Rheological behavior and microstructure of release-controlled hydrogels based on xanthan gum crosslinked with sodium trimetaphosphate

Accepted Manuscript Rheological behavior and microstructure of release-controlled hydrogels based on xanthan gum crosslinked with sodium trimetaphosphate Yongzhen Tao, Ruquan Zhang, Weilin Xu, Zikui Bai, Yingshan Zhou, Sanping Zhao, Yan Xu, Dan qing Yu PII:

S0268-005X(15)30081-3

DOI:

10.1016/j.foodhyd.2015.09.006

Reference:

FOOHYD 3129

To appear in:

Food Hydrocolloids

Received Date: 11 June 2015 Revised Date:

7 August 2015

Accepted Date: 4 September 2015

Please cite this article as: Tao, Y., Zhang, R., Xu, W., Bai, Z., Zhou, Y., Zhao, S., Xu, Y., Yu, D.q., Rheological behavior and microstructure of release-controlled hydrogels based on xanthan gum crosslinked with sodium trimetaphosphate, Food Hydrocolloids (2015), doi: 10.1016/ j.foodhyd.2015.09.006. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

Graphical abstract Rheological behavior and microstructure of release-controlled hydrogels based on xanthan gum crosslinked with sodium trimetaphosphate

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Yongzhen Tao
, Ruquan Zhang , Weilin Xu, Zikui Bai, Yingshan Zhou, Sanping Zhao,

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G' G''

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XG2-STMP XG3-STMP XG4-STMP XG5-STMP

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Rheological behavior and microstructure of release-controlled

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hydrogels based on xanthan gum crosslinked with sodium

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trimetaphosphate

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Yongzhen Taoa,∗, Ruquan Zhang b, Weilin Xua, Zikui Baia, Yingshan Zhoua, Sanping Zhaoa , Yan Xua, Dan qing Yua

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a

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Materials, Ministry of Education, Wuhan Textile University, Wuhan 430073,

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China

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Key Laboratory of Green Processing and Functional Textiles of New Textile

College of Mathematics and Computer Science, Wuhan Textile University, Wuhan 430073, China

18 19 20 21 ∗ To whom correspondence should be addressed. (Tel: +86-27-59367580, Fax: +86-27-59367578, e-mail: [email protected])

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Abstract Xanthan-based hydrogels can be used for encapsulating and controlling release of

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nutrition ingredients, therapeutic agents, and cells in food and tissue engineering applications.

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Hydroxyl groups on the xanthan gum (XG) chains permitted the formation of the hydrogels

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through crosslinking XG with sodium trimetaphosphate (STMP). Dynamical oscillation tests

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were performed to monitor the in situ crosslinking process, and to evaluate the forming

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kinetics and mechanical stability for the XG-STMP hydrogels. The results indicated that the

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transition from hydrogen bonding to chemical crosslinking for the XG chains occurred and

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reached a balance approximately at 25 and 37 ℃, respectively. The XG-STMP hydrogel

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networks with solid-like gel behavior exhibited more elastic and tougher to resist the

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deformation than the physical XG hydrogels. The XG-STMP hydrogels with porous and

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interconnected structure displayed good swelling and release-controlled properties. This work

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provides some valuable and fundamental information of the xanthan-based hydrogels for

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further application in biomaterials, medical and food engineering.

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Keywords: Xanthan gum; Hydrogel; Chemical cross-linking; Rheological behavior;

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Microstructure

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1. Introduction Polysaccharides are an important group of materials for biomedical, food and tissue

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engineering applications due to their versatile ability to form hydrogels and their

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biocompatility with tissue (Delair, 2012; Khan, & Ahmad, 2013; Koop, de Freitas, de Souza,

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Savi-Jr, & Silveira, 2015; Li, & Nie, 2015; Zhao et al, 2014). Xanthan gum (XG), an anionic

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polysaccharide, consists of a (1→4)-β-D-glucose backbone substituted on every second unit

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with a charged trisaccharide side chain containg a D-glucuronic acid between two D-mannoses.

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The inner and terminal mannoses can be substituted by an acetate and a pyruvate group

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(Jansson, Kenne, & Lindberg, 1975; Melton, Mindt, & Rees, 1976). Xanthan gum-based

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hydrogels can be formed by physical or chemical crosslinking (Bueno, Bentini, Catalani, &

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Petri, 2013; Giannouli, & Morris, 2003). However, applications of physical hydrogels are

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limited due to lack of thermal, pH, or salt stability over the long-term, since they are formed

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through hydrogen bonding or inter-molecular electrostatic (ionic) interactions. Synthetic

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chemistry has greatly broadened the scope of the formation pathways to fabricate

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polysaccharide hydrogels with satisfying and tailored properties.

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Sodium trimetaphosphate (STMP) is a non-toxic and water soluble cyclic triphosphate,

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and has been used widely as effective crosslinker for polysaccharides such as starches,

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pullulan, dextran, and xanthan gum in food, pharmaceutical, and tissue engineering

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applications (Bejenariu, Popa, Dulong, Picton, & Le Cerf, 2009; Carbinatto, de Castro, Cury,

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Magalhães, & Evangelista, 2012; Fricain et al., 2013). The coupling reaction mechanism of

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XG and STMP is indistinct, due to the complex conformational structure and transition of XG

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chains at supermolecular level. The native secondary structure of XG consists of a five-fold

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helical structure, which is stabilized by hydrogen bonds, electrostatic interactions and steric

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effects (Moorhouse, Walkinshaw, & Arnott, 1977). The order-disorder conformational

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transition can be induced by changing temperature, pH, or ionic strength (Agoub, Smith,

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Hatakeyama, 2001; Paoletti, Cesaro, & Delben, 1983). In aqueous solution, XG

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macromolecules exist in an ordered helical shape at physiologically relevant temperatures and

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salt concentrations, whereas a disordered chain conformation at elevated temperature and low

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ionic strength. Generally, the esterification and crosslinking reaction between polysaccharides

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and STMP occurs in alkaline medium. However, the investigation on conformational

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transition of XG in alkaline medium is scarcely published. We wonder to know what will

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happen for the XG chains during the formation of ester linkages between XG and STMP

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under the given synthesis condition. We have accumulated experience in modifying and

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characterizing polysaccharides (Tao et al., 2015; Tao & Feng, 2012; Tao, Zhang, Yan, & Wu,

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2007). Moreover, it is crucial to clarify the conformations and transition in the synthesis

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system, and subsequently to optimize the reaction condition for XG crosslinking.

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Dynamic oscillation testing is commonly used to provide a more explicit and detailed

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characterization of gel structure (Brenner, Tuvikene, Fang, Mastsukawa, & Nishinari, 2015;

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Da-Lozzo et al., 2013). In this work, the XG-STMP hydrogels were fabricated by

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esterification reaction in alkaline medium, and the rheological measurements of temperature

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and time sweep were performed to monitor the in situ crosslinking process. We attempt to

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demonstrate the forming kinetics and gel mechanical stability for the XG-STMP hydrogels.

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Furthermore, the chemical structure, morphology, oscillation frequency sweep, swelling and

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release properties were characterized to clarify the effect of XG concentration on the

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properties of the resultant XG-STMP hydrogels. This work provides some fundamental

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information for further application of the XG-based hydrogels in biomedical, food and tissue

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engineering.

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2. Experimental

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2.1. Materials Biological Reagent Grade xanthan gum ((C35H49O29)n, density: ρ = 1.50 g/mL at 20 ℃)

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and bovine serum albumin (BSA, 99% purity) were purchased from Shanghai Ryon

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Biological Technology. Co. Led (Shanghai, China). Chemical pure sodium trimetaphosphate

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was provided by Aladdin (Shanghai, China). NaCl, NaOH, KCl, KH2PO4, and Na2HPO4 used

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here were analytically pure, and obtained from Shanghai Chemical Co. in China.

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2.2. Preparation of XG-STMP hydrogels

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About 1, 1.5, 2, and 2.5 g of XG were dissolved individually in 50 mL of 0.1 M aqueous

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NaOH (pH=13), and were kept under magnetic stirring at room temperature (16~25 ℃) for

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24 h. A series of XG solutions with concentration of 2%, 3%, 4%, and 5% (w/v) were

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prepared, respectively. About 1.5, 2.25, 3.0, and 3.76 g of cross-linker STMP was dissolved in

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20 mL of deionized water to obtain the STMP solutions with concentration of 75, 112.5, 150,

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and 188 mg/mL, which were used to crosslink the XG solutions with different concentration

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mentioned above, respectively. The stoichiometric ratio of the system, which is defined as the

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ratio between the cross-linker moles (M = 305.89 g/mol) and the moles of repeating units of

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XG (Mrepeating unit = 933 g/mol), was set to be 3.66 in this work. To fabricate the XG-STMP

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hydrogels, 16 mL of STMP solution was added to 50 mL of XG solution, and kept under

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magnetic stirring for a few minutes. Subsequently, 3 mL of pre-gel solution was transferred

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into a cylindrical mold, and then stayed for 3 h at 37 ℃ to form a hydrogel disk (16 mm

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diameter, 15 mm thickness). After being detached from the mold, the resulting hydrogels were

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washed using deionized water for more than 10 times, and incubated in deionized water at

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37 ℃ for 24 h before being lyophilized and characterization. By means of such procedure, a

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series of hydrogels were fabricated according to the stoichiometric ratio of 3.66, and were

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coded as XG2-STMP, XG3-STMP, XG4-STMP, and XG5-STMP, respectively. The control

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sets were similarly prepared for 3% and 5% (w/v) XG, but equal volumes of deionized water

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were mixed instead of cross-linking reagent STMP. The resultant hydrogels were coded as

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XG3-water and XG5-water.

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2.3. Intrinsic viscosity measurement Intrinsic viscosity ([η]) of XG in 0.1 M aqueous NaCl and 0.1 M aqueous NaOH were

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measured at 25 ± 0.1 ℃ by using an Ubbelohde capillary viscometer, respectively. Huggins

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equation was used to estimate the [η] value by extrapolating to an infinite dilution formulated

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as (Huggins, 1942)

η sp / c = [η ] + k ' [η ] 2 c

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k′ is constant for a given polymer at a given temperature in a given solvent; ηsp/c, reduced

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specific viscosity; c, XG concentration in the range from 6.845×10-4 to 3.603×10-4 g/mL in

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0.1 M aqueous NaCl, and from 7.010×10-4 to 3.887×10-4 g/mL in 0.1 M aqueous NaOH.

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2.4. Chemical structure and morphology observation

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Infrared spectra (IR) of the samples were recorded with FTIR spectrometer (Bruker Tensior 27, German) in the range of 4000—400 cm-1 using the KBr-disk method. The cross-sections of the dried hydrogels were characterized with scanning electron

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microscopy (SEM, JSM-6510, Japan). To maintain the hydrogel structure in a dry state, the

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hydrogels were frozen at ‒ 40 ℃ for more that 12 h and then lyophilized for 48 h. The

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lyophilized samples were cut to expose their cross-sections and sputter-coated with Pt (240 s,

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40 mA), and then imaged with SEM under an accelerating voltage of 20 kV. The pore size

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measurements of the freeze-dried hydrogels were performed with Adobe Photoshop CS, and

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the major axis of the individual pore in the SEM images was measured one by one to collect

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data. Based on the statistic analysis, the average diameter of the pore for the samples was

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obtained from these measurements.

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2.5. Rheological Measurements Rheological analysis was performed on an AR 2000ex rheometer (TA Instrument, USA)

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using a 40 mm steel parallel plate geometry and 1 mm gap. The temperature was controlled

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by a water bath connected to the Peltier system in the bottom plate. A solvent trap jacket was

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used to prevent dehydration during measurements. XG and STMP solutions in given

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stoichiometric ratio were quickly mixed and transferred onto the centre of the bottom plate.

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The upper plate was immediately lowered to the desired gap size, and the dynamic oscillating

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measurement was started. Temperature sweeps for the XG3-STMP, XG3-water, XG5-STMP,

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and XG5-water hydrogels were performed at 1 Hz and 1% strain upon heating from 20 to

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60 ℃, and subsequently upon cooling from 60 to 20 ℃ at a rate of 1 ℃/min. Time sweep

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was used to monitor in situ the cross-linking process of the XG-STMP hydrogels at 37 ℃.

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Dynamic strain sweep between 0.1% and 200% at 1 Hz and at 37 ℃ was conducted after time

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sweep at 37 ℃ for 10 min and 3 h, respectively. Dynamic frequency sweep was performed in

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frequency range from 0.1 to 100 rad/s at 37 ℃ by keeping the strain constant at 1% (within

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the linear viscoelastic region) after time sweep at 37 ℃ for 10 min and 3 h, respectively. The

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storage modulus (G′), the loss modulus (G′′), loss tangent (tanδ), and complex viscosity (η*)

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were recorded as a function of temperature, time, strain%, and angular frequency (ω),

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respectively.

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2.6. Swelling and diffusion tests

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To assess the swelling property of the dried XG-STMP hydrogel samples, the hydrogels

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were frozen and lyophilized to obtain the dry weigh (W0). Then the dried XG-STMP

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hydrogels were immersed in phosphate buffer saline (PBS, pH=7.4) and deionized water at

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25 ℃ for more than 11 days. At predetermined time intervals, the swollen hydrogels were

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taken out from the immersion medium and weighed (Wt) after wiping excess surface water

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with filter paper. The swelling ratio for each hydrogel was calculated according to the

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following equation: Swelling ratio =

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All the experiments were performed in triplicate.

Wt W0

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To assess BSA release from the XG-STMP hydrogels, lyophilized hydrogels were

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swollen with 7 mL of BSA aqueous solutions (60 mg/mL) for 48 h at 25 ℃. Pipet the excess

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BSA solution, and then the swollen hydrogels were immediately rinsed with PBS for three

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times. The excess BSA solution and the washing solution were collected together for

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calculating the BSA amounts absorbed by the XG-STMP hydrogels. Subsequently, the BSA

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loaded hydrogels were immersed into 20 mL of PBS media. At different time intervals, 2.5

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mL of medium was pipetted out for analysis and 2.5 mL of fresh PBS was replenished. The

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BSA concentration in the collected aliquots was quantified with a UV-visible

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spectrophotometer (Shimadzu UV-2550, Japan) at 280 nm according to a calibration curve.

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The diffusion mechanism of BSA from the XG-STMP hydrogels was evaluated by the

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diffusional coefficient, n, which can be calculated according to (Ritger, & Peppas, 1987)

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Mt = kt n M∞

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where Mt and M∞ are the cumulative mass of BSA released from the hydrogel after t and

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infinite time (∞), respectively. The n value may be obtained from the slope of the straight line

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fitting the data of ln(Mt/M∞) vs. lnt.

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3. Results and discussion

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3.1. Intrinsic viscosity and viscosity average molecular weight of XG

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A good linear relationship between ηsp/c and c for XG has been obtained (not shown),

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The viscosity average molecular weight (Mη) is calculated to be 1.43×106 g/mol by applying

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the Mark-Houwink equation, [η ] = 1.10 ×10 −4 M w1.16± 0.03 (cm3 g −1 ) in 0.1 M aqueous NaCl at

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25 ℃ in the Mw range from 2.09×105 to 7.40×106 g/mol, which is established from the

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published data (Sato, Kojima, Norisuye, & Fujita, 1984). Sato et al. (1984) have confirmed

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that xanthan dissolves as dimers in 0.1 M aqueous NaCl, and that the dimer has the

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double-stranded helical structure. Furthermore, [η] is measured to be 1486 cm3g-1 in 0.1 M

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aqueous NaOH at 25 ℃, which is unremarkable difference from that in 0.1 M aqueous NaCl

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at 25 ℃ . Thus, XG dissolves in 0.1 M aqueous NaOH at 25 ℃ without significant

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impacting for its double-stranded helical structure.

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3.2. Chemical structure and morphology observation of the XG-STMP hydrogels

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Fig. 1 shows the FTIR spectra of XG, physical mixture of XG and STMP, and the dried

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XG-STMP hydrogels. A broad absorption peak at 3400 cm-1 is shown in the FTIR spectrum of

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XG due to the O—H stretching vibration of hydroxyl group. The peaks appeared at 1618,

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1412, and 1061 cm-1 for XG, are ascribed to asymmetrical C=O stretching vibration and

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symmetrical C=O stretching vibration of carboxylate anion (—COO¯) and C—O stretching of

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primary alcohols, respectively (Kumar & Ahuja, 2012). The characteristic peak at 1732 cm-1

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for carbonyl groups of —COOH has also been observed, indicating that salt (—COO¯) and

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acid (—COOH) structures co-exist in XG chains. Compared with the XG FTIR spectrum, the

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new absorption peaks at 1443, 1232, and 1140 cm-1 for the XG-STMP hydrogels are

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attributed to the vibration of P=O, and the peaks at 1024 and 976 cm-1 are assigned to

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stretching vibration of P—O—C (Liu et al., 2014). The physical mixture of XG and STMP

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exhibited peaks at 1298 and 997 cm-1 due to P=O and P—O bonds, respectively, but the two

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peaks disappeared in the FTIR spectra of the XG-STMP hydrogels. It indicates that the STMP

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(Shalviri, Liu, Abdekhodaie, & Wu, 2010). The reactions of STMP with XG may result in the

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formation of phosphorous species grafting onto a sugar unit or/and the cross-linkage formed

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by one phosphorous group with two sugar ring in different XG chain. Swelling tests were

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carried out, and the results showed that the XG-STMP hydrogels swell well but did not

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dissolve in deionized water for more than 11 days, suggesting the occurrence of crosslinking

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reaction. The XG-STMP hydrogels were fabricated by esterification crosslinking between

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hydroxyl groups on the XG chains and STMP. The coupling reaction can be activated in

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alkaline condition, and the preparation procedure is illustrated in Fig. 2a. Furthermore, the

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morphology observation for the XG-STMP hydrogels is shown in Fig. 2b‒e. All the

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freeze-dried hydrogels have highly porous and interconnected network structures. The pore

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sizes of the freeze-dried networks were calculated by determining the major axis of the

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individual pores on the basis of the statistic analysis. The average pore diameter of

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freeze-dried XG2-STMP hydrogel is 114.5±22.1 µm. With the increase of the XG

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concentration, the pore size decreased to 110.5±27.7, 92.2±30.4, and 31.5±5.5 µm for

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XG3-STMP, XG4-STMP, and XG5-STMP, respectively. The decrease in the pore size can be

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attributed to the increase in crosslinking density.

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3.3. Rheological behaviors

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3.3.1. Temperature effect on the in situ forming of hydrogels

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To evaluate the gel forming kinetics and gel mechanical stability, temperature evolution

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measurements were performed to monitor the in situ crosslinking process for the XG-STMP

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hydrogels. Fig. 3 shows the change of G′, G′′, and tan δ with temperature in thermal cycles for

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XG3-STMP and XG3-water. G′ remains constant up to 25 ℃, and after this temperature, G′

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decreases slowly for the XG3-STMP hydrogel upon heating over the approximate temperature

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range from 25 to 40 ℃ , whereas the corresponding value increases gradually for the 10

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lower than those of the initial one at those temperatures below 37 ℃. However, when the

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XG3-water hydrogel was cooled down, G′ increases gradually over the approximate

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temperature range from 50 to 20 ℃. In this work, the hydrogels were fabricated based on two

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adjacent XG chains crosslinked with STMP under alkaline medium (pH=13), in which

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condition, the chemical and physical crosslinkages between XG chains co-existed in the

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synthesis system under the temperature below 50 ℃. The physical crosslinkages are based on

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the hydrogen bonds between XG chains, which are ready to be destroyed by the influence of

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the basic medium and further chemical crosslinking between XG and STMP upon heating.

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The ordered aggregates of helical XG chains were progressively disrupted, and the junction

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zones formed through hydrogen bonding were replaced gradually by coupling reaction

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between XG and STMP. Thus, the XG chains in the XG3-STMP hydrogel were

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predominantly in a disordered arrangement of helical conformation after crosslinking XG and

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STMP, and the occupied space of phosphate diester linkage is larger than that of O—H



O

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linkage. Accordingly, a less compact network was created with chemically crosslinking XG

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and STMP as compared to the initial one formed mainly by hydrogen bonding (Alupei, Popa,

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& Hamcerencu, 2002). Compared with the XG3-STMP hydrogel, equal volume of water was

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mixing instead of STMP solution for preparing the XG3-water hydrogel. Under such

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circumstances, XG chain mobility increases upon heating as a result of chain reorganization,

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which leads to more physical association between adjacent XG chains and consequently

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creates more junction zones. Such reorganization also explains the increase in G′ with

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temperature rising for XG3-water system in which no chemical reaction takes place. G′′

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remains essentially constant on heating and increases slightly on cooling, further suggesting

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the change of the helical XG chains packing to ordered aggregates in XG3-water hydrogel

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(Capron, Brigand, & Muller, 1998). Nevertheless, for the XG3-STMP system, the

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reorganization on heating is no longer possible. Therefore, the junction zones based on

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ordered aggregates of the helical XG chains in the XG3-water system are more than these in

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the XG-STMP hydrogels. However, the application of physical gels is limited due to lack of

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thermal, pH, or salt stability over the long-term in aqueous solution. Therefore, it is necessary

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to fabricate hydrogels through chemical crosslinking.

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If the temperature dependency of the rheological behavior for the hydrogels is a

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consequence of the reorganization of the XG chains, it should depend on the XG

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concentration. To clarify it, the temperature sweeps of the XG5-STMP and XG5-water

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hydrogels were also preformed upon heating and cooling over the temperature range from 20

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to 60 ℃. Fig. 4 shows the change of G′, G′′, and tan δ with temperature in thermal cycles for

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XG5-STMP and XG5-water. Compared with the XG3-STMP hydrogel, G′ for the XG5-STMP

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hydrogel show the same temperature-dependent profile upon heating and cooling. It is worth

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noting that, contrary to what is observed for the XG3-water hydrogel, there is a slight

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reduction in moduli when XG5-water hydrogel is heated over the approximate temperature

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range from 20 to 40 ℃. For the XG3-water hydrogel, heating causes a G′ increase up to

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50 ℃, and the cooling curve distinctly differs from the heating curve and a large thermal

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hysteresis. However, for the XG5-water hydrogel, no significant change in the G′ profile is

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observed upon cooling, and the heating and cooling curves are almost superimposable. When

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XG concentration is as high as 5% (w/v), entanglement is more dramatic and therefore chain

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mobility extremely reduces. As a consequence, the possible reorganization arising from the

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increase in chain mobility on heating is no longer observed in XG5-water. Therefore, for the

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XG-water hydrogels, the rheological behavior is dependent on the XG concentration, whereas,

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the temperature sweep profile is similar to each other for the XG-STMP hydrogels upon

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heating and cooling. It indicates that the temperature-dependence rheological behavior results

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from reorganization of the XG chains for the XG-water hydrogels, whereas the chemical

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crosslinkage were formed between XG and STMP for the XG-STMP hydrogels. G′ of XG-water is higher than that of XG-STMP (Fig. 3a and Fig. 4a). The XG

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molecules in the XG-water hydrogels are partially ordered, and they align and stack together

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to build up a tenuous, gel-like structure. However, the relatively low values of tan δ show that

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the XG-STMP hydrogels display more solid-like gel behavior than the XG-water hydrogels

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(Fig. 3c and Fig. 4c). It indicates that the chemical crosslinking with stronger elastic property

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exists in the XG-STMP hydrogels than the hydrogen bonding in the XG-water hydrogels.

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3.3.2. Time effect on the in situ forming of hydrogels

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On the base of the results of temperature sweep, the transition happened in the

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temperature range from 20 to 40 ℃ for the XG-STMP hydrogels. 37 ℃ is close to the body

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temperature, and generally, cell culture for tissue engineering application is performed under

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this temperature. Thus, in this work, 37 ℃ is chosen in the following rheological tests. The

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time dependence of G′, G′′, tan δ, and η* for the in situ forming XG-STMP hydrogels at a low

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angular frequency (ω = 6.28 rad/s) and a small strain% (1%) at 37 ℃ over an extended

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period of time is depicted in Fig. 5. The elastic response is dominating (G′ > G′′) for all the

306

XG-STMP samples, which is approximately 5 min after mixting XG solutions with STMP

307

crosslinker. The moduli and complex viscosity decrease rapidly from the beginning of the test,

308

and reach valley after approximately 45, 38, 25, and 15 min for the XG2-STMP, XG3-STMP,

309

XG4-STMP, and XG5-STMP hydrogels, respectively. Subsequently, gradual increase of

310

moduli and complex viscosity were observed for all the XG-STMP hydrogels. Under alkaline

311

medium, STMP disrupts the xanthan ordered aggregate structure and subsequently crosslinks

312

chemically with XG chains to form the new network structure. Compared with the

313

XG5-STMP hydrogel, when deionized water was added to the XG5 solution instead of STMP,

314

it was found that the storage modulus and complex viscosity of the resultant XG5-water

AC C

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13

ACCEPTED MANUSCRIPT hydrogel increased rapidly, whereas the loss modulus decreased from the beginning of the test,

316

and slow down after approximately 25 min (Fig. 5d). The storage modulus and complex

317

viscosity for the XG5-water hydrogel increased over the sweep time range due to much more

318

junction zones forming at this temperature, which is attributed to the chain mobility and

319

subsequent ordered stack of XG chains. Furthermore, Fig. 5e illustrates the time evolution of

320

tan δ for the XG-STMP hydrogels. When the XG2-STMP and XG3-STMP hydrogels are

321

exposed under the time sweep, the tan δ values increase with G′′ growing more rapidly, and

322

subsequently, a slight decrease of tan δ is observed. However, the tan δ values for the

323

XG4-STMP and XG5-STMP hydrogels drop dramatically at the initial stage, and then

324

practically keep intact at the later stage of the time sweep range. It suggests that the

325

XG4-STMP and XG5-STMP systems become much more elastic than the initial ones and

326

shows solid-like characteristics after crosslinking for 3 h.

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327

In order to evaluate the crosslinking degree, we used the rubber elasticity theory

328

correlating the storage modulus and the average molecular weight between cross-links ( M c ).

329

For partially hydrated crosslinked networks, this equation takes the form as (Delmar, &

330

Bianco-Peled, 2015)

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EP G=

AC C

331

−

ρ RTν 2 −

Mc

−

(1 −

2M c −

)

(4)

Mn

332

where ρ is the density of polymer, R is the universal gas constant, T is the absolute

333

temperature, ν2 is the volume fraction of the polymer in the hydrogel, and M n is the number

334

average molecular weight before crosslinking. We used the G′ values at the end of the time

335

sweep experiment and Mη to estimate the M c values of the hydrogels. Based on the G′

336

values of 84.4, 174.8, 340.8, and 512.2 Pa and Eq. (4), the M c values are estimated to be

−

−

−

14

ACCEPTED MANUSCRIPT 337

329492, 273274, 212570, and 186107 g/mol for XG2-STMP, XG3-STMP, XG4-STMP, and

338

XG5-STMP, respectively. The number of repeat units between crosslinks, or the ratio between

339

M c and the molecular weight of a repeat unit (933 g/mol for XG) are further calculated to be

340

353, 293, 228, and 199 for XG2-STMP, XG3-STMP, XG4-STMP, and XG5-STMP,

341

respectively. The number of repeat units between crosslinks decreases with increasing the XG

342

concentration, indicating that more crosslinking events occur at higher XG concentration.

343

3.3.3. Strain sweep

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−

Strain sweep tests were used to determine the critical strain, characterizing the extent of

345

the linear viscoelastic response regime. Fig. 6 shows the results of strain sweep for the

346

XG-STMP hydrogels after crosslinking at 37 ℃ for 3 h. The values of G′ are constant below

347

18.2%, 22.3%, 29.3, and 34.8% strain for XG2-STMP, XG3-STMP, XG4-STMP, and

348

XG5-STMP hydrogels, respectively, and a decrease of storage modulus is observed above the

349

corresponding strain (Fig.6a). It suggests that the strain range from 0.1% to 18.2% is the

350

linear viscoelastic domain for the hydrogels with XG concentration ranged in 2% ‒ 5% (w/v).

351

The higher is the XG concentration, the more crosslinking networks of higher elasticity are

352

formed, and the linear viscoelastic modulus increases with the XG concentration. G′′ shows a

353

peak prior to the final decrease for the XG-STMP hydrogels (shown in Fig.6b). This trend is

354

typical for dispersion with network or cross-linked gels (Russ, Zielbauer, Koynov, & Vilgis,

355

2013). The increase prior to the drop of G′′ indicates an increasing use of deformation energy

356

to already deform subdomains of the gel structure before the inner structure finally breaks.

357

Such predeformation can result from relative movements between molecules not embedded in

358

network or free chain ends.

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344

359

OSC.Stress-strain curves for the XG-STMP hydrogels are shown in Fig. 6c. All the

360

XG-STMP hydrogels display a nonlinear elastic behavior. The stiffness of the hydrogel

361

networks was determined by evaluating shear modulus from the slope of the initial linear 15

ACCEPTED MANUSCRIPT region in the OSC.Stress-strain curves. The increase in XG concentration results in an

363

increase in the stiffness of the XG-STMP hydrogel network. The shear modulus is 81.8±0.2 to

364

526.0±1.3 Pa when XG concentration increased from 2% to 5% (w/v). The shear modulus

365

results of the XG-STMP hydrogels crosslinked for 3 h and for 10 min are listed and compared

366

in Table 1. The increase in the shear modulus can be attributed to the formation of dense

367

network that can sustain high mechanical deformation.

RI PT

362

The dependences of G′ and G′′ on strain%, and OSC.Stress-strain curve for the

369

XG5-STMP and XG5-water hydrogels are illustrated in Fig. 6d‒f for comparison. The linear

370

viscoelastic modulus G′ (Fig. 6d) and G′′ (Fig. 6e) of the XG5-water hydrogel are higher than

371

those of the XG5-STMP hydrogel, suggesting that much more junction zones are existed in

372

XG5-water system. However, the “strong” XG5-water hydrogel is easier to be broken than the

373

XG5-STMP hydrogel as shown in Fig. 6f. The OSC.Stress of XG5-water hydrogel becomes

374

lower than that of the XG5-STMP hydrogel as the applied strain% gets 35%. This indicates

375

that the XG5-STMP hydrogel behaves more elastic and tougher to resist the deformation than

376

the XG5-water hydrogel, that is to say, the crosslinking structure formed from XG and STMP

377

is tougher than that from hydrogen bonding between XG chains.

378

3.3.4. Frequency sweep

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368

The frequency sweep was performed to obtain the information about the hydrogel

380

structure (Clark, & Ross-Murphy, 1987; Doucet, Gauthier, & Foegeding, 2001). Fig. 7 shows

381

the frequency dependence of G′, G′′, η*, and tan δ

382

crosslinking at 37 ℃ for 3 h. The elastic modulus is significantly higher than the viscous

383

modulus for the XG-STMP hydrogels over the entire frequency sweep range, resulting a

384

relatively low values of tan δ (0.3‒0.1), and the complex viscosity decreases with increasing

385

frequency. It indicates that XG-STMP hydrogels possess a solid-like gel structure but with

386

injectable characteristics. A power-law frequency dependence of storage modulus, loss

AC C

379

for the XG-STMP hydrogels after

16

ACCEPTED MANUSCRIPT 387

modulus, and complex viscosity was observed, respectively, and can be approximated with

388

simple models as follows (Khondkar, Tester, Hudson, Karkalas, & Morrow, 2007;

389

Martinez-Ruvalcaba, Chornet, & Rodrigue, 2007; Tho, Kjøniksen, Nystrom, & Roots, 2003)

G '(ω ) = Aω n '

(5)

391

G ''(ω ) ∝ ω n ''

(6)

392

η ∗ (ω ) ∝ ω m

RI PT

390

(7)

where A is the gel strength, and ω is the oscillation frequency. The exponents of n′, n″, and m

394

are the slope in a log-log plot of G′, G′′, and η* versus ω, respectively. The resultant

395

parameters are given in Table 1. A can be interpreted as the strength of the interactions

396

between flow units, and the n′ and n″ values providing information on the variation of the

397

moduli with frequency. The XG-STMP hydrogels have relative low values of n′ and n″,

398

indicating that the storage modulus and loss modulus are slight dependent on frequency. The

399

values of gel strength (A) increase, and n′ and n″ decrease with increase of XG concentration

400

over the experimental concentration range. The n′ is much higher than n″, indicating that G′

401

increased at a higher rate than the increases in G′′ with increases in the frequency.

402

Furthermore, the m is the complex viscosity relaxation exponent of gel network. Value of m

403

close to zero announces liquidlike behavior, whereas approaching ‒1 suggests solidlike

404

response of the system. In this wok, the m values are in the rage from ‒0.863 to ‒0.928, and

405

become more negative with increasing XG concentration. These results suggest that the

406

XG-STMP hydrogels were more elastic and higher crosslinking density at increased XG

407

concentration (Sandolo, Matricardi, Alhaique, & Coviello, 2009).

408

3.4. Swelling and diffusion properties

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393

409

It is extremely important to investigate the swelling and diffusion properties of the

410

hydrogels as they can profoundly influence the controlled release behavior of drugs or

411

nutrients in medical and food engineering applications. The swelling behavior of the 17

ACCEPTED MANUSCRIPT XG-STMP hydrogels was investigated over a period of 268 h in both deionized water and

413

PBS at 25 ℃ (Fig. 8). The dynamic swelling profiles of the XG-STMP hydrogels exhibited a

414

fast swelling behavior in the first hour and achieved the equilibrium state within 28 h in PBS

415

(Fig. 8a). The initial faster swelling of the XG-STMP hydrogels was due to the osmotic

416

pressure difference. The effect of XG concentration was observed on the equilibrium swelling

417

ratios of the XG-STMP hydrogels. The swelling ratio of the XG-STMP hydrogels increased

418

with an increase in XG concentration in both deionized water and PBS. It is mainly attributed

419

to the hydrophilic nature of xanthan gum. Usually, an increase in the cross-linking density

420

leads to a lower swelling ratio of hydrogels. However, in this case of STMP cross-linked

421

xanthan gum hydrogels, the reaction with STMP introduces more anionic charges to XG

422

chains, in addition to crosslinkages. Thus, water uptake into the XG-STMP hydrogels

423

becomes greater due to a larger number of negative charges within the covalent phosphorous

424

species. Furthermore, the counter ions inside the XG-STMP hydrogels to neutralize the fixed

425

charges on the XG chains also increase osmotic pressure resulting in high swelling ratio

426

(Shalviri, Liu, Abdekhodaie, & Wu, 2010). Additionally, a relative lower equilibrium swelling

427

ratio of the XG-STMP hydrogels was observed in PBS compared to deionized water (Fig. 8b).

428

It is attributed to the salting-out effect, which is a characteristic of the aqueous solutions of

429

many polymers. (Zhang et al., 2013). The addition of salts in polymer aqueous solutions

430

results in a partial dehydration of polymer chains and decreases the hydrophilicity of the

431

polymer chains. Thus, the presence of salt reduces the hydrophilicity and equilibrium swelling

432

ratio of the XG-STMP hydrogels

AC C

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412

433

The diffusion properties were carried out in PBS at 25 ℃ using BSA as a model protein.

434

The diffusion profiles of BSA from the XG-STMP hydrogels are illustrated in Fig. 8c. The

435

results show that the BSA diffused gradually into the PBS medium in a sustained manner.

436

Moreover, the release behavior of BSA-loaded hydrogels showed a different trend with the 18

ACCEPTED MANUSCRIPT increase of XG concentration. The XG2-STMP and XG3-STMP hydrogels exhibited burst

438

release behavior for the first 8 h until reaching a maximum at 50 h and 28 h, respectively, and

439

then followed by a slower diffusion rate for the remainder of the study. Compared with the

440

XG2-STMP and XG3-STMP hydrogels, BSA diffused at a relative slow rate in the

441

XG4-STMP and XG5-STMP hydrogels and reached a maximum at 50 h, subsequently with a

442

platform. The cumulative release ratios of BSA from the XG2-STMP, XG3-STMP,

443

XG4-STMP, and XG5-STMP hydrogels were 90.0%, 64.4%, 34.8% and 28.3%, respectively.

444

To determine the diffusion kinetics and mechanism, the diffusion data was fitted according to

445

Ritger-Peppas equation (Ritger, & Peppas, 1987). For the slab, the exponent n is equal to 0.5

446

for Fick diffusion, and is between 0.5 and 1.0, or n=1.0 for non-Fickian model. In the case of

447

a cylinder, n=0.45 instead of 0.5, and 0.89 instead of 1.0 (Costa, & Loba, 2001). The fitting

448

data are listed in Table 2. The n values for the XG2-STMP and XG3-STMP hydrogels in the

449

first diffusion stage are less than 0.45, indicating that the release mechanism of BSA was

450

mainly Fickian diffusion. However, in the second stage, the n exponents are more than 0.45,

451

suggesting that the BSA release from the XG2-STMP and XG3-STMP hydrogels was Fickian

452

diffusion combined with XG chain relaxation. For the XG4-STMP and XG5-STMP hydrogels,

453

the n exponents are not more than 0.45, indicating that BSA released from the matrix follows

454

Fickian diffusion mechanism. The difference of BSA release mechanism between XG-STMP

455

hydrogels was probably influenced by the density of cross-linking and hydrogen bond

456

between XG and BSA. The XG-STMP hydrogels hold the potential for sustained release of

457

drugs or nutrients in medical and food engineering applications.

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437

458 459

4. Conclusion

460

The esterification and crosslinking reaction between xanthan gum and STMP led to the

461

creation of the XG-STMP hydrogels. Temperature and time evolution measurements were 19

ACCEPTED MANUSCRIPT performed to evaluate the forming kinetics and mechanical stability during the in situ

463

crosslinking process. Strain and frequency sweeps were carried out to obtain the information

464

about the hydrogel structure. Based on the results from oscillatory rheology, the transition

465

from hydrogen bonding to chemical crosslinking for the XG chains reached a balance

466

approximately at 37 ℃ for the XG-STMP hydrogels, which is close to the body temperature.

467

The chemical crosslinking networks existed in the XG-STMP hydrogels with stronger elastic

468

property and more solid-like gel behavior than the hydrogen bonds interaction in the physical

469

hydrogels with the same XG concentration, and exhibited more elastic and tougher to resist

470

the deformation than the physical XG hydrogel. The hydrogels with porous and

471

interconnected structure had good swelling and release-controlled properties, which were

472

tunable by changing XG concentration during the esterification and crosslinking with STMP.

473

The hydrogels fabricated from the XG and STMP exhibit the potential for sustained release of

474

drugs or nutrients in medical and food engineering applications.

475 476

Acknowledgements

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This work was supported by the National Natural Science Foundation of China

478

(21344006). Yongzhen Tao gratefully acknowledges financial support from China Scholarship

479

Council (2011842374). The authors thank Analysis and Measurement Center of Wuhan

480

Textile University, China, for supplying the instruments of the FTIR, UV, Rheological, and

481

SEM measurements.

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24

ACCEPTED MANUSCRIPT Table Captions Table 1 The experimental results of n′, n′′, m, A, and shear modulus of the XG-STMP hydrogels crosslinked for 10 min and 3 h, respectively. Table 2 BSA release kinetic data for the XG-STMP hydrogels obtained from the linear fitting

RI PT

experimental data according to Ritger-Peppas equation (n: diffusion exponent; k: kinetic

AC C

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constant; R2: correlation coefficient).

ACCEPTED MANUSCRIPT Table 1

A Crosslinking time

Sample

10 min

XG2-STMP

62.7

0.140 0.039 0.863

XG3-STMP

129.8

0.122 0.014 0.894

154.2±0.5

XG4-STMP

276.2

0.099 0.012 0.912

325.7±0.4

XG5-STMP

415.5

0.087 0.009 0.914

485.0±0.5

XG2-STMP

67.4

0.140 0.053 0.877

81.8±0.2

XG3-STMP

147.8

0.103 0.020 0.896

172.7±0.4

XG4-STMP

290.3

0.098 0.019 0.908

334.9±0.8

XG5-STMP

446.4

0.089 0.014 0.928

526.0±1.3

AC C

EP

n′′

-m

shear modulus (Pa) 77.0±0.3

SC

RI PT

n′

M AN U

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3h

(Pa⋅s n′)

ACCEPTED MANUSCRIPT Table 2

Mt = kt n M∞

Sample

R2

k

n

R2

XG2-STMP

30.5

0.22

0.947

8.7

0.47

0.981

XG3-STMP

16.5

0.24

0.996

2.8

0.63

0.964

XG4-STMP

8.8

0.38

0.982

XG5-STMP

5.3

0.45

0.974

M AN U

SC

n

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The second stageb

k

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Just for describing the diffusion stages of the XG2-STMP and XG3-STMP hydrogels.

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a,b

The first stagea

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1 7 32 34 00

14 12

1 6 18

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XG

1 06 1

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m ix tu re o f X G a n d S T M P

1298

997

M AN U

X G 2 -S T M P

X G 3 -S T M P

TE D

X G 4 -S T M P

X G 5 -S T M P

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7 27

553

1626 1443 1232

976 898 3404

1 02 4 1140

3500

2800

2100

1400

700

-1

W a ve n u m b e rs (c m )

Fig. 1. FTIR spectra of XG, mixture of XG and STMP, and the dried XG-STMP hydrogels.

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Fig. 2. Reaction scheme of the XG-STMP hydrogels (a), and SEM images for the XG2-STMP (b), XG3-STMP (c), XG4-STMP (d), and XG5-STMP (e) hydrogels crosslinked at 37 ℃ for 3 h.

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Heating for XG3-STMP Cooling for XG3-STMP Heating for XG3-water Cooling for XG3-water

a 3

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10

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30 40 50 o Temperature ( C)

Heating for XG3-STMP Cooling for XG3-STMP Heating for XG3-water Cooling for XG3-water

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G'' (Pa)

60

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Temperature ( C)

c

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Heating for XG3-STMP Cooling for XG3-STMP Heating for XG3-water Cooling for XG3-water

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20

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o

Temperature ( C)

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Fig. 3. Temperature dependence of G′(a), G″(b), and tanδ (c) for the XG3-STMP

(square) and XG3-water (circle) hydrogels measured in heating (red filled symbols) and cooling (black open symbols) processes.

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a 3

Heating for XG5-STMP Cooling for XG5-STMP Heating for XG5-water Cooling for XG5-water

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60

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Heating for XG5-STMP Cooling for XG5-STMP Heating for XG5-water Cooling for XG5-water

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30 40 50 o Temperature ( C)

60

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tan δ

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20

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o

Temperature ( C)

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Fig. 4. Temperature dependence of G′(a), G″(b), and tanδ (c) for the XG5-STMP (square) and XG5-water (circle) hydrogels measured in heating (red filled symbols) and cooling (black open symbols) processes.

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80

45 min 15

180 160 35

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30 25

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2000 4000 6000 8000 10000 12000

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G', G'' (Pa); η * (Pa.s)

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G' G'' η*

b

200

G', G'' (Pa); η * (Pa.s)

G', G'' (Pa); η* (Pa.s)

G' G'' η*

a

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tan δ

45 min 0.18

38 min

0.16

0.14

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XG2-STMP XG3-STMP XG4-STMP XG5-STMP

0

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Time (s)

Fig. 5. Time dependence of G′, G″, and η* for the hydrogels: XG2-STMP (a),

XG3-STMP (b), XG5-STMP (c) and XG5-water (d), and tanδ (e) for the XG-STMP hydrogels.

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3

10

a

b

18.2%

XG2-STMP XG3-STMP XG4-STMP XG5-STMP

22.3% 2

10

34.8%

2

10

XG2-STMP XG3-STMP XG4-STMP XG5-STMP 0.1

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G'' (Pa)

G' (Pa)

29.3%

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Strain%

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osc.stress (Pa)

osc.stress (Pa)

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XG5-STMP XG5-water

d

XG2-STMP XG3-STMP XG4-STMP XG5-STMP

c

G'' (Pa)

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SC

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100 0

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10 0.01

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Strain%

AC C

Strain%

Fig. 6. G′ (a) and G′′ (b) as a function of strain%, and OSC.Stress-strain curves (c) with frequency of 1 Hz at 37 ℃ for the XG-STMP hydrogels, in comparison with the XG5-water hydrogel (d‒f).

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G' G'' G' G'' G' G'' G' G''

G',G'' (Pa)

a

2

10

XG2-STMP XG2-STMP XG3-STMP XG3-STMP XG4-STMP XG4-STMP XG5-STMP XG5-STMP

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ω (rad/s)

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tan δ

XG2-STMP XG3-STMP XG4-STMP XG5-STMP

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-2

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ω (rad/s)

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XG2-STMP XG3-STMP XG4-STMP XG5-STMP

c

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EP

∗

η (Pa s)

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1

10

AC C

0

10

-2

10

-1

10

0

10

1

10

ω (rad/s)

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10

3

10

Fig. 7. Variation of (a) G′ (filled symbol) and G″ (open symbol), (b) tanδ, and (c) η* as a function of angular frequency with strain%=1% for the XG2-STMP (black square), XG3-STMP (red circle), XG4-STMP (green triangle), and XG5-STMP (blue inverted triangle) hydrogels.

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a 9 6 XG2-STMP XG3-STMP XG4-STMP XG5-STMP

3 0

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b

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15 in water in PBS

12 9

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Equilibrium Sweling Ratio

Time (h)

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Swelling Ratio

12

6 3 0

TE D

XG2-STMP XG3-STMP XG4-STMP XG5-STMP

c

80 60 40

AC C

EP

Cumulative Release (%)

100

XG2-STMP XG3-STMP XG4-STMP XG5-STMP

20

0

0

30

60

90

120

150

180

Time (h)

Fig. 8. Swelling ratio (a,b) and diffusion profiles (c) for the XG-STMP hydrogels crosslinked at 37 ℃ for 3 h.

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Highlights


The esterification reaction between xanthan gum and STMP led to create hydrogels. The hydrogen bonding was disrupted and replaced gradually by coupling

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reaction.

The XG-STMP hydrogels exhibited elastic and tough to resist the deformation.




The XG-STMP hydrogels displayed good swelling and release-controlled

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properties.