2:1, v/v). Soon after, 15 μL of ferrous iron solution and 15 μL of 3.94 M ammonium thiocyanate were blended with the above mixture. The ferrous iron solution was freshly fabricated by reacting equal amounts of 0.144 M FeSO4 and 0.132 M BaCl2 solution in 0.4 M HCl. The final solution (3 mL) was vortexed and maintained in the dark at room temperature for 20 min. The absorbance was determined at 510 nm by a UV spectrophotometer and peroxide values were calculated based on the standard curve of cumene hydroperoxide. Thiobarbituric acid reactive substances (TBARs) were determined according to the method reported by Qiu et al. with some modifications (Qiu, Zhao, Decker, & McClements, 2015). 0.375 g of TBA and 15 g of trichloroacetic acid were dissolved into 82.9 mL of distilled water to be TBA reagent. Liposome and dry hydrogel bead with 20 mg of linseed oil were added into the TBA reagent to keep the total volume at 4 mL in a tube. Lately, the tube was vortexed and putted in an ultrasound cleaner to extract the total malondialdehyde. Soon after, the tube was placed in a boiling water bath for 15 min. The mixture was cooled down in an ice water bath and centrifuged at 1000 × g for 15 min. Finally, the absorbance was determined at 532 nm by a UV spectrophotometer. TBARs values were calculated according to the calibration curve of 1, 1, 3, 3tetraethoxypropane.

2.10. In vitro release study The in vitro release behaviors of quercetin and ɑ-linolenic acid in liposome and hydrogel beads were performed by dialysis bag method. Samples were placed in dialysis bags with Molecular Weight Cut Off 12000–14000 respectively. The mixture composed of ethanol and distilled water (35:65, v/v) was applied as the dissolution medium. The dialysis bag was immersed in a beaker containing 200 mL of dissolution medium (37 °C) stirred at 100 rpm 1 mL of dissolution medium was withdrawn and analyzed by UV at 374 nm (quercetin) and 300 nm (ɑlinolenic acid) at predetermined time intervals. An equal volume of fresh pre-warmed medium was added to compensate for the loss due to sampling immediately. Kinetics of quercetin and ɑ-linolenic acid released from hydrogel beads were examined based on the magnitude of correlation coefficients obtained after application of zero order (1), first order (2), Higuchi (3), Rigter-Peppas (4) and Weibull (5) models applying the following set of equations:

Mt = at + b Mi

(1)

M Ln ⎛1 − t ⎞ = at + b Mi ⎠ ⎝

(2)

Mt = at 1/2 + b Mi

(3)

M Ln ⎛ t ⎞ = aLnt + b ⎝ Mi ⎠

(4)

M Log ⎡−Ln ⎛1 − t ⎞ ⎤ = aLogt + b ⎢ Mi ⎠ ⎥ ⎝ ⎦ ⎣

(5)

⎜

⎜

⎟

⎟

⎜

⎟

2.11. In vitro simulated digestive study 2.13. Photostability of quercetin The in vitro digestive behaviors of liposome and hydrogel beads were evaluated by a two-step experiment according to the reported method with some modifications (Huang, Wang, Li, Xia, & Xia, 2018b). Simulated stomach and intestinal digestive solutions were fabricated according to the method reported by Minekus et al. (2014). 10 mL of liposome and equal amount of hydrogel beads were added to the simulated stomach digestive solution to keep the total volume fixed at 17.5 mL. Soon afterwards, 5 μL of 0.3 M CaCl2 solution and 1.6 mL of pepsin solution (25000 U/mL) prepared by simulated stomach digestive solution were incorporated. Finally, after being adjusted to pH 3.0, the mixture was incubated at 37 °C for 2 h with continuous stirring at 100 rpm. After stomach digestion step, 11 mL of simulated intestinal digestive solution was incorporated into the above stomach digestive solution. Soon after, 2.5 mL of bile extract solution (69 mg/mL) and 40 μL of

The chemical stability of quercetin against ultraviolet light was assessed by sunshine exposure. Both liposome and hydrogel beads were exposed to sunlight. The samples were withdrawn at fixed time interval and diluted for the quantitation of quercetin.

2.14. Statistical analysis All the experiments were carried out in triplicate, and values were expressed as average. Statistical significance was determined by analysis of variance (ANOVA) and Student's t-test and a value of p < 0.05 was considered to be statistically significant.

3

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Table 1 Particle size, quercetin and α-linolenic acid contents of liposomes and hydrogel beads. Formulation Liposomes Hydrogel bead (Wet) Hydrogel bead (Dry)

Particle Size

Quercetin content (%) a

262.2 ± 2.5 nm 1760 ± 112 μm 1063 ± 105 μm

0.158 ± 0.003 0.194 ± 0.010b 2.280 ± 0.015c

α-linolenic acid content (%) 0.804 ± 0.012a 0.986 ± 0.037b 11.407 ± 0.052c

Different letters in the same column indicate statistically significant differences (p < 0.05) of quercetin and α-linolenic acid contents. Results are displayed as mean ± SD (n = 3).

3. Results and discussion

uniformly distributed throughout the chitosan matrix. Generally, several factors may influence the structural property of hydrogel beads prepared by the injection method, including biopolymer concentration, crosslinking agent concentration, hardening time, needle diameter and injection flow rate. The above results indicate that the preparation conditions of 2.5% chitosan, 1.5% sodium tripolyphosphate, 30 min of hardening time, needle diameter 0.6 mm and injection distance 15 cm were suitable for the preparation of hydrogel beads with stable structure. The quercetin and ɑ-linolenic acid contents of liposomes and hydrogel beads were different (Table 1). After ionic crosslinking, the contents of quercetin and ɑ-linolenic acid were significantly increased (p < 0.05). Ion crosslinking is a process of dynamic equilibrium. The outer layer of the hydrogel beads was first crosslinked, and then the phosphate ions entered the interior of the hydrogel beads, while water and a small amount of liposomes emerged from the hydrogel beads. Since the size of liposomes was much bigger than that of water, the emerging rate of water from the hydrogel beads was greater than that of liposomes, which might result in higher concentration of quercetin after crosslinking. Wang et al. also found that the drug loading of the hydrogel beads increased rapidly with the increase of loading time from 0 to 90 min (Wang, Zhang, & Wang, 2009). After hydrogel beads were dried, the contents of quercetin and ɑ-linolenic acid were also significantly increased due to the removal of moisture.

3.1. Physicochemical characterizations of liposomes and hydrogel beads In this study, lvinseed oil and quercetin co-loaded liposomes and hydrogel beads were fabricated. The hydrogel beads were prepared by injection of a mixture of liposomes and chitosan hydrochloride solution’ into a sodium tripolyphosphate solution. The hydrogel beads fabricated by this method had a smooth surface with a spherical shape (Figs. 1), and had diameters between 1.5 and 2 mm (Table 1). Upon drying the particle size of hydrogel beads seemed to have reduced by 50% (Table 1). Besides, no surface indentations could be observed suggesting that drying did not lead to uneven shrinkage of the hydrogel beads. Therefore, after drying, the hydrogel beads can still maintain their initial internal structure. The SEM images gave further understanding of the inner structure of hydrogel beads as well as the oil distribution inside the beads. After hydrogel beads were dissected, the uneven structure was observed and lipids were adsorbed on the surface of hydrogel beads (Fig. 1A–B). Hexane had been used previously to wash away all the oil droplets within the hydrogel beads. Fig. 1D shows the chitosan network (white) around the empty holes where the liposomes previously resided (darker colour). The white chitosan layer noticed around the empty holes proved that the liposomes were physically bound to the chitosan gel matrix. The enlarged photo also indicated that the liposomes were

Fig. 1. Scanning electron micrographs of hydrogel bead particle (A), inner distribution of oil droplets within hydrogel beads (B), hexane washed hydrogel bead particle (C) and inner distribution of oil droplets within hexane washed hydrogel beads (D). 4

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Fig. 2. XRD patterns of quercetin, physical mixture and hydrogel beads.

3.2. XRD XRD experiment was carried out to analyze the crystalline state of quercetin in hydrogel beads. From Fig. 2, we can observe strong characteristic peaks of quercetin at 6.2°, 10.8°, 12.5°, 15.8°, 16.2°, 17.9°, 23.9°, 24.8° and 27.4° which indicated the high crystalline state of pure quercetin. Meanwhile, the characteristic peaks of quercetin were found in the XRD spectra of physical mixture. Due to the low concentration of quercetin in physical mixture, the intensity of characteristic peaks was weaker than that of pure quercetin. However, there was no characteristic peak of quercetin in the XRD spectra of hydrogel beads, which meant that quercetin in hydrogel beads was in molecular dispersion or soluble state.

Fig. 3. DSC thermograms of physical mixture and hydrogel beads.

indicated that the three-dimensional network structure of the hydrogel beads could delay the release of bioactives and improve its absorption and utilization. At the end of 8 h, quercetin was released from the wet and dry hydrogel beads by 28.2 ± 2.0% and 26.0 ± 1.8% respectively. The drying process could reduce the release of quercetin to some extent, but the difference was not significant (p > 0.05). The release profile of αlinolenic acid was similar with that of quercetin. The above results showed that drying had little effect on the release of bioactives from hydrogel beads. Five kinetic models were applied to fit the release data of quercetin and α-linolenic acid from hydrogel beads. The release mechanisms of quercetin and α-linolenic acid from hydrogel beads were further investigated. The linear correlation coefficient (r2) of fitting equation is shown in Table 2. Based on the linearity values, the Higuchi model showed the highest linearity for hydrogel beads. Hence, in vitro release behavior of chitosan hydrogel beads can be explained by Higuchi equation model. For Higuchi model, the release mechanism can be explained as Fickian diffusion. Therefore, quercetin and α-linolenic acid were released from the skeleton of the hydrogel beads by the mechanism of Fick diffusion.

3.3. DSC The melting behaviors of PC60 and chitosan were examined by DSC which was helpful to understand the physical state of liposomes in hydrogel beads. The DSC thermograms of physical mixture (composed of PC60 and chitosan) and hydrogel beads are shown in Fig. 3. Physical mixture performed two sharp endothermic peaks at 252.25 °C and 278.04 °C which indicated the crystalline transition of solid-to-liquid. When PC60 and chitosan were formulated in hydrogel beads, these two endothermic peaks disappeared which illustrated that the thermodynamic properties of chitosan and PC60 in hydrogel beads changed after ionic cross-linking. This may be due to the electrostatic adsorption interaction between chitosan and sodium tripolyphosphate. In addition, the amino group of chitosan and phosphate group of PC60 may also interact by electrostatic adsorption. The above two reasons may lead to the change of thermodynamic properties of chitosan and PC60 after ion cross-linking. 3.4. In vitro release study The cumulative release percentages of quercetin and α-linolenic acid from different formulations were represented in Fig. 4. The cumulative release percentage of quercetin from liposomes was 65.3 ± 1.3% in 8 h. However, after loaded in chitosan hydrogel beads, the release rate and degree of quercetin slowed down. Quercetin in wet and dry hydrogel beads released only 28.2 ± 2.0% and 26.0 ± 1.8%. The release of hydrogel beads showed a two-stage pattern. Due to the fact that some of the liposomes were absorbed around or on the surface of the hydrogel beads, the resistance to release was small, and quercetin was released fast in the initial stage. With the prolongation of release time, the release rate of quercetin gradually slowed down. Similar to the release curve of quercetin, α-linolenic acid loaded in hydrogel beads also showed significant sustained release effect. The above result

3.5. In vitro simulated digestion experiment Photographs of liposomes and hydrogel beads after simulated digestion of stomach and intestine are shown in Figs. 2. The stability of liposomes in simulated gastrointestinal digestive solution was poor. Flocculation instability occurred after simulated stomach digestion, and stratification occurred after simulated intestinal digestion. These phenomena were not benefit for the digestion and absorption of bioactives in liposomes, and could reduce the oral bioavailability of bioactives. After loaded in hydrogel beads, these instability phenomena did not occur. Figs. 3 is the general appearance of hydrogel beads after 5

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Fig. 4. (A) Quercetin release profiles of liposome, wet and dry hydrogel beads. (B) α-linolenic acid release profiles of liposome, wet and dry hydrogel beads. Results are expressed as mean ± SD (n = 3).

Table 2 The release kinetics parameters (r2) of wet and dry hydrogel beads. Kinetics mode

Zero-order First-order Higuchi Rigter-Peppas Weibull

α-linolenic acid

Quercetin Wet

Dry

Wet

Dry

0.9425 0.9593 0.9926 0.8904 0.9038

0.9136 0.9351 0.9804 0.7757 0.7929

0.9221 0.9421 0.9842 0.9044 0.9181

0.9376 0.9511 0.9779 0.9570 0.9644

simulated intestinal digestion. Hydrogel beads retained complete skeleton structure after simulated gastrointestinal digestion, which brought beneficial conditions for the slow release of bioactives. After simulated intestinal digestion, the particle size of wet hydrogel beads changed from 1760 ± 112 μm to 1482 ± 113 μm (Fig. 5). The particle size became smaller significantly. This may be due to the dissolution of the liposomes from the hydrogel beads, which made the position of the liposomes vacant and the hydrogel beads shrank. The particle size of dry hydrogel beads changed from 1063 ± 105 μm to 1408 ± 94 μm (Fig. 5). After digestion by simulated gastrointestinal tract, there was no significant difference between the particle size of dry hydrogel beads and that of wet hydrogel beads (p > 0.05). A pH titration method was applied to evaluate the effect of delivery system type on the rate and extent of lipid digestion. The volume of NaOH that had to be titrated into the digestive solution to keep a constant pH (7.0) can represent the free fatty acids (FFAs) released from liposomes and hydrogel beads during the intestine digestion. The free oil droplets were rapidly digested during the initial 20 min, followed by a more gradual release for the remainder of the incubation time (Fig. 6). The rapid initial digestion rate of lipids in liposomes can be attributed to the large specific surface area of lipids exposed to lipase. After loaded in hydrogel beads, the release rate of lipids slowed down and the total

Fig. 6. NaOH consumptions of liposome, wet and dry hydrogel beads during the intestinal digestion for 2 h. Results are displayed as mean ± SD (n = 3).

free fatty acids released at the end of digestion decreased. There were many possible reasons for the decrease of lipid digestion rate after loaded in hydrogel beads. First, lipase molecules must be adsorbed onto the surface of oil droplets to begin lipid digestion (Singh, Ye, & Horne, 2009). For liposomes, lipase molecules can easily be adsorbed on the surface of lipids and promote their digestion. However, when liposomes were loaded in hydrogel beads, lipase molecules must penetrate the polymer network structure of the hydrogel beads before reaching the lipid surface, which slowed down the digestion. Secondly, the layer of long-chain fatty acids accumulated on the surface of the droplet can inhibit the contact between lipase and lipid inside the droplet, which also slows down digestion (Li, Hu, Yumin, Xiao, & Mcclements, 2011). Generally, long-chain fatty acids do not accumulate on the droplet surface. They are dissolved in mixed micelles or formed as insoluble salts with calcium ions (Devraj et al., 2013). For hydrogel beads, calcium ions and mixed micelles cannot easily penetrate through the crosslinked polymer network inside the hydrogel beads. Thirdly, mixed micelles and insoluble salts with free fatty acids must leave the surface of droplets after lipid digestion. When liposomes were loaded in hydrogel beads, this process was also delayed. Compared with the wet state, the digestion rate and degree of lipid in the dry hydrogel beads decreased to a certain extent, but the decrease was small and the difference was not significant. This result is consistent with the phenomenon of 3.4, which indicated that the drying process has little effect on the release of bioactives from hydrogel beads.

3.6. Accelerated oxidation study of linseed oil The PV and TBARs values of linseed oil in liposomes and chitosan hydrogel beads during accelerated oxidation study are shown in Fig. 7A and B. The peroxide and malondialdehyde levels of linseed oil in

Fig. 5. Particle size of wet and dry hydrogel beads after in vitro digestion experiment (initial and intestine). Results are expressed as mean ± SD (n = 3). 6

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Fig. 7. (A) PV values of linseed oil liposomes, linseed oil hydrogel beads and linseed oil + quercetin hydrogel beads during the storage of 15 days at 55 °C. (B) TBARs of linseed oil liposomes, linseed oil hydrogel beads and linseed oil + quercetin hydrogel beads during the storage of 15 days at 55 °C. Results are displayed as mean ± SD (n = 3).

4. Conclusion In the present study, linseed oil and chitosan co-loaded liposomechitosan hydrogel beads were successfully fabricated by the injectiongelation method. The prepared spheres had obvious three-dimensional network structure, which indicated the successful fabrication of the chitosan hydrogel beads. The binding effect of chitosan hydrogel beads enhanced the physical stability of liposomes in the gastrointestinal tract, delayed the release of functional bioactives in liposomes, and eventually increased the oral bioavailability. At the same time, due to the encapsulation of chitosan hydrogel beads, the contact between the oxygen in the air and bioactives in liposomes was isolated, which improved the chemical stability of the bioactives. This study has provided a novel and effective strategy for the delivery of quercetin and linseed oil, and laid a foundation for the future application in functional food development. Funding

Fig. 8. Quercetin remained in liposomes and hydrogel beads exposed to natural sunlight for 6 months. Results are displayed as mean ± SD (n = 3).

This work was supported by Prospective Applied Research Project of Suzhou (SYG201829) and the National Key Research and Development Program of China (2017YFD0400704).

liposomes increased sharply, which indicated that the unsaturated fatty acids underwent serious oxidation. However, the PV and TBARs values of linseed oil in hydrogel beads increased slightly. Compared with liposomes, the oxidation products of hydrogel beads decreased significantly. These results indicated that the oxidation of bioactives can be slowed down by loading liposomes into chitosan hydrogel beads. This was due to the fact that second encapsulation isolated the contact between liposome and oxygen, and had a good protective effect on the stability of the bioactives. In the presence of quercetin, there was little change in the PV or TBARS values of hydrogel beads throughout storage, indicating that quercetin can protect linseed oil against oxidation.

Declarations of interest None. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.lwt.2019.108615. References

3.7. Photostability of quercetin

Ahmed, E. M. (2015). Hydrogel: Preparation, characterization, and applications: A review. Journal of Advanced Research, 6, 105–121. Balanč, B., Trifković, K., Ðorđević, V., Marković, S., Pjanović, R., Nedović, V., et al. (2016). Novel resveratrol delivery systems based on alginate-sucrose and alginatechitosan microbeads containing liposomes. Food Hydrocolloids, 61, 832–842. Bhattarai, N., Gunn, J., & Zhang, M. (2010). Chitosan-based hydrogels for controlled, localized drug delivery. Advanced Drug Delivery Reviews, 62, 83–99. Chen, B., Hou, M., Zhang, B., Liu, T., Guo, Y., Dang, L., et al. (2017). Enhancement of the solubility and antioxidant capacity of α-linolenic acid using an oil in water microemulsion. Food & Function, 8, 2792–2802. Devraj, R., Williams, H. D., Warren, D. B., Mullertz, A., Porter, C. J. H., & Pouton, C. W. (2013). In vitro digestion testing of lipid-based delivery systems: Calcium ions combine with fatty acids liberated from triglyceride rich lipid solutions to form soaps and reduce the solubilization capacity of colloidal digestion products. International Journal of Pharmaceutics, 441, 323–333. Fathi, M., Mozafari, M. R., & Mohebbi, M. (2012). Nanoencapsulation of food ingredients

The results of photostability of quercetin are shown in Fig. 8. The retention rate of quercetin in liposomes was 75.21 ± 2.06% after 6 months of storage under natural sunlight. At the same time, the retention rate of quercetin in hydrogel beads was 89.75 ± 2.21% after 6 months storage. The stability of quercetin in hydrogel beads was better than that of liposomes. The possible reason was that liposomes encapsulated in hydrogel beads inhibited the contact of liposomes with oxygen in aqueous solution and ultraviolet light in air. The results of photostability study showed that the chemical stability of the bioactives could be improved by loading the liposomes into hydrogel beads.

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Mooren, F. C., Berthold, A., Domschke, W., & Kreuter, J. (1998). Influence of chitosan microspheres on the transport of prednisolone sodium phosphate across HT 29 cell monolayers. Pharmaceutical Research, 15, 58–65. Nakajima, M., Wang, Z., Chaudhry, Q., Park, H. J., & Juneja, L. R. (2015). Nano-scienceengeering-technology applications to food and nutrition. Journal of Nutritional Science & Vitaminology, 61, S180–S182. Obara, K., Ishihara, M., Ishizuka, T., Fujita, M., Ozeki, Y., Maehara, T., et al. (2003). Photocrosslinkable chitosan hydrogel containing fibroblast growth factor-2 stimulates wound healing in healing-impaired db/db mice. Biomaterials, 24, 3437–3444. Piornos, J. A., Burgos-Díaz, C., Morales, E., Rubilar, M., & Acevedo, F. (2017). Highly efficient encapsulation of linseed oil into alginate/lupin protein beads: Optimization of the emulsion formulation. Food Hydrocolloids, 63, 139–148. Qiu, C., Zhao, M., Decker, E. A., & McClements, D. J. (2015). Influence of protein type on oxidation and digestibility of fish oil-in-water emulsions: Gliadin, caseinate, and whey protein. Food Chemistry, 175, 249–257. Shin, G. H., Kim, J. T., & Park, H. J. (2015). Recent developments in nanoformulations of lipophilic functional foods. Trends in Food Science & Technology, 46, 144–157. Singh, H., Ye, A., & Horne, D. (2009). Structuring food emulsions in the gastrointestinal tract to modify lipid digestion. Progress in Lipid Research, 48, 92–100. Torchilin, V. P. (2007). Targeted pharmaceutical nanocarriers for cancer therapy and imaging. The AAPS Journal, 9, E128–E147. Torres, O., Murray, B., & Sarkar, A. (2017). Design of novel emulsion microgel particles of tuneable size. Food Hydrocolloids, 71, 47–59. Wang, W., Sun, C., Mao, L., Ma, P., Liu, F., Yang, J., et al. (2016). The biological activities, chemical stability, metabolism and delivery systems of quercetin: A review. Trends in Food Science & Technology, 56, 21–38. Wang, Q., Zhang, J., & Wang, A. (2009). Preparation and characterization of a novel pHsensitive chitosan-g-poly (acrylic acid)/attapulgite/sodium alginate composite hydrogel bead for controlled release of diclofenac sodium. Carbohydrate Polymers, 78, 731–737. Yuan, D., Jacquier, J. C., & O'Riordan, E. D. (2018). Entrapment of proteins and peptides in chitosan-polyphosphoric acid hydrogel beads: A new approach to achieve both high entrapment efficiency and controlled in vitro release. Food Chemistry, 239, 1200–1209.

using lipid based delivery systems. Trends in Food Science & Technology, 23, 13–27. Huang, J., Wang, Q., Li, T., Xia, N., & Xia, Q. (2018a). Multilayer emulsions as a strategy for linseed oil and α-lipoic acid micro-encapsulation: Study on preparation and in vitro characterization. Journal of the Science of Food and Agriculture, 98, 3513–3523. Huang, J., Wang, Q., Sun, R., Li, T., Xia, N., & Xia, Q. (2018b). A novel solid self-emulsifying delivery system (SEDS) for the encapsulation of linseed oil and quercetin: Preparation and evaluation. Journal of Food Engineering, 226, 22–30. Illum, L. (1998). Chitosan and its use as a pharmaceutical excipient. Pharmaceutical Research, 15, 1326–1331. Jumaa, M., Furkert, F. H., & Muller, B. W. (2002). A new lipid emulsion formulation with high antimicrobial efficacy using chitosan. European Journal of Pharmaceutics and Biopharmaceutics, 53, 115–123. Kajla, P., Sharma, A., & Sood, D. R. (2015). Flaxseed—a potential functional food source. Journal of Food Science & Technology, 52(4), 1857–1871. Kamaly, N., Xiao, Z., Valencia, P. M., Radovic-Moreno, A. F., & Farokhzad, O. C. (2012). Targeted polymeric therapeutic nanoparticles: Design, development and clinical translation. Chemical Society Reviews, 41, 2971–3010. Kosaraju, S. L., Weerakkody, R., & Augustin, M. A. (2009). In-vitro evaluation of hydrocolloide based encapsulated fish oil. Food Hydrocolloids, 23, 1413–1419. Liang, L., Line, V. L. S., Remondetto, G. E., & Subirade, M. (2010). In vitro release of atocopherol from emulsion-loaded b-lactoglobulin gels. International Dairy Journal, 20, 176–181. Li, Y., Hu, M., Yumin, D., Xiao, H., & Mcclements, D. J. (2011). Control of lipase digestibility of emulsified lipids by encapsulation within calcium alginate beads. Food Hydrocolloids, 25, 122–130. Li, Y., Yao, J., Han, C., Yang, J., Chaudhry, M. T., Wang, S., et al. (2016). Quercetin, inflammation and immunity. Nutrients, 8, 167. Maurer, N., Fenske, D. B., & Cullis, P. R. (2001). Developments in liposomal drug delivery systems. Expert Opinion on Biological Therapy, 1, 923–947. Minekus, M., Alminger, M., Alvito, P., Balance, S., Bohn, T., Bourlieu, C., et al. (2014). A standardised static in vitro digestion method suitable for food —an international consensus. Food & Function, 5, 1113–1124. Montembault, A., Tahiri, K., Korwin-Zmijowska, C., Chevalier, X., Corvol, M. T., & Domard, A. (2006). A material decoy of biological media based on chitosan physical hydrogels: Application to cartilage tissue engineering. Biochimie, 88, 551–564.

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