Synthesis, structure, and properties of N-2-hydroxylpropyl-3-trimethylammonium-O-carboxymethyl chitosan derivatives

Journal Pre-proof Synthesis, structure, and properties of N-2-hydroxylpropyl-3-trimethylammonium-O-carboxymethyl chitosan derivatives

Qun Liu, Jialiang Chen, Xiaodeng Yang, Congde Qiao, Zhi Li, Chunlin Xu, Yan Li, Jinling Chai PII:

S0141-8130(19)38034-1

DOI:

https://doi.org/10.1016/j.ijbiomac.2019.12.125

Reference:

BIOMAC 14152

To appear in:

International Journal of Biological Macromolecules

Received date:

5 October 2019

Revised date:

23 November 2019

Accepted date:

14 December 2019

Please cite this article as: Q. Liu, J. Chen, X. Yang, et al., Synthesis, structure, and properties of N-2-hydroxylpropyl-3-trimethylammonium-O-carboxymethyl chitosan derivatives, International Journal of Biological Macromolecules(2019), https://doi.org/ 10.1016/j.ijbiomac.2019.12.125

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© 2019 Published by Elsevier.

Journal Pre-proof

Synthesis, Structure, and Properties of N-2-Hydroxylpropyl-3-Trimethylammonium-O-Carboxymethyl Chitosan Derivatives

Qun Liua, Jialiang Chena,1, Xiaodeng Yanga,*, Congde Qiaoa, Zhi Lia, Chunlin Xub, Yan Lia,c, Jinling Chaic Shandong Provincial Key Laboratory of Molecular Engineering, Qilu University of TechnologyShandong Academy of Science, Ji’nan 250353, PR China. b

Process Chemistry Centre, Laboratory of Wood and Paper Chemistry, Åbo Akademi University,

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Turku, FI-20500 Finland. c

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a

Collaborative Innovation Center of Functionalized Probes for Chemical Imaging in Universities of

Jialiang Chen is an undergraduate student enrolled in 2016, and majors in chemical engineering.

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1

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Shandong, Shandong Normal University, Jinan, Shandong Province, 250014, China.

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* Corresponding Author: Xiaodeng Yang, E-mail: [email protected]

1

Journal Pre-proof Abstract N-2-hydroxylpropyl-3-trimethylammonium-O-carboxymethyl

chitosan

(HTCMCh) was synthesized through homogeneous reaction. The effects of different reaction condition on the properties of HTCMCh were characterized by FTIR, NMR, SEM, TEM, DLS, XRD, TGA, and DSC. The results of FTIR spectra, 1H NMR, and 13

C NMR proved the successful synthesis of HTCMCh. The DS was dependent upon

reaction time and pretreated pH of the starting material, independent of temperature and nepoxy⁄n-NH2. With increasing reaction time, the crystallinity of HTCMCh

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decreased, and the intermolecular interactions transformed from hydrogen bonding to

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strong electrostatic interactions, which enhanced HTCMCh thermal stability. SEM observations showed smooth cross section morphologies of HTCMCh films. With the

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increase in reaction time, the tensile strength significantly increased. The

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viscoelasticity transformed from viscous to elastic with aging time, confirming the formation of polyelectrolyte complexes. The optimum reaction conditions: reaction

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time of 2 h, an initial material pH of 9.47, nepoxy⁄n-NH2 of 2/1.

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Keywords: N-2-hydroxylpropyl-3-trimethylammonium-O-carboxymethyl chitosan;

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synthesis; physicochemical properties

2

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1. Introduction Polyelectrolyte complexes (PECs) are composed of polyelectrolytes with opposite charge, primarily driven by hydrogen bonding, electrostatic, and hydrophobic interactions(Dautzenberg, 1997). They have garnered increased research attention for their non-toxicity and convenient structure adjustment(Ben Messaoud, Promeneur, Brennich, Roelants, Le Griel, & Baccile, 2018; Luo & Wang, 2014). Chitosan (CS) can form PECs with anion-based biomacromolecules including

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DNA (Bravo-Anaya, Fernández-Solís, Rosselgong, Nano-Rodríguez, Carvajal, & Rinaudo, 2019), sodium alginate(Komoto, Furuike, & Tamura, 2019; Kulig,

ro

Zimoch-Korzycka, Król, Oziembłowski, & Jarmoluk, 2017; Sæther, Holme, Maurstad,

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Smidsrød, & Stokke, 2008; Zhang, Wang, Hu, Feng, Xiong, Guo, et al., 2019), xanthan gum and gum ghatti(Lal, Dubey, Gaur, Verma, & Verma, 2017),

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pectin(Maciel, Yoshida, & Franco, 2015; Yao, Tu, Cheng, Zhang, & Liu, 1997),

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gelatin(Cai, Hou, Luo, Han, Fu, Zeng, et al., 2016), heparin sodium(Bueno, Souza, Follmann, Pereira, Martins, Rubira, et al., 2015), and k-carrageenan(Rassas, Braiek,

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Bonhomme, Bessueille, Rafin, Majdoub, et al., 2019), and biomacromolecules after being grafted by anions including carboxymethyl gum katira(Minkal, Ahuja, & Bhatt,

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2018), carboxymethyl cellulose(Argüelles-Monal, Gárciga, & Peniche-Covas, 1990; Belluzo, Medina, Cortizo, & Cortizo, 2016), carboxymethyl starch(Assaad, Wang, Zhu, & Mateescu, 2011), carboxymethyl cashew gum(Maciel, Silva, Paula, & de Paula, 2005), and dextran sulfate sodium(Ye, An, Wu, Zhao, Zheng, & Wang, 2019). CS-based PECs have potential applications in drug delivery(Bravo-Anaya, Fernández-Solís, Rosselgong, Nano-Rodríguez, Carvajal, & Rinaudo, 2019; Bueno, et al., 2015; Kim, Lee, Oh, Shin, Oh, Park, et al., 1999; Kulig, Zimoch-Korzycka, Król, Oziembłowski, & Jarmoluk, 2017; Minkal, Ahuja, & Bhatt, 2018), artificial tissue(Belluzo, Medina, Cortizo, & Cortizo, 2016; Han, Zhou, Yin, Yang, & Nie, 2010; Komoto, Furuike, & Tamura, 2019), smart packaging(Maciel, Yoshida, & Franco, 2015), heavy metal removal(Ye, An, Wu, Zhao, Zheng, & Wang, 2019; Zhang, et al., 2019), textiles(Cheng, Guan, Yang, Tang, & Yao, 2019; Yin, Weng, Han, Liu, Tan, 3

Journal Pre-proof Chen, et al., 2018), medical treatment(Rassas, et al., 2019), and other fields. Due to insolubility in neutral solution, CS must be protonated by adding hydrochloric acid or acetic acid, which subsequently affect the properties of PECs. To overcome the drawbacks of CS and expand their application field, CS chemical modifications are commonly performed. O-carboxyl substituted(Dong, Wen, Junxia, & Yigang, 2017; Ruza, Macedo, Marques, Paulucci, Cunha, Villetti, Castro, et al., 2019; Pan, Chen, Yang, Wu, He, Yin, et al., 2019; Wataru, Tatsuya, Yoshinari, & Masahiro, 2016) and N-ammonium substituted CS derivatives(J. Cai, Dang, Liu,

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Wang, Fan, Yan, et al., 2015; de Oliveira Pedro, Schmitt, & Neumann, 2016; Li, Wei,

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Zhang, Gu, & Guo, 2019; Martins, Nasário, Silva-Gonçalves, de Oliveira Tiera, Arcisio-Miranda, Tiera, et al., 2018; Wei, Li, Chen, Zhang, Mi, Dong, et al., 2019) are

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two kinds of CS derivatives. Chemical modifications often take place in aqueous

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NaOH solution(de Oliveira Pedro, Schmitt, & Neumann, 2016; Kalliola, Repo,

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Srivastava, Zhao, Heiskanen, Sirviö, et al., 2018), acetic acid solution(Ibrahim, Saleh, Elsharma, Metwally, & Siyam, 2019), isopropyl alcohol(Ruza, Macedo, et al., 2019;

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Ruza Gabriela Medeiros de Araújo Macedo, Marques, Tonholo, & Balaban, 2019), thionyl chloride(Cai, et al., 2015), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide,

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and N-hydroxysuccinimide(Jung, Wen, & Sun, 2019) because of the poor solubility of CS in neutral water solution. The above mentioned chemical modifications were heterogeneous reactions, which usually cause environmental pollution and potential health problems. Moreover, the heterogeneous reaction requires long reaction times and may lead to numerous byproducts, which can be cumbersome. Ionic

liquids

(ILs)

are

considered

as

potential

green

solvents

for

biomacromolecules (Chen, Xu, Li, Wang, & Zhang, 2011; Li, Asikkala, Filpponen, & Argyropoulos, 2010; Richard, Scott, John, & Rogers, 2002; Silva, Mano, & Reis, 2017; Yang, Qiao, Li, & Li, 2016). More importantly, ILs are stable over a wide temperature window and show good chemical and thermal stability and negligible vapor pressure, allowing for ease in purification through water or solvent evaporation in mixed liquid solutions and recyclability. CS derivatives have been synthesized in 4

Journal Pre-proof ILs, and their utilization in wound healing, biology and medicine, biomaterials, wastewater treatment, tissue engineering, drug and gene delivery, textiles, and food protection were investigated(Khan, Ullah, & Oh, 2016; Li, Wang, Guo, Huang, & Sun, 2012; Pei, Cai, Shang, & Song, 2014; Shamshina, Zavgorodnya, Berton, Chhotaray, Choudhary, & Rogers, 2018; Wang, Zheng, Li, Zhang, Xiao, Guan, et al., 2013, 2015; Wei, Huang, Zhou, Zhang, Hua, & Zhu, 2013). To our knowledge, there has no article about synthesis and characterization of N-quaternary ammonium-O-carboxyl anion chitosan, a kind of amphoteric chitosan derivative, in AmimCl. a

CS

of

As

derivative,

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N-2-hydroxylpropyl-3-trimethylammonium-O-carboxymethyl chitosan (HTCMCh) tends to form polyelectrolyte complexes due to its polyampholyte character. However,

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to our knowledge, there has no article about synthesis and characterization of such an

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amphoteric chitosan derivative in AmimCl. In the current paper, HTCMCh was synthesized in AmimCl by a nucleophilic substitution reaction. The effects of reaction

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time, temperature, 𝑛𝑒𝑝𝑜𝑥𝑦 ⁄𝑛−𝑁𝐻2, and pretreated pH of O-carboxymethyl chitosan on the degree of substitution were investigated. The structure and thermal properties of

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HTCMCh were characterized by FTIR, 1H NMR,

13

C NMR, XRD, DSC, and TGA.

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The HTCMCh was used to prepare films and hydrogels, and the intermolecular interactions were characterized by SEM, tensile testing and viscoelasticity. The intermolecular interactions were also studied by molecular simulation. The present study aims to investigate the influences of different reaction conditions on the properties of HTCMCh. The observations provide theoretical basis in promoting the utilization of chitosan derivatives in artificial tissue engineering, packaging materials, and so on.

2. Experimental 2.1 Materials O-Carboxymethyl chitosan (O-CMCh) was purchased from Shanghai Macklin Biochemical Co., Ltd. The deacetylation degree is 91.2%, determined by 1H NMR 5

Journal Pre-proof method, the substitution degree of carboxylation degradation is ≥ 80%, and the viscosity is 80 mPa·s. Glycidyl trimethyl ammonium chloride (GTAC) was supplied by Jiangsu Jianglai biotechnology Co. Ltd. The epoxy content was 95.0%. The determining method is similar to that reported in our previous work(Yang, Zhang, Qiao, Mu, Li, Xu, et al., 2015). 1-Allyl-3-methylimidazole chloride (AmimCl) was provided by Lanzhou Institute of Chemical Physics (Lanzhou, China). Ethanol and acetone (A.R.) were purchased from Sinopharm Chemical Reagent Co., Ltd. All the reagents were used without further purification.

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2.2 Synthesis of HTCMCh

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The synthesis of HTCMCh was similar to that of HTCC(Yang, et al., 2015). Typically, 0.4 g O-CMCh was mixed with 20 g AmimCl in a three-neck 100 mL flask.

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The three-neck flask was heated to 80 ºC with an oil bath and kept for 4 h under

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stirring. Secondly, 0.58 g GTAC (𝑚𝑒𝑝𝑜𝑥𝑦 ⁄𝑚−𝑁𝐻2 =2/1) was weighed into the flask.

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After the mixture of O-CMCh and GTAC was stirred for another 4 h, and naturally cooled to 25 ºC, precipitant of ethanol/acetone (volume ratio, 1/4) was used to

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precipitate crude HTCMCh. The crude HTCMCh was refined with above mentioned precipitant for three times. Thirdly, the refined HTCMCh was vacuum dried at 80 ºC

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for 30 h. The modification reaction procedure is shown in Scheme 1. 7

6

8

5 4

1 3

2

a

b c

d

Scheme 1 Modification reaction procedure of HTCMCh. To investigate the effect of pH on nucleophilic substitution reaction, the initial pH of O-CMCh solution was adjusted to 7.13 and 5.68, respectively, using 1.0 mol/L HCl solution. Then the O-CMCh with pH of 7.13 or 5.68 was freeze-dried and used to synthesize HTCMChs in AmimCl. The HTCMChs synthesized under different conditions were labelled with 6

Journal Pre-proof different subscripts, such as HTCMCh0.5h, which represented a reaction time of 0.5 h, or HTCMChpH5.68, which denoted synthesis from a 5.68 pH O-CMC freeze-dried solution. 2.3 Characterization of HTCMCh FTIR spectra were recorded with KBr pellets on a Nicolet iS10 infrared spectrometer (Thermo Fisher Scientific Inc., Waltham, MA) from 4000 to 400 cm-1 64 times. 1

H NMR spectra of O-CMCh and HTCMCh were detected on a Bruker Advance

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II 400 spectrometer (Bruker, Switzerland). D2O was used as solvent. HTCMCh

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synthesized at 2 and 8 h were also detected by 13C NMR.

The crystallinity of HTCMCh was determined by XRD (AXS D8-ADVANCE diffractometer,

Bruker,

Germany)

with

-p

X-ray

graphite

monochromatized

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a 2θ of 5 to 50 ºC range were used.

re

high-intensity Cu/kα radiation (λ=1.5406 Ǻ, 40 kV, 20 mA). A 2 ºC/min scan rate and

DSC data was conducted on a Q2000 differential scanning calorimeter (TA

na

instrument). The heating rate was 10 ºC/min. To eliminate the thermal history, samples of O-CMCh, HTCMCh0.5h and HTCMCh2.0h were heated to 100 ºC and kept

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for 300 s in aluminum microcapsules before recording the data. The DSC data was collected during the second heating process from -10 to 150 ºC. High purity nitrogen with a flow rate of 50 mL/min was used. Thermodynamic stability of O-CMCh and HTCMChs were evaluated by an SDT Q600 simultaneous thermal analyzer (TA Instruments, USA) from ca. 25 to 650 ºC with a heating rate of 10 ºC/min. Nitrogen was used and a flow rate was 100 mL/min. TEM images of O-CMCh aggregates in solutions with different pH were carried out on a JEM-100CXII electron microscope. The size and its distribution of O-CMCh or HTCMCh aggregates were obtained on a Zetasizer Nano ZS90 (Malvern). 2.4 Preparation and characterization of HTCMCh films HTCMCh films were manufactured by a solvent casting technique. For each film, 7

Journal Pre-proof 0.15 g of HTCMCh with various DS were solubilized in 20 mL distilled water for 4 h at 45 ºC under stirring. After the film-forming solutions were degassed, they were transformed into Teflon panes, which are 3.5 cm in diameter and 0.7cm in height. The solutions in the Teflon pane were dried by water volatilization in an air drying oven. The inner temperature of the air drying oven was fixed at 45 ºC. The HTCMCh0.5h, HTCMCh4.0h and HTCMCpH5.68 were chosen. The O-CMCh film was also prepared as a control. The morphologies of O-CMCh and HTCMCh films were observed on a JEOL

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emission source. The accelerating voltage was 5 kV.

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JSM6700F field-emission scanning electron microscope (FF-SEM) fitted with a field

The mechanical properties (tensile strength (TS) and elongation at break (EB)) of

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the O-CMCh and HTCMCh films were detected on an electronic universal testing

re

machine (Ji’nan Tenson Machinery Co. Ltd., Ji’nan). The tested films were cut into

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5.0 cm × 1.0 cm strips, and the crosshead speed was 0.2 cm/min. The thickness of the films was measured 5 points and averaged for every film. Each sample was measured

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for three film strips, and the data were averaged. 2.5 Preparation and characterization of HTCMCh Hydrogels

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To clearly present the change of the intermolecular interactions, a highly-concentrated HTCMCh system was prepared. Typically, HTCMCh0.5h aqueous solution with concentration of 2.5wt% was prepared at 40 ºC. The aqueous solution was transferred into a 50 mL single-mouth round bottom flask and vacuum rotary evaporated at 40 ºC. The samples were cooled to 25 ºC and observed every 5 min, until the weak gel was formed. The samples were weighted and the concentrations of HTCMCh0.5h (148 g/L) were calculated. Five samples were prepared. Five weak gels were placed in an incubator at 25 ºC, and the effect of aging time on the gel properties was studied. The aging times were 14h, 6, 10, 15 and 24 days. The viscoelasticity of HTCMCh gels were detected with a parallel-plate geometry (20.0 mm in diameter and 1000.0 μm in gap) on a DHR-2 rheometer (TA Instrument, USA). Before the viscoelasticity measurements, an amplitude sweep at a 8

Journal Pre-proof fixed angular frequency was performed to guarantee the used strain was in the linear viscoelastic region. The linear viscoelastic region was defined as the region where the storage modulus (G’) is unrelated to the strain. Then the storage (G’), and loss (G’’) moduli were detected within the angular frequency of 0.1 to100 rad/s at 25 ºC. 2.6 Molecular simulations The size of the simulation box was 50.0 Å × 50.0 Å × 50.0 Å and periodic boundary conditions were employed in three dimensions. Four HTCC molecules were

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placed in the simulation box to study the intermolecular hydrogen-bonding and electrostatic interactions. The consistent valence force field (CVFF) and

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corresponding atomic charges were used to describe HTCC molecules. The standard

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Lorentz-Berthelot mixing rules were used to calculate the Lennard-Jones (LJ) parameters between different types of atoms. All electrostatic interactions were

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handled using the Ewald summation technique.

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The simulation systems were first energy-minimized to determine the optimized conformations. Then molecular dynamic simulation in the NPT (T=298 K, P=1 atm)

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ensemble was employed to simulate the dynamic process of HTCC molecules in vacuum. The whole simulation process was conducted for 500 ps. During the

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simulation process, a Nosé-Hoover thermostat and barostat were chosen to maintain temperature and pressure. The damping constants of temperature and pressure were 0.1 ps.

3. Results and discussion 3.1 Synthesis of HTCMCh Fig. 1A shows the FTIR spectra of O-CMCh and HTCMCh. The spectrum of O-CMCh shows the characteristic absorption peaks at 3433 cm-1 vs(–O–H and –N–H), 2952 cm-1 v(–C–H), 1620 cm-1 δ(–NH2) or vas(–C=O), 1418 cm-1 vs(–C=O) and δ(– CH2)(Kalliola, et al., 2018; C. Xu, Cao, Zhao, Zhou, Cao, Li, et al., 2018). New peaks at 1476 cm-1 and 1388 cm-1 are observed in HTCMCh spectra. They are assigned to 9

Journal Pre-proof vs(–CH3) and vs(–NH2) in quaternary ammonium ions, respectively, indicating the successful introduction of GTAC on O-CMCh skeleton. Meanwhile, the broad peak at ca. 3400 cm-1 and the red-shifted peak from 1620 cm-1 to 1595 cm-1 are ascribed to the formation of hydrogen bonds(Xu, et al., 2018), confirming the synthesis of HTCMCh. (A) a

CMCh HTCMCh0.5h HTCMCh2.0h HTCMCh8.0h

a

(B)

b c

1418

d b

1620

3433 3540

c d

Hc

of

3468

H1,b

2852 2925

1598 1388 1476

4000

178.1

5.5

800

CMCh

c

HTCMCh2.0h

c

d

HTCMCh8.0h

90

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100

80

70

(ppm)

3.0

2.5

2.0

na

70.3

74.8

78.2

102.6

a

3.5

 (ppm)

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200

c

4.0

HCOCH3

54.2 51.7

190

/ppm

4.5

H2

60.1 56.5 55.8

180

66.3

170

62.9

160

H3,4,5,6,6'

H1

5.0

Ha

re

a

d

a

110

a

1112

3200 2400 1600 -1 Wavenumber (cm )

(C)

d

c b

-p

3429

CMCh HTCMCh0.5h HTCMCh2.0h HTCMCh8.0h

a b c d

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d

Hd

60

50

40

Fig.1 FTIR (A), 1H NMR (B) and 13C NMR (C) spectra of O-CMCh and HTCMChs. The reaction time is 0.5 h (HTCMCh0.5h), 2.0 h (HTCMCh2.0h) and 8.0 h (HTCMCh8.0h).

In the 1H NMR spectrum of O-CMCh(Fig.1B), the resonance peaks at 4.3, 3.8-3.2, 2.9, and 2.5 ppm correspond to H1, H3,4,5,6,6’, H2 and H-COCH3(Kalliola, et al., 2018), respectively. The features of the HTCMCh spectrum and that of O-CMCh are analogous, except for four new resonance peaks at 4.3, 3.1, 3.0 and 2.9 ppm. According to Wei, et al. (2019) and Xu, et al. (2011), the four resonance peaks are assigned to Hb, Hc, Hd, and Ha in the 2-hydroxylpropyl-3-trimethylammonium group, 10

Journal Pre-proof respectively. For the 13C NMR spectrum of O-CMCh(Fig.1C), the resonance peaks at 178.1, 102.6, 78.2, 74.8, 70.3, 60.1, 56.5, and 55.8 ppm correspond to C8, C1, C4, C5, C3, C7, C6, and C2(Wang, 2008), respectively. Based on our previous result(Yang, et al., 2015), the new peaks in the HTCMCh spectrum at 66.3, 62.9, 54.2, and 51.7 ppm are assigned to Cc, Cb, Cd, and Ca in the 2-hydroxylpropyl-3-trimethylammonium group, respectively. Both the 1H NMR and 13C NMR results confirm the presence of HTCMCh.

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3.2 Effect of reaction conditions on the degree of substitution According to Mourya & Inamdar (2009), the degree of GTAC substitution (DS)

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on O-CMCh could be calculated from Equation(1). The results are listed in Table 1. ICH3 ⁄9 ×100% ⁄ H2-6,6' 6

DS= I

-p

(1)

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where 𝐼𝐶𝐻3 and 𝐼𝐻2−6;6′ are the integrals of –CH3 in quaternary ammonium group in HTCMCh and the skeleton of CS, respectively.

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At a fixed nepoxy ⁄n-NH2 (2/1) and reaction temperature (80 ºC), the DS

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significantly increased from 22.0% to 41.9% with increasing reaction time from 0.5 h to 2.0 h. Then, it increased slightly to 57.6% with a prolonged reaction time of 12 h

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(Table 1). This might be ascribed to the hydrophobic interactions between 2-hydroxylpropyl-3-trimethylammonium groups and the aggregation of HTCMCh in AmimCl, which is similar to that of other carboxyl-containing chitosan derivatives(Elsabee, Morsi, & Al-Sabagh, 2009; Sui, Changqing, Yanjing, Zhiguo, & Xiangzheng, 2006; Sui, Wang, Chen, & Xu, 2004; Sui, Wang, Dong, & Chen, 2008; C.-C. Wang, Lin, Lee, & Ye, 2011). The critical aggregation concentrations of N-carboxyl-O-succinyl

chitosan

derivatives,

(2-hydroxypropyl-3-butoxy)

propyl-succinyl-chitosan, (2-hydroxyl-3-butoxyl)-propyl carboxymethyl-chitosan, and (2-hydroxyl-3-butoxyl) propyl carboxymethyl chitosans in aqueous solutions are 5.72-8.09 mmol/L (depending on the alkyl chain length of the succinyl tails)(Wang, Lin, Lee, & Ye, 2011), 16 g/L(Sui, Wang, Dong, & Chen, 2008), 0.5 g/L(Sui, Changqing, Yanjing, Zhiguo, & Xiangzheng, 2006) and 10 g/L(Sui, Wang, Chen, & 11

Journal Pre-proof Xu,

2004),

respectively.

The

–NH2

free

groups

accompanied

by

the

2-hydroxylpropyl-3-trimethylammonium groups are wrapped inside the micelle (Fig. 2A), decreasing the opportunity for the nucleophilic substitution reaction. The DLS results show that the hydrodynamic diameters of HTCMCh0.5h and HTCMCh2.0h (pH = 9.5) are ca. 400 nm and 450 nm, respectively, confirming the self-aggregation of HTCMCh in aqueous solution (Fig. 2B). The hydrodynamic diameters depend on the pH of the HTCMCh solution, which is ascribed to the decreased repulsion between carboxyl groups at low pH(de Oliveira, Hoffmann, Pereira, Goycoolea, Schmitt, &

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Neumann, 2018; Xin, Xu, Wang, Mao, & Zhang, 2008).

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At a fixed reaction time (2 h) and temperature (80 ºC), the DSs increase from 41.7% to 46.5 % with 𝑛𝑒𝑝𝑜𝑥𝑦 ⁄𝑛−𝑁𝐻2 increases from 1/2 to 2/1. This ascribes to the

-p

increase of the collision chance between the epoxy and –NH2 groups when more

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GTAC is used. In contrast, the temperature ranges from 60 to 100 ºC showed slight

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na

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influence on the nucleophilic substitution reaction.

12

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(A)

aggregation

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-p

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of

Intermolecular interactions

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Hydrogen bonding Electrostatic interaction

Fig.2 Scheme for the construction of HTCMCh micelle (A) and the particle size distribution of HTCMCh obtained at different pH and reaction time (B).

With a pH decrease of O-CMCh from 9.47 to 7.13 and 5.68, the DS decreased to 27.14% and 20.0 %, respectively, while the reaction temperature (80 ºC), reaction time (2 h) and 𝑛𝑒𝑝𝑜𝑥𝑦 ⁄𝑛−𝑁𝐻2 (2/1) were held constant. It is known that O-CMCh is a kind of pH-sensitive polysaccharide(Kalliola, et al., 2018; Xu, et al., 2018). The carboxyl group can be neutralized at a pH less than 7.2, accompanied by decreased solubility and self-aggregation in aqueous solutions(Kalliola, Repo, Srivastava, Heiskanen, Sirviö, Liimatainen, et al., 2017). In addition, the HTCMCh as described 13

Journal Pre-proof above is self-aggregating. The self-aggregation behaviors of both O-CMCh and HTCMCh reduced the opportunity that epoxy groups reacted with –NH2, leading to the decrease of DS.

Table 1 The DS and other physicochemical parameters of HTCMCh synthesized under different

(h)

(ºC)

𝑛𝑒𝑝𝑜𝑥𝑦 𝑛−𝑁𝐻2

0

-

-

-

-

-

11.44

265.8

281.4

41.44

1

0.5

80

2/1

9.47

22.02

7.84

269.1

282.1

49.88

2

1

80

2/1

9. 47

22.68

-

-

-

-

3

1.5

80

2/1

9. 47

39.13

-

-

-

-

4

2

80

2/1

9. 47

41.93

8.68

268.2

281.7

45.44

5

4

80

2/1

9. 47

45.98

7.64

268.7

281.6

44.83

6

5

80

2/1

9. 47

49.62

-

-

-

-

7

6

80

2/1

9. 47

-p

reaction conditions.

45.15

-

-

-

-

8

8

80

2/1

9. 47

58.31

8.20

268.9

282.5

51.40

9

10

80

2/1

9. 47

54.20

-

-

-

-

10

12

80

2/1

9. 47

57.64

-

-

-

-

11

2

60

2/1

9. 47

40.73

7.39

269.3

282.2

46.86

12

2

70

13

2

90

14

2

100

15

2

16

2

17

2

18

2

w1

Ti

Tm

w2

(%)

(%)

(ºC)

(ºC)

(%)

ro

of

DS

re

pH

lP

T

na

t No.

9. 47

41.07

8.58

269.3

282.5

47.02

2/1

9. 47

43.10

8.26

268.5

281.9

45.71

2/1

9. 47

43.10

7.36

267.4

281.4

45.58

80

1/1

9. 47

43.10

6.25

267.2

280.6

44.39

80

1/2

9. 47

41.75

7.54

267.5

281.4

44.62

80

2/1

7.13

27.14

-

-

-

-

80

2/1

5.68

20.00

-

-

-

-

Jo ur

2/1

Note: “-”- data not determined; w1- weight loss at the first stage; T-reaction temperature; Ti-initial decomposition temperature for O-CMCh and HTCMCh; Tm-the temperature corresponds to the maximum weight loss rate; w2-weight loss at the second stage.

3.3 Effect of reaction time on the microstructure of HTCMCh The influence of the 2-hydroxylpropyl-3-trimethylammonium group on the crystallinity of HTCMCh was investigated by XRD analysis. The XRD pattern of O-CMCh (contrast sample) was similar to those of HTCMCh, namely, there was only 14

Journal Pre-proof one obvious diffraction peak at ca. 2θ=20º (Fig. 3A). This peak is caused by the (101) and (002) planes, which agrees well with our previous results(Yang, et al., 2015). Contrast to O-CMCh, the peak intensity (PI, relative value) of HTCMCh0.5h and HTCMCh2.0h decreased with longer reaction time, and the full width at half maximum (FWHM) narrowed (inset in Fig. 3A). With even longer reaction time, the PI increased and the FWHM was relatively constant. It is known that the peak intensity indicates the number of crystal planes arranged in the same direction, and the XRD peak width indicates the crystalline degree or the crystal size. The integral area

of

(relative values) of the O-CMCh, HTCMCh0.5h, HTCMCh2.0h, HTCMCh4.0h, and

ro

HTCMCh8.0h diffraction peaks are 9448, 8150, 8118, 10523, and 12446, respectively. The results agreed well with the trend in peak intensity. For HTCMCh0.5h and the

decrease

of

crystallinity

indicated

-p

HTCMCh2.0h,

that

the

introduced

re

2-hydroxylpropyl-3-trimethylammonium group broke hydrogen bonds in O-CMCh

lP

molecules, resulting in significant weakening of the intermolecular interactions(Yin, Dang, Liu, Yan, Cha, Yu, et al., 2017). That is, more amorphous regions appeared in

na

the HTCMCh molecules with longer reaction time from 0.5 to 2.0 h. With even longer reaction time, more 2-hydroxylpropyl-3-trimethylammonium groups were introduced

Jo ur

in the O-CMCh molecules. According to Sun et al.(Sun, Du, Fan, Chen, & Yang, 2006), the crystalline structure of O-CMCh can be severely destroyed, where crystallinity disappeared with longer reaction time. In contrast, the PIs of HTCMCh4.0h and HTCMCh8.0h are higher than O-CMCh. This is ascribed to the large number of introduced 2-hydroxylpropyl-3-trimethylammonium groups that promote strong intermolecular electrostatic interactions, resulting in more crystalline regions. The molecular dynamic simulation molecular structures are shown in Fig. 3B, in which the blue dot lines indicate the hydrogen bonding, and the green circles indicate the electrostatic interactions. The simulation results indicate that the hydrogen bonds are predominant in the intermolecular interactions for O-CMC, while the electrostatic interaction are predominant in the intermolecular interactions for HTCMCh (the DS is 50%). This confirms the results of the XRD diffractogram. 15

Journal Pre-proof

(A)

CMCh HTCMCh0.5h HTCMCh2.0h HTCMCh4.0h HTCMCh8.0h

PI FWHM 177 1.16 128 0.86 119 1.01 220 0.94 257 0.98

(B1)

(B2)

10

20

2



30

(B4)

y

y

50

(B6)

(B5)

y

y

z

na

x

lP

re

-p

ro

of

(B3)

40

x

z

Fig.3 XRD patterns of O-CMCh and HTCMCh obtained at different reaction time(A), and

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schematic structures of molecular stimulation(B). Figs.B1 and B2 are original molecular structures of O-CMCh and HTCMCh, Figs. B3 and B4 are stimulated structures of O-CMCh, and B5 and B6 are stimulated structures of HTCMCh, respectively. The dots correspond the atoms: ●-N, ●-O, ●(white dot)-H, ●-C, ●-Na and ●-Cl, respectively.

3.4 Effect of reaction time on thermal properties TGA was used evaluate the thermostability of HTCMCh and O-CMCh. TGA curves of O-CMCh and HTCMCh synthesized at different reaction times are shown in Fig.4(A). There are two distinct weight loss stages at ca. 35-150 ºC and ca. 200-300 ºC, respectively. The weight loss stage at ca. 35-150 ºC corresponds to the evaporation of bonding water(Bukzem, Signini, dos Santos, Lião, & Ascheri, 2016). The weight loss of O-CMCh was 11.4%, which was much larger than those of HTCMCh0.5h 16

Journal Pre-proof (7.84%), HTCMCh2.0h (7.68%), HTCMCh4.0h (7.64%), and HTCMCh8.0h (8.20%), respectively. This implied less binding water in HTCMCh molecules compared with O-CMCh, due to the introduction of the 2-hydroxylpropyl-3-trimethylammonium group. The amount of bonded water is independent of HTCMCh DS, which might be +

due to the inter-molecular interactions between the –COO– and –N(CH3 ) groups. 3

For the second weight loss stage (200-300 ºC), both O-CMCh and HTCMCh showed larger weight losses. They were 41.44 (O-CMCh), 49.88 (HTCMCh0.5H),

of

45.44 (HTCMCh2.0H), 44.83 (HTCMCh4.0H), and 51.40% (HTCMCh8.0H), respectively. This weight loss corresponded to the dehydration, depolymerization, and

ro

decomposition of polysaccharide structure(Yin, et al., 2017). The onset temperatures

-p

at this stage were 265.8 (O-CMCh), 269.1 (HTCMCh0.5), 268.2 (HTCMCh2.0), 268.7 (HTCMCh4.0), and 268.9 ºC (HTCMCh8.0), respectively, and the maximum weight

re

loss rates appeared at 281.4 (O-CMCh), 282.1 (HTCMCh0.5), 281.7 (HTCMCh2.0),

lP

281.6 (HTCMCh4.0), and 282.5 ºC (HTCMCh8.0), respectively. These results implied that the introduction of the 2-hydroxylpropyl-3-trimethylammonium group enhanced

na

the thermostability of HTCMCh slightly due to the hydrogen-bonding or strong electrostatic interactions between HTCMCh molecules(Hu, Wang, & Wang, 2016).

(A)

o

265 C o

Weight loss (%)

269 C o 267 C

CMCh HTCMCh0.5h HTCMCh2.0h HTCMCh4.0h HTCMCh8.0h

o

269 C o

269 C

0.0

(B)

O-CMCh HTCMCh0.5h HTCMCh2.0h

-0.5

Heat flow (W/g)

Jo ur

However, the thermostability was independent of HTCMCh DS(Table 1).

-1.0 -1.5 -2.0 o

-2.5

114 C o

Ex: Up

124 C

-3.0

0

100

200

o

t/ C

300

400

o

116 C

0

500

50

o

100

150

t/ C

Fig.4 TG (A) and DSC (B) curves of O-CMCh and HTCMCh synthesized in AmimCl with different reaction time.

The DSC curves of O-CMCh, HTCMCh0.5h and HTCMCh2.0h are shown in Fig. 4(B), where a broad endothermic peak is observed at 124, 114 and 116 ºC, 17

Journal Pre-proof respectively. According to Kittur and coworkers(Kittur, et al., 2002), carboxymethyl derivatives display no glass transition phenomena. The endothermic peak position and endothermic enthalpy (ΔH) are due to the interruption and rearrangement of the chitosan chains(Zimet, Mombrú, Mombrú, Castro, Villanueva, Pardo, et al., 2019) and the evaporation of polymer-enclosed water. The endothermic enthalpies (ΔH) are 145 (O-CMCh), 141 (HTCMCh0.5h), and 170 W/g (HTCMCh2.0h), respectively. Kittur et al. (Kittur, et al., 2002) revealed that a high degree of substitution (DS) corresponded to a high ΔH for the enhanced water holding capacity at high DS. Also, the glass transition

of

temperature of agarose-maleoylagarose copolymer increased after the maleoyl groups

ro

were introduced, which restricted the chain movement and intermolecular interaction(Ortiz, Matsuhiro, Zapata, Corrales, & Catalina, 2018). In the current paper,

-p

both the endothermic temperature and ΔH of HTCMCh0.5h were lower than those of

chain

polymeric

separation

induced

by

the

introduction

of

lP

high

re

O-CMCh, which could be ascribed to the decreased intermolecular interactions and a

2-hydroxylpropyl-3-trimethylammonium groups. Both values increased for reaction

na

time (HTCMCh2.0h). Combining the first weight loss stages in the TGA curves, the water holding capacities of HTCMCh0.5h and HTCMCh2.0h were approximated. One

Jo ur

could conclude the water holding strength of HTCMCh2.0h and the electrostatic intermolecular interaction were stronger than that of HTCMCh0.5h. This result agrees well with XRD data.

3.5 Effect of reaction time on the mechanical properties of HTCMCh films The HTCMCh2.0h film showed excellent transmission of light (not shown). SEM images provided a better understanding of the micro-structures of HTCMCh films. As shown in Fig. 5, the cross-sectional SEM images of HTCMCh0.5h (Fig. 4B) and HTCMCh2.0h (Fig. 6C) were smooth and homogeneous without obvious deposit sediments or imperfections, compared to those of O-CMCh (blue arrows in Fig. 5A) and HTCMCh2.0h(pH5.68) films (red arrows in Fig. 5D). This indicated that the HTCMCh0.5h and HTCMCh2.0h molecules were well-dispersed in solution compared to O-CMCh and HTCMCh2.0h(pH5.68) molecules. This was ascribed to the strong 18

Journal Pre-proof intermolecular interactions between HTCMCh molecules. The DS of HTCMCh0.5h and HTCMCh2.0h are 22% and 42%, respectively. For HTCMCh0.5h, a small amount of +

introduced -N(CH3 ) groups were inclined to screen the –COO- groups. While for 3

+

HTCMC2.0h, a large amount of -N(CH3 ) groups were prone to interact with –COO3

groups through electrostatic interactions(Wang, Yang, Qiao, Li, Li, & Xu, 2018). The results are consistent with those of the XRD patterns (Fig. 3). However, the morphology of HTCMCh2.0h(pH5.68) showed several imperfections (the red arrows in

of

Fig. 5D), which were due to the decreased intermolecular interactions of the

ro

protonated carboxyl groups, which, in turn, induced aggregation behavior of

-p

HTCMCh molecules(Liu, Wang, Zou, Wei, & Tong, 2012). B

Jo ur

C

na

lP

re

A

D

19

Journal Pre-proof 10 (E)

Stress /MPa

8 6 4 2 0

O-CMCh HTCMCh0.5h HTCMCh2.0h

0

20

40

60

80

Strain /%

ro

HTCMCh2.0h(pH5.68) (D), and their stress-strain curves (E).

of

Fig.5 Cross-section SEM images of O-CMCh (A), HTCMCh0.5h (B), HTCMCh2.0h (C),

-p

The typical hydrogen bonding and intermolecular electrostatic interactions could be associated with the increased mechanical properties and contact angles and

re

decreased water vapor permeability(Hasheminya, Rezaei Mokarram, Ghanbarzadeh,

lP

Hamishekar, & Kafil, 2018). The tensile strengths of the HTCMCh films were 8.68 (HTCMCh0.5h) and 9.25 MPa (HTCMCh2.0h), respectively, which were 1.15 and 1.21

na

times that of the O-CMCh film (7.57 MPa) (Fig. 5E). This was ascribed to the strong electrostatic interactions between the HTCMCh molecules and the formed network

Jo ur

microstructures(Akhtar, Riaz, Hamed, Abdin, Chen, Wan, et al., 2018). The elongation at break decreased from 67.7 (O-CMCh) to 63.8 (HTCMCh0.5h) and then 45.8% (HTCMCh2.0h) for the tested films. This trend was similar to a common phenomenon for composite polymer films(Li, Wu, Song, Cheng, Suzuki, & Lei, 2016). The contact angles of HTCMCh0.5h (81º) and HTCMCh2.0h (104º) films increased, compared with that of the O-CMCh film (61º) (inserts in Fig.5), indicating more – +

CH3 in – N(CH3 ) was enriched on the surface of the HTCMCh film. However, the 3

contact angle is smaller than that of HTCC film (117.2º) (Wang, Yang, Qiao, Li, Li, & Xu, 2018), due to the electrostatic interaction between the -COO- and +

– N(CH3 ) groups. The results are similar to those reported by Noshirvani, et al. 3

(2017) and Salari, et al. (2018). 20

Journal Pre-proof 3.6 Viscoelasticity of HTCMCh hydrogels Rheology is a commonly used method to study intermolecular interactions. The plots of the storage (G’) and loss (G’’) moduli of HTCMCh0.5h samples are shown in Fig. 6. Both the G’ and G’’ were frequency dependent. Interestingly, for the hydrogel aged at 14 h, a predominantly viscous behavior (G’ < G’’) was shown, which was similar to that of N-(carboxymethyl) chitosan(Delben, Lapasin, & Pricl, 1999). As aging time increased to 6 d, the G’ and G’’ intersected, where, prior, the hydrogel was dominated by elasticity (G’>G’’). The crossover frequencies increased from 0.22 (6 d)

of

to 1.88 (10 d) rad/s, showing a close aging time dependence. When the aging time

ro

was longer than 15 d, the hydrogel was dominated by elasticity, and the G’ increased more notably at a low angular frequency than at a high one. This indicated the

-p

transition of the intermolecular interaction mode between the HTCMCh0.5h molecules.

re

At the initial aging stage, hydrogen bonding dominated the intermolecular interactions,

lP

which transformed into predominant electrostatic intermolecular interactions with aging time in aqueous systems. According to Pang, et al. (2007), these changes in properties

were

ascribed

na

rheology

to

the

introduction

of

3

10

2

(A)

G'

10

10

0.22

1

0

10

-1

10

0

 (rad/s)

10

1

, , , , ,

10

aging G'' time 14h 6d 10d 15d 25d

10

3

(B)

G', G'' (Pa)

10

Jo ur

G', G'' (Pa)

2-hydroxylpropyl-3-trimethylammonium group.

10

10.26 rad/s

2

1.88 rad/s

10 d G' G''

10

2

1

10

21

-1

10

0

 (rad/s)

10

1

10

2

Journal Pre-proof

3

(C)

10

3

(D)

G', G'' (Pa)

10

G', G'' (Pa)

10

10

2

2

25 d G' G''

15d G' G''

10

-1

10

0

 (rad/s)

10

1

10

10

2

1

10

-1

10

0

 (rad/s)

10

1

10

2

Fig.6 Plots of storage (G’, filled symbols) and loss (G’’, open symbols) moduli versus angular

of

frequency (ω) of HTCMCh0.5h hydrogels at 25 ºC. Figs. B, C and D are separated lines from Fig.

ro

A.

-p

4. Conclusions

highly

water-soluble

re

The

N-2-hydroxylpropyl-3-trimethylammonium-O-carboxymethyl chitosan (HTCMCh)

lP

with various DS has been successfully synthesized through homogeneous reaction. And the solubility is independent of aqueous pH. The DS of the HTCMCh ranged

na

from 22% to 58%, depending on the reaction time, temperature, 𝑛𝑒𝑝𝑜𝑥𝑦 ⁄𝑛−𝑁𝐻2 , and pH of the O-CMCh pretreating solution. The O-CMCh pretreated with aqueous

Jo ur

solution showed a decrease in the possibility of modification, due to the aggregation of O-CMCh in AmimCl. The optimum reaction conditions were: reaction time of 2 h, initial material pH of 9.47, 𝑛𝑒𝑝𝑜𝑥𝑦 ⁄𝑛−𝑁𝐻2 =2/1 and 80 ºC. For HTCMCh with low DS, the hydrogen bonding predominated the intermolecular interactions, whereas, the electrostatic interactions dominated the intermolecular interactions for HTCMCh with high DS, and form PEC. The results were confirmed by TG/DSC, XRD, mechanical properties of HTCMCh films, viscoelasticity of HTCMCh hydrogels, and molecular dynamic simlation. Acknowledgments The authors acknowledge the financial support from National Natural Science Foundation of China (21908116), and Natural Science Funds of Shandong Province (ZR2018BB050). Conflicts of Interest: The authors declare no conflict of interest. 22

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Author statement

Qun Liu: Synthesis and characterization(FTIR, NMR, DSC) of chitosan derivatives; Jialiang Chen: Preparation and characterization (SEM, tensile) of chitosan derivative films; Xiaodeng Yang: Design the experiment, analyze the experimental data, and

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writing-editing the manuscript; Congde Qiao: Characterization of chitosan derivatives (DSC, and TGA);

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Yan Li: Characterization of chitosan derivatives (TEM, and size and distribution

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determination); Zhi Li: Molecular simulations;

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Jinling Chai: Review the manuscript.

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Chunlin Xu: Preparation and characterization of HTCMCh hydrogels;

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Highlights of the work:

● Optimum reaction conditions of synthesizing HTCMCh were obtained. ● Effect of reaction conditions on structure and properties of HTCMCh were studied. ● Inter- and intra- molecular interactions between HTCMCh molecules were

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demonstrated.

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