Formation of self-assembled polyelectrolyte complex hydrogel derived from salecan and chitosan for sustained release of Vitamin C

Carbohydrate Polymers 234 (2020) 115920

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Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol

Formation of self-assembled polyelectrolyte complex hydrogel derived from salecan and chitosan for sustained release of Vitamin C

T

Xinyu Hua,b,c,d,*, Yongmei Wanga, Liangliang Zhanga, Man Xua a

Key Lab. of Biomass Energy and Material, Jiangsu Province, Institute of Chemical Industry of Forestry Products, Chinese Academy of Forestry, Nanjing, 210042, China Co-Innovation Center of Efficient Processing and Utilization of Forest Resources, Jiangsu Province, Nanjing, 210042, China c Key Lab. of Chemical Engineering of Forest Products, National Forestry and Grassland Administration, Beijing, 100714, China d National Engineering Lab. for Biomass Chemical Utilization, Nanjing, 210042, China b

A R T I C LE I N FO

A B S T R A C T

Keywords: Vitamin C protection Polyelectrolyte complex hydrogel Self-assembly Salecan Electrostatic interactions pH-dependent release Release mechanism

Vitamin C (VC) is an indispensable nutrient for human health. However, poor chemical stability in gastric environment restricts its full assimilation by intestine. It is important to construct a safe carrier that can protect VC from the gastric fluid and sustainably release it in intestine. Herein, we designed a novel polyelectrolyte complex (PEC) hydrogel through self-assembly of salecan and chitosan. PEC structure formed by electrostatic interactions was confirmed by FT-IR, XRD, XPS and TGA. Their swelling, morphology, rheology, cytocompatibility and biodegradation were well investigated. In particular, VC released in a controlled and pH-dependent manner. The release amount in simulated intestinal fluid (SIF) was significantly higher than simulated gastric fluid (SGF), and can be maintained at high level in blood after 6 h. Release mechanism agreed well with RitgerPeppas model. The purpose of this study was to develop a smart nutrient delivery platform for targeted release of VC in intestinal condition.

1. Introduction Hydrogels are three-dimensional polymeric networks capable of imbibing and retaining considerable large amounts of water or biological fluids even under certain pressure. Due to their rubbery consistency and propensity to soak up water, hydrogels have been developed for diverse applications including tissue engineering, soft machines, nutrients and drug delivery (Al-Sabah et al., 2019; Apopei Loghin, Biliuta, Coseri, & Dragan, 2017; Dinu, Cocarta, & Dragan, 2016; Dragan & Cocarta, 2016; Dragan, Cocarta, & Gierszewska, 2016; Dragan, Humelnicu, & Dinu, 2019). Usually, hydrogels can be formed by either natural polymers or fully synthetic polymers. Compared to the synthetic ones, the advantage of natural polymers such as polysaccharides is their excellent biocompatibility and biodegradability (Kennedy & Knill, 2016; Singh, Kaur, & Kennedy, 2019; Wang, Hu, Du, & Kennedy, 2010). However, a considerable part of polysaccharidebased hydrogels are prepared by chemical crosslinking or graft copolymerization with organic crosslinkers. The construction of chemical structures may bring toxic components to the hydrogels, which limited their further application as drug or nutrient carriers (Chen, Remondetto, & Subirade, 2006). The self-assembly of anionic and

cationic polysaccharides into polyelectrolyte complex (PEC) hydrogels has been recognized as an effective method to address this drawback (Wu et al., 2018). Polysaccharide-based PEC hydrogels, defined as physical hydrogels, have inspired great interest as drug or nutrient carriers. Generally speaking, this kind of hydrogels is fabricated simply by the fusion of two oppositely charged polysaccharides in aqueous solutions without any chemical covalent crosslinking agents (Bhattarai, Gunn, & Zhang, 2010; Tentor et al., 2017). The formation mechanism is based on the strong electrostatic interactions between oppositely charged functional groups of polysaccharides, together with other forces like hydrogen bonding or hydrophobic interactions between polysaccharides, which perform cooperatively to maintain a stable PEC network structure (Doi & Kokufuta, 2011; Hugerth, Caram-Lelham, & Sundelöf, 1997). For each delivery system, PEC structuration makes it possible to self-assemble and construct a nutrient carrier that is safe, effective and environmentally friendly. Besides that, the polysaccharide-based PEC hydrogels are capable of undergoing desired, programmable shape transformations by changing their volume reversibly under certain external stimuli, which is very important for the controlled release of nutrients in specific regions of the gastrointestinal tract (Luo & Wang,

⁎ Corresponding author at: Key Lab. of Biomass Energy and Material, Jiangsu Province, Institute of Chemical Industry of Forestry Products, Chinese Academy of Forestry, Nanjing, 210042, China. E-mail address: [email protected] (X. Hu).

https://doi.org/10.1016/j.carbpol.2020.115920 Received 7 November 2019; Received in revised form 20 January 2020; Accepted 26 January 2020 Available online 03 February 2020 0144-8617/ © 2020 Elsevier Ltd. All rights reserved.

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2. Experimental section

2014; Neufeld & Bianco-Peled, 2017). Salecan, a novel water-soluble anionic polysaccharide, is harvested from the fermentation medium of a salt-tolerant strain Agrobacterium sp. ZX09. Its structure contains a backbone built up by a (1→3)-β-Dglucan composed of β-1-3-linked glucopyranosyl units with a small number of α-1-3-linked (Xiu et al., 2010). As an extracellular β-glucan, salecan displays favorable biological features such as antioxidation and nontoxicity, which are often needed in pharmaceutical and food industries (Chen et al., 2012; Xiu, Zhan et al., 2011; Xiu, Zhou, Zhu, Wang, & Zhang, 2011; Zhang et al., 2012; Zhou et al., 2013). Moreover, the succinyl and pyruvyl substituent groups (succinate/pyruvate is approximately 1/1) on salecan tend to be ionized in aqueous media, which imparts negative charge to the polysaccharide chains (Xu et al., 2017). This characteristic makes it possible for salecan to form PEC hydrogels with other cationic polyelectrolytes. Like chitosan, obtained through the deacetylation of chitin, is a widely-used cationic linear polysaccharide consisting of β-(1-4)-2-acetamido-D-glucose and β-(1-4)2-amino-D-glucose units. The other features of chitosan as a biopolymer include biocompatibility, biodegradability and antibacterial activity (Dragan, Apopei Loghin, & Cocarta, 2014; Kennedy & Knill, 2016). Due to its high positive charge density, chitosan is recognized for the excellent interactions with polyanionic polysaccharides and the created PEC materials can be widely applied in food, agricultural and biomedical fields (Rasente et al., 2016; Wang, Yang, Ju, Udenigwe, & He, 2018). Vitamin C (VC) is an indispensable water-soluble nutrient needed to preserve healthy physiological processes in humans. It promotes collagen biosynthesis, provides photoprotection, causes melanin reduction, scavenges free radical and enhances the immunity (anti-viral effect) (Aguilera, de Gálvez, Sánchez, & Herrera-Ceballos, 2012; Chikvaidze & Khachatryan, 2011; Sahoo & Mukherjee, 2003; Wintergerst, Maggini, & Hornig, 2006). Moreover, VC can help to prevent scurvy, reduce the risk of cancers and cardiovascular diseases, and enhance the absorption of iron to prevent anaemia (Aride et al., 2010; Hutchinson, Burley, Greenwood, Thomas, & Cade, 2010; Talaulikar & Manyonda, 2011). However, VC is easily oxidized and decomposed in acid environments such as the stomach (Kameshima, Sasaki, Isobe, Nakajima, & Okada, 2009). If VC can be protected from the gastric fluid and then delivered intact to the intestines, its consumption and therapeutic effects would be significant. For this reason, it is necessary to encapsulate VC in the safe and stimuli-responsive PEC hydrogel carrier (Peng et al., 2016). Additionally, there are many cases where conventional drug or nutrient administration methods do not provide satisfactory pharmacokinetic profiles because the drug or nutrient concentration rapidly falls below desired levels. To solve these problems, advanced controlled delivery systems have been designed. These systems utilize carriers that sustained release their contents in order to maintain drug concentrations at the desired levels for a longer period of time (Chen et al., 2018; Gao et al., 2013). Here, we present a self-assembled PEC hydrogel prepared from two oppositely charged natural polysaccharides: salecan and chitosan for controlled VC delivery. To the best of our knowledge, this is the first report on the synthesis of salecan/chitosan PEC hydrogels as nutrient carriers. Their chemical structure, thermal stability, swelling capacity, interior morphology, rheological properties, cytocompatibility and biodegradation were well investigated. Our hypothesis was that VC could be retained within the hydrogels in gastric condition, but released purposefully under intestinal conditions using pH as a trigger. So the VC release from the hydrogels was studied systematically in simulated gastric and intestinal pH media, as well as in vivo. Finally, the releasing profiles were analyzed by a power law equation to reveal the release mechanisms. The findings of this work could be promising to create new hydrogel formulations to fulfill nutrient delivery needs.

2.1. Materials Salecan (Mw ∼ 1900 kDa) was obtained from Sichuan Synlight Biotech Ltd (China). Its purity was about 99.40 % (Table S1 in supporting information). Chitosan (Mw ∼ 1700 kDa, deacetylation degree was 94.04 %, Table S2 and viscosity ∼ 100 mPa s) and acetic acid were purchased from Sigma Aldrich (Shanghai, China). Vitamin C (purity: 99.97 %, Fig. S1), simulated intestinal fluid (SIF) and simulated gastric fluid (SGF) were kindly provided by Nanjing KeyGen Biotech Co., Ltd (Nanjing, China). 2.2. Preparation of salecan/chitosan PEC hydrogels Salecan/chitosan PEC hydrogels were prepared using an environmental friendly self-assembly method. Preparation was rapid and reliable, and hydrogels were obtained spontaneously under very mild conditions, which were superior to other VC carriers (Gao et al., 2013; Garcia et al., 2018; Yasaei, Khakbiz, Zamanian, & Ghasemi, 2019). Briefly, salecan solution (2 %, w/v) was prepared by dissolving 2 g of salecan in 100 mL deionized water. A certain amount of chitosan was added to salecan solution under magnetic stirring at room temperature for 3 h to form a slurry solution (precursor solution). The final volume was made up to 10 mL with deionized water. Then the obtained slurry solution was poured into a circular glass mold and placed in a desiccator as shown in Fig. 1, and PEC hydrogels were formed upon exposure to acetic acid atmosphere for 3 h. Afterwards, the PEC hydrogels were washed thoroughly with deionized water to remove acetic acid residues, frozen at −20 °C for 12 h and later lyophilized (Christ, Alpha 1–2 LD Plus, Germany). The feed compositions of the hydrogels are listed in Table 1. 3. Characterization 3.1. Chemical characterization and thermal gravimetric analysis Fourier transform infrared spectroscopy (FT-IR) analysis was carried out on a Nicolet IS-10 spectrometer by attenuated total reflection (ATR) method in the 550–4000 cm−1 wavenumber region, using a 16 scan per sample cycle and a resolution of 4 cm−1. X-ray diffraction (XRD) patterns were obtained by using an X-ray diffractometer (Bruker D8 Focus) with a Cu Kα radiation source (wavelength λ = 1.54 Å) at a voltage of 30 kV and a current of 20 mA. The scanning scope of 2θ was from 5° to 70°. Thermogravimetric analysis (TGA) was recorded using a Netzsch TG209F1 thermogravimetric analyzer in a temperature range of 50 and 700 °C under constant nitrogen purge of 100 mL/min at a heating rate of 10 °C/min. 3.2. X-ray photoelectron spectroscopy (XPS) analysis XPS analysis was performed on a Kratos Axis Ultra XPS instrument using a monochromatic Al Kα X-ray source (1486.8 eV) operating at 15 kV and 10 mA. The take-off angle was fixed at 90° and the chamber pressure was maintained between 1 × 10−9 and 2 × 10−9 Torr. Elemental high-resolution scans were conducted with a 23.5 eV pass energy for the O1s and N1s core levels. Data were processed using Casa XPS software v 2.3.15. 3.3. Water uptake (WU) The WU of the PEC hydrogels was evaluated gravimetrically. The freeze-dried samples were first weighed (Wd) and then placed in SGF and SIF at 37 °C. At a certain time, swollen sample weights (Wt) were measured after removing the excess of surface water with filter paper. The WU was calculated as following Eq. (1): 2

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Fig. 1. (A) Scheme showing the preparation of salecan/chitosan PEC hydrogel, (B) loading mechanism of VC in PEC hydrogel.

3.6. In vitro VC release experiments

Table 1 Composition of initial reaction mixtures used for the preparation of hydrogels. Ingredient

Salecan solution (2 %, w/v) (mL) Chitosan (g) Deionized water (mL)

3.6.1. Preparation of VC-loaded PEC hydrogels VC was loaded onto the PEC hydrogels using an equilibrium partitioning method according to our previous study (Hu et al., 2017). Specifically, the freeze-dried hydrogels were fully immersed in VC solution (1 %, w/v) and shaken at room temperature in dark to reach the equilibrium state. After 24 h, VC-loaded samples were gently taken out from the solution and washed thoroughly with deionized water to remove excess VC molecules on the surface. The supernatant solutions containing unloaded VC and washing solutions were collected together. The concentration of VC in the withdrawn solution was analyzed by UV–Vis spectrophotometer at 265 nm using a standard calibration curve. The loading efficiency (LE) was calculated by the following Eq. (2):

Weight ratio (salecan/chitosan) 7/3

6/4

5/5

4/6

7 0.06 3

6 0.08 4

5 0.1 5

4 0.12 6

WU = (Wt – Wd)/Wd

(1)

All measurements were made in triplicate.

3.4. Scanning electron microscopy (SEM)

LE (%) = (M0 – Me)/M0 × 100

The morphology of the PEC hydrogels was examined by SEM (JEOLJSM-6380LV) with an accelerating voltage of 30 kV. Prior to analysis, the freeze-dried samples were cut into small pieces, fixed onto an aluminum stub with double-sided conductive tape and sputtered with a layer of gold to improve electrical conductivity for SEM observation. The average pore size of the hydrogels was measured by Nano Measurer 1.2.5 (Fudan University, China).

Where M0 and Me is the total amounts of VC in the initial solution and remnant solution, respectively. All measurements were made in triplicate.

(2)

3.6.2. VC release In vitro release studies were performed by immersing VC-loaded hydrogel samples into 50 mL of SGF (pH 1.2) for 2 h and then in 50 mL of SIF (pH 6.8) until maximum release. The experiments were carried out in a thermostatic shaker at shaking speed of 50 r/min at 37 °C. At pre-determined time intervals, 1 ml of aliquots were withdrawn followed by replacement of the withdrawn volume by fresh media to ensure the consistency of the volume in the release condition. The released amount of VC was determined from the absorbance at 265 nm by UV with the help of a calibration curve. The cumulative percentage of VC release was determined by the following Eq. (3):

3.5. Rheology measurements The rheological experiments were performed on an Anton Paar MCR 101 rheometer using a Peltier Plate with 35 mm diameter and a gap value of 1 mm. Prior to the measurements, dynamic strain sweep was carried out to ascertain the linear viscoelasticity region. Then dynamic frequency sweep test was conducted under a frequency ranging from 0.1 to 10 Hz and constant strain of 0.1 %. The storage modulus (G’) and the loss modulus (G”) were recorded as a function of frequency. All measurements were performed in duplicate at 25 °C.

n−1

Cumulative release(%) =

Ve ∑1

Ci + V0 Cn

mVC

× 100

(3)

Where mVC represents the amount of VC in the hydrogel, V0 is the volume of the release medium (V0 = 50 mL), and Cn represents the 3

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concentration of VC in the nth sample. All release data were averaged over three measurements.

Where M0 and Mt is the initial VC amount and the amount at a specific time interval, respectively. All assays were conducted in triplicate.

3.7. Cytocompatibility of the PEC hydrogels

3.9. Biodegradation study

3.7.1. Cell culture The L929 and 3T3-L1 cell lines were used to investigate the cytocompatibility of the PEC hydrogels. The cells were routinely cultured in DMEM containing 10 % fetal bovine serum (FBS), penicillin (5000 U ml−1) and streptomycin (50 μg ml−1) on tissue culture polystyrene dishes at 37 °C in 5 % CO2 for 24 h.

The biodegradation study of the PEC hydrogels was carried out in pH 7.4 PBS solution containing lysozyme (2 mg/mL) (Hirano, Tsuchida, & Nagao, 1989). Briefly, the pre-weighted swollen samples were soaked in PBS and incubated at 37 °C under continuous shaking. The PBS solution was replaced every day by a fresh solution. At the indicated time point, samples were carefully withdrawn from the medium and weighed after being wiped with soft paper tissue. The weight remaining was calculated by using Eq. (5):

3.7.2. Cytotoxicity assay The PEC hydrogels were cut into small circular pieces, immersed in 75 % ethanol for 1 h and rinsed repeatedly with sterilized PBS to remove residual ethanol. Hydrogel pieces were placed in 24-well plates and L929 and 3T3-L1 cells were seeded at a density of 1 × 104 cells/ well. The culture medium was changed every day. After incubation for 48 h, 750 μL of MTT reagent (5 mg/mL) was added to each well and further incubated for 4 h. Then, dimethyl sulfoxide (DMSO) was subsequently added to dissolve the purple formazan salts. The solution was swirled homogeneously about 10 min by the shaker. The absorbance of the formazan solutions was then read at 490 nm (BioTek ELx800). Cells treated with medium served as the negative control. All assays were conducted in triplicate and mean values and their standard deviations were calculated.

Weight remaining (%) = Wt/W0 × 100

(5)

Where W0 and Wt are the weights of the samples before and after degradation for a specific time interval, respectively. The experiments were conducted in triplicate. 3.10. Statistical analysis Statistical analysis was carried out by origin software and one-way analysis of variance (ANOVA) was used to evaluate the statistical differences between groups. The difference between two groups with p < 0.05 and p < 0.01 was considered to be statistically significant. 4. Results and discussion

3.7.3. Live/Dead staining assay A Live/Dead assay was used to observe live and dead cells on the PEC hydrogels. The sterile hydrogel surfaces were statically seeded with L929 and 3T3-L1 cells at a density of 1 × 104 cells per well in 12-well culture plates and incubated at 37 °C in 5 % CO2 for 48 h. After that, the culture medium was removed and the cell-entrapped hydrogels were rinsed with sterile PBS, and incubated for 45 min with 2 μM calcein AM and 8 μM propidium iodide (PI). Live cells have intracellular esterase activity and are stained green with calcein AM, whereas PI enters the dead cells through damaged membrane and becomes integrated in the DNA, staining the cells red. After rinsing with PBS, cells were visualized using a fluorescent microscope (Olympus IX51, Japan). Cells cultured on tissue culture polystyrenes (TCPS) with same medium were set as a control.

4.1. Synthesis and characterization of salecan/chitosan PEC hydrogel Salecan/chitosan PEC hydrogel was fabricated by self-assembly of two polysaccharides bearing opposite charges via strong electrostatic interactions (Fig. 1A). In this process, chitosan was uniformly dispersed in the salecan solution to form a slurry solution, rather than being dissolved in an acid solution directly. It should be noted that the slurry solutions were placed in a gaseous acetic acid atmosphere. In this way, the amino groups of chitosan were gradually protonated to form positively charged –NH3+, accompanied by the dissolve of chitosan in the salecan solution. These –NH3+ then immediately interacted with the negatively charged carboxyl groups (−COO–) of salecan to form a three-dimensional network hydrogel, which ensured a homogeneous and stable structure of the prepared hydrogel. This method also avoided undesirable precipitation and agglomerate due to the direct mixture of salecan and chitosan acid solution (Dautzenberg, 1997; Zhao et al., 2009). The FT-IR spectra of materials, including salecan, chitosan and PEC hydrogels are shown in Fig. 2A. For salecan, a broad band in the region around 3500–3200 cm−1 was related to the stretching vibrations of OeH. The characteristic peaks at 1610 and 1381 cm−1 were due to the asymmetrical and symmetrical stretching vibrations of −COO– (Hu, Wang, & Xu, 2019). Around 3500–3100 cm−1, a wide band can be observed in the spectrum of chitosan, referring to the combined peaks of NeH and OeH stretching vibration. The characteristic absorption peaks at 1648 and 1585 cm−1 corresponded to the C]O stretching of the secondary amide (amide I) and NeH bending of amino and amide II, respectively (Jiang et al., 2014). The peaks obtained at 1375 cm−1 originated from the CeH bending vibration of −CH2– and −CH3 (Vinceković et al., 2016). The spectra of PEC hydrogels showed obvious changes in the salecan functional groups region. For example, the characteristic peak for asymmetric −COO– stretching vibrations shifted from 1610 cm−1 in salecan to 1599 cm−1 in 5/5 PEC hydrogel, and the peak for symmetric −COO– stretching vibrations shifted from 1381 cm−1 in salecan to 1390 cm−1 in 5/5 PEC hydrogel. Similar shifts were also observed in 7/3, 6/4 and 4/6 samples. Meanwhile, new peaks appearing at around 1572 cm−1 (7/3 and 5/5 samples), 1571 cm−1 (6/

3.8. In vivo VC release experiments All animal procedures were approved and carried out according to our institutional guidelines for the care and utilization of laboratory animals. Male mice (18 − 22 g) were housed in an environmentally controlled room with temperature maintained at 22 ± 3 °C, 30 %–60 % relative humidity and 12 h light/dark cycle. They were fed with standard laboratory food and water ad libitum. Prior to the experiments, mice were quarantined and allowed to acclimate for 1 week. For release studies, 15 mice were evenly and randomly divided into five groups (A, B, C, D and E). Group A was orally administered with free VC at a dose of 500 mg/kg and set as control. Group B, C, D and E were orally administered with 4/6, 5/5, 6/4 and 7/3 VC-loaded hydrogels (suspended in 0.9 % physiological saline) at a dose of 500 mg/kg. At pre-determined time intervals, 150 μL of blood was collected from the rim of mice eye and coagulated at room temperature for 30 min, centrifuged for 20 min at a speed of 2500 r/min and the supernatants were collected. The VC amount was determined by kit (Qincheng Biotech Co., Ltd, Shanghai, China) using the ascorbic acid/fast blue salt B test method (Wende & Hanguo, 1993). The VC release percentage was determined by the following Eq. (4): VC release (%) = Mt/M0 × 100

(4) 4

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Fig. 2. (A) FT-IR spectra, (B) XRD patterns, (C) TGA thermograms and (D) DTGA curves of salecan, chitosan and PEC hydrogels (salecan/chitosan ratio = 7/3, 6/4, 5/5 and 4/6).

Fig. 3. High-resolution XPS spectra for O1s of salecan and PEC hydrogels, N1s of chitosan and PEC hydrogels (salecan/chitosan ratio = 7/3, 6/4, 5/5 and 4/6).

bending vibration of −CH2– and −CH3 at around 1372–1375 cm-1 were also identified. The XRD spectra of salecan, chitosan and PEC hydrogels are presented in Fig. 2B. Salecan had a broad diffraction peak positioned at around 2θ = 20.3°, while chitosan exhibited two distinct diffraction peaks at around 2θ = 10° and 20.1°. These observed diffraction peaks can be attributed to the crystal regions that existed in the

4 sample) and 1570 cm-1 (4/6 sample) were attributed to the NeH deformation vibrations in –NH3+, which overlapped with the NeH bending of amide II (Martins, de Oliveira, Garcia, Kipper, & Martins, 2018). These results revealed the strong electrostatic interactions between negatively charged carboxyl groups (−COO–) on salecan and the positively charged amino groups (–NH3+) on chitosan (Vinceković et al., 2016). In addition, absorption bands assigned to the CeH 5

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salecan/chitosan ratio, and was much higher than that of salecan (298 °C) or chitosan (307 °C) (Fig. 2D), indicating that the assembly of two polysaccharides via electrostatic interactions was helpful for the improvement of thermal stability (Juntapram, Praphairaksit, Siraleartmukul, & Muangsin, 2012; Şen, Uzunsoy, Baştürk, & Kahraman, 2017). A typical survey XPS spectrum for salecan, chitosan and PEC hydrogels is shown in Fig. 3. The high-resolution scan for O1s of salecan revealed that the peak was well fitted into two sub-peaks centered at 531.9 and 532.7 eV, corresponding to −COO– and –C−OH/−COC– oxygens, respectively. The N1s peak of chitosan was decomposed to two peaks with binding energy of 398.8 eV (–NH2) and 400.6 eV (–NHCOCH3) (Coutinho et al., 2012). In contrast, the binding energy showed obvious change in the spectra of PEC hydrogels. Meanwhile, new separated peaks were also observed. For example, the binding energy of oxygen in −COO– of 5/5 PEC hydrogel was decreased by 0.9 eV, from 531.9 eV in salecan to 531.0 eV in PEC hydrogel, indicating that −COO– groups were involved in self-assembly process. Compared with that of chitosan, a new peak at 401.4 eV can be clearly identified in N1s spectrum of 5/5 PEC hydrogel, which was related to the protonated amino groups (–NH3+) (Romero et al., 2015). Similar phenomena were also observed in 7/3, 6/4 and 4/6 samples. These results further confirmed the occurrence of electrostatic interactions between salecan and chitosan, and the successful formation of the PEC hydrogel. 4.2. Swelling properties It is extremely important to investigate the swelling properties of the hydrogels as they have great influence on the controlled release behavior of drugs or nutrients in medical and food engineering applications. The time-dependent swelling curves for the PEC hydrogels swollen at SIF is shown in Fig. 4A. The WU increased significantly with increasing salecan/chitosan ratio from 4/6–7/3. The sample prepared with salecan/chitosan ratio of 7/3 exhibited the maximum WU of 16.3 g/g, compared to 5.3 g/g in the case of 4/6. This was because that increasing the proportion of hydrophilic salecan in the hydrogels enhanced the affinity of the PEC network to water molecules (Hu, Wang, Zhang et al., 2019). Conversely, with the increase of chitosan proportion in the hydrogels, the molar charge ratio of salecan/chitosan (nCOO–/nNH3+) decreased from 3.73 to 1.12, the electrostatic attractions between salecan and chitosan became stronger and thus enhanced the molecular entanglement between polysaccharide chains. In this way, the PEC network became tighter with less hydrodynamic free volume and can absorb less of the solvent resulting in lower swelling (Gierszewska, Ostrowska-Czubenko, & Chrzanowska, 2018). Fig. 4B displays the WU values of the PEC hydrogels in SGF and SIF. Obviously, the WU of the hydrogels in SGF were significantly lower (p < 0.01) than in SIF. The observed results may be attributed to the reason that in SGF (pH = 1.2), the residual carboxyl groups existing in the PEC hydrogels were protonated (pKa of pyruvate acid ∼ 2.49, pKa1 and pKa2 of succinate acid is 4.21 and 5.64, respectively), the strong hydrogen bonds among them could be established. Such hydrogen bonds constructed a stiffer network and restricted the polysaccharide chains relaxation process, suppressing the penetration of water molecular into the PEC network (Wang, Chen, An, Chang, & Song, 2018). Overall, the pH-sensitive property of the PEC hydrogels demonstrates their potential applications in the controlled delivery of nutrients to specific regions through the gastrointestinal tract.

Fig. 4. (A) Water uptake (WU) in SIF of the PEC hydrogels plotted as a function of time and (B) equilibrium water uptake (EWU) values at 660 min in SGF and SIF of the PEC hydrogels. ** indicates significant difference with p < 0.01.

polysaccharide structure, which was due to the hydrogen bonding interactions among functional moieties of polysaccharide chains, such as hydroxyl groups of salecan, and hydroxyl and amino groups of chitosan (Hu, Wang, Xu, 2019; Zhang et al., 2015). However, only broad and weak peaks centered at around 2θ = 20.0°–20.2° were observed in the diffractogram of PEC hydrogels. The peak intensity was gradually decreased with the decrease of salecan/chitosan ratio. These indicated that the electrostatic interactions between salecan and chitosan resulted in destruction of the original crystalline structure of the polysaccharides. The thermogravimetric analysis of salecan, chitosan and PEC hydrogels are displayed in Fig. 2C and D. All samples have a slight weight loss near 100 °C due to the vaporization of the adsorbed moisture. The weight loss of salecan mainly occurred at 152–700 °C with approximately 68.4 %, which was attributed to the depolymerization and rupture of CeO and CeC bonds of the saccharide ring (Hu, Wang, Zhang, & Xu, 2019). For chitosan, thermal degradation between 154–700 °C with 55.9 % weight loss can be assigned to the decomposition of the main skeleton of chitosan (Xie, Zhang, & Li, 2006). After the formation of PEC hydrogels, all samples exhibited similar thermal degradation behavior. The major and rapid thermal degradation starting around 160–167 °C with about 65.9–69.6 % weight loss can be associated with the degradation of the polysaccharides backbone, cleavage of numerous functional groups in the glucopyranose ring and advanced degradation of PEC chains. However, it should be noted that the Tm of PEC hydrogels increased from 348 to 364 °C as the decrease of

4.3. Morphological analysis The microstructures of the lyophilized PEC hydrogels are shown in Fig. 5. It was clearly observed that the hydrogels exhibited an interconnected highly porous architecture. As the salecan/chitosan ratio increased, the pore size was found to increase accordingly. The sample prepared with salecan/chitosan ratio of 4/6 had an average pore size of 6

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Fig. 5. SEM images of the cross-sectional morphology of freeze-dried hydrogels at 400 × magnification. Scale bars represent 50 μm.

Fig. 6. (A) Storage modulus G’ (closed symbols) and loss modulus G” (open symbols) of the PEC hydrogels as a function of frequency, (B) cumulative VC release from the PEC hydrogels in SGF and SIF, plots of ln (Mt/M∞) against ln t for VC release in (C) SGF and (D) SIF.

10.1 μm, and this value increased to 17.5 μm for 5/5, 34.6 μm for 6/4 and 50.8 μm for 7/3. This trend could be related to the WU of the hydrogels discussed above. The increase of salecan/chitosan ratio enhanced the WU of the hydrogels and hence promoted the water migration in PEC network. Larger ice crystals were thus formed within the hydrogels and the following sublimation extracts the water which left place for relatively large cavities. This highly porous microstructure

will facilitate the fast and adequate diffusion of nutrients through the PEC network (Bearat, Lee, & Vernon, 2012; Dinu, Přádný, Drăgan, & Michálek, 2013).

4.4. Rheological properties Rheological measurements were performed to characterize the 7

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strong hydrogen bond interactions occurred among them. These hydrogen bonds played the role of barriers that densified the PEC network, and thus prevented the release of VC from the hydrogels (Lima et al., 2018). In contrast, when the release environment changed to SIF, the release rate increased markedly and the maximum of VC release amount could reach 92.3 % after 10 h, which was higher than that of other VC carriers (Alishahi et al., 2011; Gao et al., 2014; Voss et al., 2018; Yasaei et al., 2019). In this case, the higher WU would benefit the water transfer, which provided a driving force promoting the diffusion of VC molecules from the hydrogels (Park, Shin, & Park, 2018). In addition, the release properties of the PEC hydrogels prepared at different salecan/chitosan ratios were also studied. As the increase of salecan/ chitosan ratio from 4/6 to 7/3, the release amount showed an increasing trend from 4.1% to 10% in SGF after 2 h, and from 74.9% to 92.3% in SIF in the next 8 h. This result further demonstrated the release profile was directly correlated with their swelling capacity as mentioned above. Therefore, this kind of PEC hydrogel can be utilized as a nutrient delivery carrier to overcome its gastrointestinal irritation side effects and improve its treatment effects.

Table 2 Parameters n, ln k and correlation coefficients (R2) according to Ritger-Peppas model for VC release in SGF and SIF. Salecan/chitosan

7/3 6/4 5/5 4/6

SGF

SIF

n

ln k

R2

n

ln k

R2

0.391 0.408 0.427 0.435

−0.181 −0.221 −0.278 −0.347

0.998 0.996 0.995 0.994

0.603 0.630 0.672 0.717

−0.638 −0.743 −0.867 −1.045

0.994 0.995 0.993 0.996

4.6. Release kinetics To further understand the release mechanism, the results were analyzed on the basis of Ritger-Peppas Eq. (6) up to an initial 60 % of release (Ritger & Peppas, 1987). Mt/M∞ = ktn

(6)

Where Mt/M∞ represents the fractional VC release at time t, Mt and M∞ are the cumulative amounts of VC released at time t and at infinite time, respectively, k is a constant characteristic of the VC-loaded hydrogel system and n is the release (diffusion) exponent, which depends on the release mechanism. For a release system having cylindrical shape, n < 0.45 corresponds to Fickian diffusion (Case I), whereas 0.45 < n < 0.89 indicates that diffusion is non-Fickian or anomalous diffusion, and for n > 0.89, a super Case II release kinetics is operative. The constant k, the exponent n and the corresponding coefficient of determination (R2) are listed in Table 2. The n values for all the hydrogels were below 0.45 in SGF, revealing that the VC release was indeed Fickian. However, the n value ranged between 0.603 and 0.717 in SIF, being indicative of non-Fickian diffusion with a tendency to macromolecular relaxation due to the higher WU and looser PEC network (Wang, Liu, Shuai, Cui, & Nie, 2014). Fig. 6C and D show a typical plot of ln (Mt/M∞) versus ln t in various release medium. It can be found that all the correlation coefficient values approached unity (R2 > 0.99), suggesting that the kinetic data was well-fitted with the Ritger-Peppas model.

Fig. 7. Cell viability of L929 and 3T3-L1 cells cultured in the PEC hydrogels for 48 h (salecan/chitosan ratio = 7/3, 6/4, 5/5 and 4/6).

mechanical properties of the PEC hydrogels. It can be found from Fig. 6A that G’ was not sensitive to changes in frequency and always greater than G” for all PEC hydrogels over the entire frequency range, indicating a typical hydrogel characteristics with the elastic response as the predominant property (Dong, Snyder, Williams, & Andzelm, 2013). G’ increased as the decrease of the salecan/chitosan ratio and finally reached to maximum value at the ratio of 4/6. The possible reason was that with the increment of chitosan content, the molar charge ratio of salecan/chitosan (nCOO–/nNH3+) decreased gradually, more physical junctions were formed by ion pairs between negative carboxyl groups of salecan and positive amino groups of chitosan due to the stronger electrostatic attractions existing in the hydrogels. This contributed to the formation of a more compact and rigid network structure, accompanied with a higher G’ value (Dong et al., 2013). 4.5. In vitro controlled release of VC For practical nutrient delivery applications, the release of loaded substance to a specific site in gastrointestinal tract is very important. VC-loaded PEC hydrogels were prepared by equilibrium partitioning method and the highest LE can even reach 94.1 % (Fig. S2). This was attributed to the highly interconnected three-dimensional and porous network structures of the PEC hydrogels and their good loading capacity for VC. As shown in Fig. 1B, the intramolecular hydrogen-bonding interactions among the functional groups of PEC hydrogel and VC could be formed, which behaved cooperatively to maintain the high LE (Wen, Cao, Yin, Wang, & Zhao, 2009). The in vitro release profiles were studied in SGF (first 2 h) and SIF (following 8 h), and the results are shown in Fig. 6B. It can be clearly seen that VC was released in a sustained manner and the cumulative release amount can be controlled by changing the pH of release media. The release amount was very low in SGF: less than 10 % of VC was liberated from different salecan/chitosan ratios of the PEC hydrogels within 2 h. This was because that in SGF, the residual carboxyl groups of the PEC hydrogels were protonated and the

4.7. Cytocompatibility of the PEC hydrogels Cytotoxicity is one of the most important factors to be considered in selecting materials as nutrient carrier. As revealed in Fig. 7, the cell viabilities of L929 and 3T3-L1 cells cultured in different hydrogels were all above 95 %. What’s more, there was no obvious difference (p > 0.05) in cell viability between the PEC hydrogels and negative control group indicating that these PEC hydrogels were non-toxic to L929 and 3T3-L1 cells. The cell viability of L929 and 3T3-L1 cells cultured in the PEC hydrogels was further evaluated by using Live/Dead staining. The green fluorescence in Fig. 8A revealed that the cells encapsulated in the PEC hydrogels were mostly alive and hardly any red fluorescence could be seen after 48 h of incubation. The results of cytotoxicity and Live/Dead assay strongly demonstrated excellent cytocompatibility of these PEC hydrogels.

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Fig. 8. (A) Live/Dead stain of L929 and 3T3-L1 cells on the PEC hydrogels after 48 h of culture. Scale bars represent 50 μm, (B) VC release percentage in blood after 2, 6 and 10 h of oral administration (salecan/chitosan ratio = 7/3, 6/4, 5/5 and 4/6), * indicates significant difference with p < 0.05 when compared to group A (control), (C) biodegradation of the PEC hydrogels in the presence of lysozyme.

4.8. In vivo VC release experiments

VC content in blood.

VC is usually assimilated in the small intestine and then delivered to the blood through the portal vein. So it is very important for a VC carrier to maintain the high level of VC content in blood after release in small intestine (Gao et al., 2013; Wang, Zhang et al., 2018). In vivo release profile of VC from the PEC hydrogels was investigated in mice (Fig. 8B). For all groups, almost no VC can be detected in blood after 2 h due to its release and assimilation in intestinal condition. At 6 h after administration, the VC was detected and its amount gradually increased from group B to E. This trend was consistent with in vitro release. In particular, it should be noted that the release amount of VC from PEC hydrogels was much higher than that of free VC (p < 0.05). This result may be attributed to the reason that free VC was easily decomposed in gastric environment (Kameshima et al., 2009), which decreased the release and assimilation capacity in intestinal condition, and thus greatly affected its delivery to blood. In sharp contrast, after the loading on our PEC hydrogels, the VC can be protected from the gastric fluid and then delivered intact to the intestine, which guaranteed high content of VC in blood. Moreover, group E showed the maximum release amount, 86.4 % of VC was released from 7/3 sample after 6 h, and the release quantity could reach 90.0 % when the delivery time ran up to 10 h. This result was very close to that of in vitro release. Similar phenomenon was observed in other three groups, as shown in Table S3. Taken together, these results further demonstrated that the VC loaded PEC hydrogels not only followed a pH-controlled and sustained manner under gastrointestinal conditions, but also maintained the high level of

4.9. Biodegradation study Biodegradability is an important factor in designing hydrogels for applications in nutrient delivery. The biodegradability of the PEC hydrogels was investigated in PBS solution containing lysozyme. As shown in Fig. 8C, after 66 h of incubation, the degradation of 7/3 sample was the fastest with a remaining weight of about 0.9 %. For 6/4, 5/5 and 4/6 sample, the weight remaining was 4.2 %, 7.9 % and 11.4 %, respectively. This result could be attributed to the reason that the increase in WU of the hydrogel accelerated the diffusion of enzyme through the hydrogel matrix and consequently increased the degradation rate (El-Sherbiny, 2010). 5. Conclusions In this study, we have successfully developed a novel PEC hydrogel that could be easily prepared by self-assembly of two oppositely charged polysaccharides salecan and chitosan. The formation of PEC network structures via electrostatic interactions was confirmed by FTIR, XRD, XPS and TGA. Variation in salecan/chitosan ratio had a clear influence on the WU and pore size of the PEC hydrogels. The 7/3 salecan/chitosan hydrogel presented a maximum WU of 16.3 g/g and the largest pore size of 50.8 μm. While the general trend of G’ increased following the decrease of salecan/chitosan ratio. The in vitro release profile of the VC-loaded hydrogels showed a pH-triggered and sustained 9

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release property. Very inhibited release was observed for VC in SGF. After transferred to SIF for 8 h, the maximum release amount could even reach 92.3 %. In vivo release experiment revealed that the maximum release amount from PEC hydrogel achieved 90.0 % in blood after 10 h, much higher than that of free VC. The release profiles were well fitted with the Ritger-Peppas model. The n value obtained in SGF indicated a diffusion controlled, Fickian type mechanism while in SIF a swelling controlled, non-Fickian type mechanism occurred. Moreover, all hydrogels showed excellent cytocompatibility and biodegradability. Based on the results of this study, salecan/chitosan PEC hydrogel, as a potential delivery system, has a great prospect for the site-specific nutrient delivery in the intestine.

Dinu, M. V., Cocarta, A. I., & Dragan, E. S. (2016). Synthesis, characterization and drug release properties of 3D chitosan/clinoptilolite biocomposite cryogels. Carbohydrate Polymers, 153, 203–211. Dinu, M. V., Přádný, M., Drăgan, E. S., & Michálek, J. (2013). Ice-templated hydrogels based on chitosan with tailored porous morphology. Carbohydrate Polymers, 94(1), 170–178. Doi, R., & Kokufuta, E. (2011). Conductometric and Light Scattering Studies on the Complexation between Cationic Polyelectrolyte Nanogel and Anionic Polyion. Langmuir, 27(1), 392–398. Dong, H., Snyder, J. F., Williams, K. S., & Andzelm, J. W. (2013). Cation-Induced Hydrogels of Cellulose Nanofibrils with Tunable Moduli. Biomacromolecules, 14(9), 3338–3345. Dragan, E. S., & Cocarta, A. I. (2016). Smart Macroporous IPN Hydrogels Responsive to pH, Temperature, and Ionic Strength: Synthesis, Characterization, and Evaluation of Controlled Release of Drugs. ACS Applied Materials & Interfaces, 8(19), 12018–12030. Dragan, E. S., Apopei Loghin, D. F., & Cocarta, A. I. (2014). Efficient Sorption of Cu2+ by Composite Chelating Sorbents Based on Potato Starch-graft-Polyamidoxime Embedded in Chitosan Beads. ACS Applied Materials & Interfaces, 6(19), 16577–16592. Dragan, E. S., Cocarta, A. I., & Gierszewska, M. (2016). Designing novel macroporous composite hydrogels based on methacrylic acid copolymers and chitosan and in vitro assessment of lysozyme controlled delivery. Colloids and Surfaces B, Biointerfaces, 139, 33–41. Dragan, E. S., Humelnicu, D., & Dinu, M. V. (2019). Development of chitosan-poly (ethyleneimine) based double network cryogels and their application as superadsorbents for phosphate. Carbohydrate Polymers, 210, 17–25. El-Sherbiny, I. M. (2010). Enhanced pH-responsive carrier system based on alginate and chemically modified carboxymethyl chitosan for oral delivery of protein drugs: Preparation and in-vitro assessment. Carbohydrate Polymers, 80(4), 1125–1136. Gao, X., Chen, L., Xie, J., Yin, Y., Chang, T., Duan, Y., et al. (2014). In vitro controlled release of vitamin C from Ca/Al layered double hydroxide drug delivery system. Materials Science and Engineering C, 39, 56–60. Gao, X., Lei, L., O’Hare, D., Xie, J., Gao, P., & Chang, T. (2013). Intercalation and controlled release properties of vitamin C intercalated layered double hydroxide. Journal of Solid State Chemistry, 203, 174–180. Garcia, V. A. S., Borges, J. G., Maciel, V. B. V., Mazalli, M. R., Lapa-Guimaraes, J. G., Vanin, F. M., et al. (2018). Gelatin/starch orally disintegrating films as a promising system for vitamin C delivery. Food Hydrocolloids, 79, 127–135. Gierszewska, M., Ostrowska-Czubenko, J., & Chrzanowska, E. (2018). pH-responsive chitosan/alginate polyelectrolyte complex membranes reinforced by tripolyphosphate. European Polymer Journal, 101, 282–290. Hirano, S., Tsuchida, H., & Nagao, N. (1989). N-acetylation in chitosan and the rate of its enzymic hydrolysis. Biomaterials, 10(8), 574–576. Hu, X., Wang, Y., Zhang, L., Xu, M., Dong, W., & Zhang, J. (2017). Redox/pH dual stimuliresponsive degradable Salecan-g-SS-poly(IA-co-HEMA) hydrogel for release of doxorubicin. Carbohydrate Polymers, 155, 242–251. Hu, X., Wang, Y., & Xu, M. (2019). Study of the cell responses in tantalum carbide nanoparticles-enriched polysaccharide composite hydrogel. International Journal of Biological Macromolecules, 135, 501–511. Hu, X., Wang, Y., Zhang, L., & Xu, M. (2019). Design of a novel polysaccharide-based cryogel using triallyl cyanurate as crosslinker for cell adhesion and proliferation. International Journal of Biological Macromolecules, 126, 221–228. Hugerth, A., Caram-Lelham, N., & Sundelöf, L.-O. (1997). The effect of charge density and conformation on the polyelectrolyte complex formation between carrageenan and chitosan. Carbohydrate Polymers, 34(3), 149–156. Hutchinson, J., Burley, V. J., Greenwood, D. C., Thomas, J. D., & Cade, J. E. (2010). Highdose vitamin C supplement use is associated with self-reported histories of breast cancer and other illnesses in the UK Women’s Cohort Study. Public Health Nutrition, 14(5), 768–777. Jiang, C., Wang, Z., Zhang, X., Zhu, X., Nie, J., & Ma, G. (2014). Crosslinked polyelectrolyte complex fiber membrane based on chitosan–sodium alginate by freeze-drying. RSC Advances, 4(78), 41551–41560. Juntapram, K., Praphairaksit, N., Siraleartmukul, K., & Muangsin, N. (2012). Electrosprayed polyelectrolyte complexes between mucoadhesive N,N,N,-trimethylchitosan-homocysteine thiolactone and alginate/carrageenan for camptothecin delivery. Carbohydrate Polymers, 90(4), 1469–1479. Kameshima, Y., Sasaki, H., Isobe, T., Nakajima, A., & Okada, K. (2009). Synthesis of composites of sodium oleate/Mg–Al-ascorbic acid-layered double hydroxides for drug delivery applications. International Journal of Pharmaceutics, 381(1), 34–39. Kennedy, J. F., & Knill, C. J. (2016). Carbohydrate Polymers, 139, 177. Lima, D. S., Tenório-Neto, E. T., Lima-Tenório, M. K., Guilherme, M. R., Scariot, D. B., Nakamura, C. V., et al. (2018). pH-responsive alginate-based hydrogels for protein delivery. Journal of Molecular Liquids, 262, 29–36. Luo, Y., & Wang, Q. (2014). Recent development of chitosan-based polyelectrolyte complexes with natural polysaccharides for drug delivery. International Journal of Biological Macromolecules, 64, 353–367. Martins, J. G., de Oliveira, A. C., Garcia, P. S., Kipper, M. J., & Martins, A. F. (2018). Durable pectin/chitosan membranes with self-assembling, water resistance and enhanced mechanical properties. Carbohydrate Polymers, 188, 136–142. Neufeld, L., & Bianco-Peled, H. (2017). Pectin–chitosan physical hydrogels as potential drug delivery vehicles. International Journal of Biological Macromolecules, 101, 852–861. Park, S. H., Shin, H. S., & Park, S. N. (2018). A novel pH-responsive hydrogel based on carboxymethyl cellulose/2-hydroxyethyl acrylate for transdermal delivery of naringenin. Carbohydrate Polymers, 200, 341–352. Peng, H., Chen, S., Luo, M., Ning, F., Zhu, X., & Xiong, H. (2016). Preparation and self-

CRediT authorship contribution statement Xinyu Hu: Conceptualization, Methodology, Software, Validation, Investigation, Writing - original draft, Writing - review & editing. Yongmei Wang: Formal analysis, Data curation. Liangliang Zhang: Visualization. Man Xu: Writing - review & editing, Supervision. Acknowledgement This work was supported by the Key Lab. of Biomass Energy and Material, Jiangsu Province (KLBEM) under the Grant JSBEM-S-201904 and the National Natural Science Foundation of China under the Grant 31700500. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.carbpol.2020.115920. References Aguilera, J., de Gálvez, M. V., Sánchez, C., & Herrera-Ceballos, E. (2012). Changes in photoinduced cutaneous erythema with topical application of a combination of vitamins C and E before and after UV exposure. Journal of Dermatological Science, 66(3), 216–220. Alishahi, A., Mirvaghefi, A., Tehrani, M. R., Farahmand, H., Shojaosadati, S. A., Dorkoosh, F. A., et al. (2011). Shelf life and delivery enhancement of vitamin C using chitosan nanoparticles. Food Chemistry, 126(3), 935–940. Al-Sabah, A., Burnell, S. E. A., Simoes, I. N., Jessop, Z., Badiei, N., Blain, E., et al. (2019). Structural and mechanical characterization of crosslinked and sterilised nanocellulose-based hydrogels for cartilage tissue engineering. Carbohydrate Polymers, 212, 242–251. Apopei Loghin, D. F., Biliuta, G., Coseri, S., & Dragan, E. S. (2017). Preparation and characterization of oxidized starch/poly(N,N-dimethylaminoethyl methacrylate) semi-IPN cryogels and in vitro controlled release evaluation of indomethacin. International Journal of Biological Macromolecules, 96, 589–599. Aride, P. H. R., Ferreira, M. S., Duarte, R. M., De Oliveira, A. M., De Freitas, D. V., Dos Santos, A. L. W., et al. (2010). Ascorbic Acid (Vitamin C) and Iron Concentration in Tambaqui, Colossoma macropomum, Iron Absorption. Journal of the World Aquaculture Society, 41(s2), 291–297. Bearat, H. H., Lee, B. H., & Vernon, B. L. (2012). Comparison of properties between NIPAAm-based simultaneously physically and chemically gelling polymer systems for use in vivo. Acta Biomaterialia, 8(10), 3629–3642. Bhattarai, N., Gunn, J., & Zhang, M. (2010). Chitosan-based hydrogels for controlled, localized drug delivery. Advanced Drug Delivery Reviews, 62(1), 83–99. Chen, L., Remondetto, G. E., & Subirade, M. (2006). Food protein-based materials as nutraceutical delivery systems. Trends in Food Science & Technology, 17(5), 272–283. Chen, P., Wang, Z., Zeng, L., Wang, S., Dong, W., Jia, A., et al. (2012). Protective effects of salecan against carbon tetrachloride-induced acute liver injury in mice. Journal of Applied Toxicology, 32(10), 796–803. Chen, Y., Yan, X., Zhao, J., Feng, H., Li, P., Tong, Z., et al. (2018). Preparation of the chitosan/poly(glutamic acid)/alginate polyelectrolyte complexing hydrogel and study on its drug releasing property. Carbohydrate Polymers, 191, 8–16. Chikvaidze, E., & Khachatryan, I. (2011). ESR study of photoinduced free radicals by visible light in hair and the effects of ascorbic acid (vitamin C). International Journal of Cosmetic Science, 33(4), 322–327. Coutinho, D. F., Sant, S., Shakiba, M., Wang, B., Gomes, M. E., Neves, N. M., et al. (2012). Microfabricated photocrosslinkable polyelectrolyte-complex of chitosan and methacrylated gellan gum. Journal of Materials Chemistry, 22(33), 17262–17271. Dautzenberg, H. (1997). Polyelectrolyte Complex Formation in Highly Aggregating Systems. 1. Effect of Salt: Polyelectrolyte Complex Formation in the Presence of NaCl. Macromolecules, 30(25), 7810–7815.

10

Carbohydrate Polymers 234 (2020) 115920

X. Hu, et al.

Nanoparticles from Chitosan and Acylated Rapeseed Cruciferin Protein for Curcumin Delivery. Journal of Agricultural and Food Chemistry, 66(11), 2685–2693. Wang, Q., Zhang, H., Huang, J., Xia, N., Li, T., & Xia, Q. (2018). Self-double-emulsifying drug delivery system incorporated in natural hydrogels: A new way for topical application of vitamin C. Journal of Microencapsulation, 35(1), 90–101. Wen, X., Cao, X., Yin, Z., Wang, T., & Zhao, C. (2009). Preparation and characterization of konjac glucomannan–poly(acrylic acid) IPN hydrogels for controlled release. Carbohydrate Polymers, 78(2), 193–198. Wende, Z., & Hanguo, H. (1993). Spectrophotometric determination of ascorbic acid with fast blue salt B in pharmaceutical preparation. CHINESE JOURNAL of ANALYTICAL CHEMISTRY, (5), 28. Wintergerst, E. S., Maggini, S., & Hornig, D. H. (2006). Immune-enhancing role of vitamin C and zinc and effect on clinical conditions. Annals of Nutrition & Metabolism, 50(2), 85–94. Wu, T., Huang, J., Jiang, Y., Hu, Y., Ye, X., Liu, D., et al. (2018). Formation of hydrogels based on chitosan/alginate for the delivery of lysozyme and their antibacterial activity. Food Chemistry, 240, 361–369. Xie, H., Zhang, S., & Li, S. (2006). Chitin and chitosan dissolved in ionic liquids as reversible sorbents of CO2. Green Chemistry, 8(7), 630–633. Xiu, A., Kong, Y., Zhou, M., Zhu, B., Wang, S., & Zhang, J. (2010). The chemical and digestive properties of a soluble glucan from Agrobacterium sp. ZX09. Carbohydrate Polymers, 82(3), 623–628. Xiu, A., Zhan, Y., Zhou, M., Zhu, B., Wang, S., Jia, A., et al. (2011). Results of a 90-day safety assessment study in mice fed a glucan produced by Agrobacterium sp. ZX09. Food and Chemical Toxicology, 49(9), 2377–2384. Xiu, A., Zhou, M., Zhu, B., Wang, S., & Zhang, J. (2011). Rheological properties of Salecan as a new source of thickening agent. Food Hydrocolloids, 25(7), 1719–1725. Xu, L., Cheng, R., Li, J., Wang, Y., Zhu, B., Ma, S., et al. (2017). Identification of substituent groups and related genes involved in salecan biosynthesis in Agrobacterium sp. ZX09. Applied Microbiology and Biotechnology, 101(2), 585–598. Yasaei, M., Khakbiz, M., Zamanian, A., & Ghasemi, E. (2019). Synthesis and characterization of Zn/Al-LDH@SiO2 nanohybrid: Intercalation and release behavior of vitamin C. Materials Science and Engineering C, 103, 109816. Zhang, D., Zhou, W., Wei, B., Wang, X., Tang, R., Nie, J., et al. (2015). Carboxyl-modified poly(vinyl alcohol)-crosslinked chitosan hydrogel films for potential wound dressing. Carbohydrate Polymers, 125, 189–199. Zhang, Y., Xia, L., Pang, W., Wang, T., Chen, P., Zhu, B., et al. (2012). A novel soluble β1,3-d-glucan Salecan reduces adiposity and improves glucose tolerance in high-fat diet-fed mice. The British Journal of Nutrition, 109(2), 254–262. Zhao, Q., Qian, J., An, Q., Gao, C., Gui, Z., & Jin, H. (2009). Synthesis and characterization of soluble chitosan/sodium carboxymethyl cellulose polyelectrolyte complexes and the pervaporation dehydration of their homogeneous membranes. Journal of Membrane Science, 333(1), 68–78. Zhou, M., Jia, P., Chen, J., Xiu, A., Zhao, Y., Zhan, Y., et al. (2013). Laxative effects of Salecan on normal and two models of experimental constipated mice. BMC Gastroenterology, 13(1), 52.

assembly mechanism of bovine serum albumin–Citrus peel pectin conjugated hydrogel: A potential delivery system for vitamin C. Journal of Agricultural and Food Chemistry, 64(39), 7377–7384. Rasente, R. Y., Imperiale, J. C., Lázaro-Martínez, J. M., Gualco, L., Oberkersch, R., Sosnik, A., et al. (2016). Dermatan sulfate/chitosan polyelectrolyte complex with potential application in the treatment and diagnosis of vascular disease. Carbohydrate Polymers, 144, 362–370. Ritger, P. L., & Peppas, N. A. (1987). A simple equation for description of solute release II. Fickian and anomalous release from swellable devices. Journal of Controlled Release, 5(1), 37–42. Romero, R., Chubb, L., Travers, J. K., Gonzales, T. R., Ehrhart, N. P., & Kipper, M. J. (2015). Coating cortical bone allografts with periosteum-mimetic scaffolds made of chitosan, trimethyl chitosan, and heparin. Carbohydrate Polymers, 122, 144–151. Sahoo, P. K., & Mukherjee, S. C. (2003). Immunomodulation by dietary vitamin C in healthy and aflatoxin B1-induced immunocompromised rohu (Labeo rohita). Comparative Immunology, Microbiology and Infectious Diseases, 26(1), 65–76. Şen, F., Uzunsoy, İ., Baştürk, E., & Kahraman, M. V. (2017). Antimicrobial agent-free hybrid cationic starch/sodium alginate polyelectrolyte films for food packaging materials. Carbohydrate Polymers, 170, 264–270. Singh, R. S., Kaur, N., & Kennedy, J. F. (2019). Pullulan production from agro-industrial waste and its applications in food industry: A review. Carbohydrate Polymers, 217, 46–57. Talaulikar, V. S., & Manyonda, I. T. (2011). Vitamin C as an antioxidant supplement in women’s health: A myth in need of urgent burial. European Journal of Obstetrics, Gynecology, and Reproductive Biology, 157(1), 10–13. Tentor, F. R., de Oliveira, J. H., Scariot, D. B., Lazarin-Bidóia, D., Bonafé, E. G., Nakamura, C. V., et al. (2017). Scaffolds based on chitosan/pectin thermosensitive hydrogels containing gold nanoparticles. International Journal of Biological Macromolecules, 102, 1186–1194. Vinceković, M., Jalšenjak, N., Topolovec-Pintarić, S., Đermić, E., Bujan, M., & Jurić, S. (2016). Encapsulation of Biological and Chemical Agents for Plant Nutrition and Protection: Chitosan/Alginate Microcapsules Loaded with Copper Cations and Trichoderma viride. Journal of Agricultural and Food Chemistry, 64(43), 8073–8083. Voss, G. T., Gularte, M. S., Vogt, A. G., Giongo, J. L., Vaucher, R. A., Echenique, J. V. Z., et al. (2018). Polysaccharide-based film loaded with vitamin C and propolis: A promising device to accelerate diabetic wound healing. International Journal of Pharmaceutics, 552(1), 340–351. Wang, J., Liu, C., Shuai, Y., Cui, X., & Nie, L. (2014). Controlled release of anticancer drug using graphene oxide as a drug-binding effector in konjac glucomannan/sodium alginate hydrogels. Colloids and Surfaces B, Biointerfaces, 113, 223–229. Wang, Q., Hu, X., Du, Y., & Kennedy, J. F. (2010). Alginate/starch blend fibers and their properties for drug controlled release. Carbohydrate Polymers, 82(3), 842–847. Wang, Y.-W., Chen, L.-Y., An, F.-P., Chang, M.-Q., & Song, H.-B. (2018). A novel polysaccharide gel bead enabled oral enzyme delivery with sustained release in small intestine. Food Hydrocolloids, 84, 68–74. Wang, F., Yang, Y., Ju, X., Udenigwe, C. C., & He, R. (2018). Polyelectrolyte Complex

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