Preparation and characterization of chitosan physical hydrogels with enhanced mechanical and antibacterial properties

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Preparation and characterization of chitosan physical hydrogels with enhanced mechanical and antibacterial properties Peng Li a,1 , Jian Zhao a,1 , Yu Chen a,∗ , Bin Cheng b , Ziyang Yu a , Ying Zhao a , Xiaoting Yan a , Zongrui Tong a , Shaohua Jin a a b

School of Materials Science and Engineering, Beijing Institute of Technology, Beijing 100081, PR China Department of Clinical Laboratory, Pudong New Area People’s Hospital, Shanghai 201200, PR China

a r t i c l e

i n f o

Article history: Received 12 July 2016 Received in revised form 28 October 2016 Accepted 3 November 2016 Available online xxx Keywords: Chitosan CTS-Ag+ /NH3 physical hydrogels Mechanical property Antibacterial activity

a b s t r a c t The chitosan physical hydrogels formed under gaseous ammonia atmospheres usually have poor mechanical properties and low antibacterial activities, which limit its application as biomaterials. In the current study, CTS-Ag+ /NH3 physical hydrogels with great comprehensive properties were prepared by the gelation of chitosan in the presence of AgNO3 under a gaseous ammonia atmosphere. Compared with the previously reported hydrogels made with chitosan and AgNO3 , the CTS-Ag+ /NH3 hydrogels were more homogeneous and transparent. In addition, the AgNO3 content in the hydrogels was decreased to 0.064–0.424 wt.%. The formation mechanism and the influence of reaction conditions on the structures and properties of CTS-Ag+ /NH3 physical hydrogels were characterized by FT-IR, SEM, XPS, XRD and rheological measurement. Tensile testing suggested that CTS-Ag+ /NH3 physical hydrogels had a higher tensile strength than the CTS/NH3 hydrogel. Moreover, the CTS-Ag+ /NH3 physical hydrogels showed excellent antibacterial activities against both gram positive and negative bacteria. © 2016 Published by Elsevier Ltd.

1. Introduction Chitosan, a linear biopolymer constituted by d-glucosamine and N-acetyl-d-glucosamine units, with the percent of d-glucosamine units exceeds 50%, has shown great potentials as a biomaterial for wound care due to its biocompatibility, nontoxicity, and antimicrobial properties (Boucard et al., 2007; Ong, Wu, Moochhala, Tan, & Lu, 2008; Ribeiro et al., 2009). Particularly, the chitosan hydrogels can follow the geometry of the wound to ensure a good superficial contact, enabling them a promising wound dressing for a variety of skin wounds, such as third-degree burns (Boucard et al., 2007; Jayakumar et al., 2011; Ribeiro et al., 2009; Tamura, Furuike, Nair, & Jayakumar, 2011). In the past decade, there has been a growing interest in the pure physical hydrogels of chitosan without any residual cross-linkers due to their higher safety and lower cost. Physical hydrogels are held together by molecular entanglements or non-covalent bonds ˙ nska, ´ (Ostrowska-Czubenko & Gierszewska-Druzy 2009). Gener-

∗ Corresponding author. E-mail address: [email protected] (Y. Chen). 1 Joint first authors.

ally, there are two ways to prepare chitosan physical hydrogels. One is to form rigid chitosan hydrogels by evaporating a chitosan acetate salt solution in a hydroalcoholic medium (Boucard, Viton, & Domard, 2005; Montembault, Viton, & Domard, 2005a). The other method first reported by Montembault, Viton and Domard (2005b) is to fabricate physical hydrogels of chitosan by the gelation of a chitosan solution under a gaseous ammonia atmosphere. Nie et al. (2015) proposed that the orientation in and formation of these chitosan hydrogels were due to the entanglements of polymer chains. Due to their good biocompatibility, the physical hydrogels of chitosan have great potentials in tissue engineering and wound dressing (Montembault et al., 2005b; Ribeiro et al., 2009). However, pure physical hydrogels of chitosan formed under a gaseous ammonia atmosphere (CTS/NH3 hydrogels) are limited by their poor mechanical properties. CTS/NH3 hydrogels are usually composited with rigid protective gels to form bi-layered physical hydrogels for its application as a wound dressing (Boucard et al., 2007), which makes the preparation more complicated and reduces the antibacterial activities of the hydrogels. Therefore, CTS/NH3 hydrogels with improved mechanical and antibacterial properties without composition needed is a promise, yet few studies carried out, for the applications of such hydrogels in biomedical field.

http://dx.doi.org/10.1016/j.carbpol.2016.11.016 0144-8617/© 2016 Published by Elsevier Ltd.

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Ag+ is active against a wide range of pathogens including multidrug resistant strains with a far lower propensity for resistance development (Ong et al., 2008) and has been widely used in many antibacterial biomaterials (Ma, Zhou, & Zhao, 2008; Raghavendra, Jung, kim, & Seo, 2016; Wei, Sun, Qian, Ye, & Ma, 2009; Yadollahi, Farhoudian, & Namazi, 2015). Recently, a hydrogel made with chitosan and AgNO3 was reported by Kozicki et al. (2016). However, the AgNO3 concentration had to be ten times as much as or even higher than that of chitosan to form a continuous hydrogel with adequate mechanical stability. These hydrogels could be used for the functional finishing of textiles but are inappropriate as wound dressings due to their high AgNO3 contents that could irritate the wound. In addition, the hydrogel shape is hardly controllable and simply adding AgNO3 into the solutions of chitosan to prepare hydrogel cannot guarantee a uniform composition. In the current work, AgNO3 was added into chitosan solutions, followed by the gelation of the chitosan solution under an ammonia atmosphere, to achieve a novel chitosan-AgNO3 (CTS-Ag+ /NH3 ) physical hydrogel with controllable shapes and visible sol-gel transitions. The ammonia was used as the gas phase to promote the formation of the chitosan-AgNO3 physical hydrogels. The addition of AgNO3 was aimed to enhance the mechanical properties of the hydrogels and endowed them a better antibacterial property. The strategy not only greatly reduced the AgNO3 content in the hydrogels but also resulted in more uniform composites.

2. Experimental 2.1. Materials Chitosan with a deacetylation degree of 90% and an average molecular weight of 2.3 × 106 was purchased from Zhejiang Aoxing Biotechnology Co., Ltd (Shanghai, China). Silver nitrate of analytical grade was purchased from Tianjin Fuchen Chemical Reagents Factory (Tianjin, China). Analytical grade ammonia solution and acetic acid were purchased from Xilong Chemical Co., Ltd (Guangdong, China).

2.2. Preparation of CTS-Ag+ /NH3 hydrogels Homogeneous chitosan solutions at various concentrations ranging from 0.5 wt.% to 3.0 wt.% were prepared by dissolving a certain amount of chitosan in 2% (v/v) acetic acid aqueous solutions. A certain amount of AgNO3 (Table 1) was then added into the chitosan solutions under stirring to form chitosan-AgNO3 solutions. The chitosan-AgNO3 solution (30 mL) was poured into a Petri dish, which was then placed in a sealed environment containing 100 mL of ammonia solution (in order to create ammonia atmosphere) and kept for 24 h to form a hydrogel. The hydrogel was then taken out from the Petri dish and washed thoroughly with distilled water until the pH of the wash was neutral. By this method, a series of CTS-Ag+ /NH3 hydrogels were prepared with different concentrations of chitosan at different mass ratios of chitosan to Ag+ . Table 1 shows the reaction parameters of these CTS-Ag+ /NH3 hydrogels. The studies were divided to two groups. Group 1 includes samples from CTS11 to CTS15, with which the concentration of Ag+ was increased gradually and the mass fraction of chitosan was kept at constant. This group was aimed to investigate the influence of the concentration of Ag+ on the properties of the hydrogels. Moreover, in order to study the influence of the concentration of chitosan on the properties of hydrogels, group 2 was adopted. This group includes samples from CTS21 to CTS26, with which the concentration of chitosan was increased and the mass ratio of AgNO3 to chitosan was kept at constant.

2.3. Characterization of CTS-Ag+ /NH3 hydrogels 2.3.1. Fourier transform infrared spectroscopy (FT-IR) The hydrogel sample was pre-coated on a KBr window, completely dried and analyzed on an FTIR spectrometer (NEXUS-470 FTIR, Nicolet Instrument Co. USA) in the range of 400–4000 cm−1 at a resolution of 6 cm−1 and 32 scans.

2.3.2. X-ray photoelectron spectroscopy (XPS) The XPS analysis of freeze-dried hydrogel samples was carried out on a PHI Quanter-II spectrometer (Ulvac-PHI Inc., Japan) using a monochromatic Al K␣ X-Rays source with a fixed excitation energy of 1500 eV. The analyzed area was 300 ␮m × 700 ␮m. The power of anode was set to 150 W and the hemispherical electron energy analyzer was operated at a pass energy of 20 eV for all high resolution measurements. A charge neutralizer was used for the spectrum recording due to the insulating characters of the samples. The XPS data analysis was conducted with the XPS-peek41 software. Background subtraction was performed by the Shirley algorithm and C C peak (C1s, binding energy 284.8 eV) was used for the final calibration of each spectrum. Relative contents of different forms of N and Ag+ in CTS/NH3 hydrogel and CTS-Ag+ /NH3 hydrogel were calculated from their absorption peak areas.

2.3.3. Wide angle X-ray scattering Wide angle X-ray diffraction (XRD) of samples was recorded on a X’pert Pro MPD type X-ray diffractometer (PaNalystical Co., Holand) with Cu K␣ characteristic radiation (wavelength ␭ = 0.154 nm) at a voltage of 40 kV and a current of 50 mA. The scanning rate was 5◦ /min and the scanning scope of 2␪ was from 5◦ to 60◦ .

2.3.4. Solid state 13 C NMR measurement Solid state 13 C NMR test was performed on a 13 C NMR spectrometer (UltraShield 600 PLUS, Germany Bruker). Freeze-dried hydrogels were used and operated at a recording frequency of 100.63 MHz and spinning rate of 8 kHz.

2.3.5. Rheological measurements The rheological properties of the hydrogel samples were determined by measuring their storage modulus (G ) and loss modulus (G ) with an Anton Paar instrument (Physica MCR 301, Germany) equipped with a parallel-plate geometry (25 mm diameter). The measurements of G and G as a function of angular frequency at strain = 0.5% were carried out at 25 ◦ C in a sweeping frequency range from 0.1 to 700 rad/s. The effects of temperature were determined by conducting the tests at strain = 0.5% and angular frequency = 1 rad/s as the relative temperature increased from 0 to 100 ◦ C at a rate of 5 ◦ C/min.

2.3.6. Determination of swelling properties of CTS-Ag+ /NH3 hydrogels The freeze-dried hydrogels (∼1 mm thick) were cut into 15 mm × 15 mm pieces, weighed and immersed in 50 mL of distilled deionized water at 25 ◦ C for a certain period of time (specific time was shown in Fig. 3C) to determine the water absorption kinetics. As the saturated water absorption in PBS is important for biological applications, the equilibrium swelling rate of the freeze–dried hydrogel were determined in PBS (pH = 7.4) at 25 ◦ C for 12 h. At the end of immersing, the sample was taken out and wiped with filter papers to completely remove the surface water from the hydrogel and weighed immediately. The hydrogel sample was put back to the distilled deionized water again to repeat the above process

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Table 1 Compositions of the CTS/NH3 and CTS-Ag+ /NH3 hydrogels. Sample

Mass fraction of chitosan (wt.%)

Mass fraction of AgNO3 (wt.%)

Mass ratio of AgNO3 to chitosan

CTS0 CTS11 CTS12 CTS13 CTS14 CTS15 CTS21 CTS22 CTS23 CTS24 CTS25 CTS26

2.00 2.00 2.00 2.00 2.00 2.00 0.50 1.00 1.50 2.00 2.50 3.00

0 0.085 0.170 0.255 0.340 0.424 0.064 0.128 0.192 0.255 0.320 0.384

0 0.043 0.085 0.128 0.170 0.212 0.128 0.128 0.128 0.128 0.128 0.128

Fig. 1. Digital photographs of hydrogels (A) and schematic diagram for the formation mechanism of CTS-Ag+ /NH3 hydrogels (B).

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A)

B) 1.8

CTS0

CTS11

ratio of peak intensity

1.6

CTS12 CTS13 CTS14 CTS15

1410

1640

1.4

1.2

1.0

0.8

1570

0.6 3500

3000

2500

2000

1500

1000

CTS0

-1

CTS11

CTS12

CTS13

CTS14

CTS15

Wavenumber (cm )

C)

D)

O 1s

CTS0 wide

CTS12 wide

O 1s

Intensity (a.u.)

Intensity (a.u.)

C 1s C 1s

Ag 3d

N 1s

1100 1000

900

800

700

600

500

400

N 1s

300

200

100

0

1100 1000

900

800

700

Binding Energy (eV)

E)

F)

-NH2

NH-acetyl

408

406

404

402

400

398

200

100

0

-NH2

N-Ag

NH-acetyl

400

300

CTS12 N 1s

396

394

392

390

410

408

406

404

Binding Energy (eV)

G)

500

Intensity (a.u.)

Intensity (a.u.)

CTS0 N 1s

410

600

Binding Energy (eV)

402

400

398

396

394

392

390

Binding Energy (eV)

H)

Intensity (a.u.)

CTS14 N 1s

-NH2 N-Ag +

CTS-Ag /NH3

NH-acetyl

CTS/NH3

CTS

410

408

406

404

402

400

398

396

394

392

390

5

10

15

20

25

30

35

40

45

50

55

60

Binding Energy (eV) Fig. 2. FT-IR, XPS and XRD spectra of CTS/NH3 and CTS-Ag+ /NH3 hydrogels. (A) FT-IR spectra of CTS/NH3 hydrogel (CTS0) and CTS-Ag+ /NH3 hydrogels (from CTS11 to CTS15). (B) The ratio of peak intensity of the peak at 1570 cm−1 and 1640 cm−1 . The XPS wide-scan spectra of: CTS0 (C); CTS12 (D), and XPS high resolution N1 s spectra of: CTS0 (E); CTS12 (F); CTS14 (G). (H) The XRD patterns of CTS, CTS/NH3 hydrogel and CTS-Ag+ /NH3 hydrogel.

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4

10

3

10

B) 10

Storage Modulus CTS0 CTS11 CTS12 CTS13 CTS14 CTS15

2

10

-1

Loss Modulus CT S0 CTS11 CTS12 CTS13 CTS14 CTS15

10

0

10

1

10

10

4

10

3

10

2

10

2

5

Storage Modulus CTS0 CTS11 CTS12 CTS13 CTS14 CTS15

1

0

20

2000

CTS11 CTS12 CTS13 CTS14 CTS15

1800 1600

80

100

1100

D)

1000

1200 1000 800 600 400

PBS deionized water

900

Swelling ratio (%)

1400

Swelling ratio (%)

60

Relative Temperature (ć)

Angular Frequency (rad/s)

C)

40

Loss Modulus CTS0 CTS11 CTS12 CTS13 CTS14 CTS15

Loss Modulus G'' (Pa)

5

Loss Modulus G'' (Pa) Storage Modulus G' (Pa)

Storage Modulus G' (Pa)

A) 10

5

800 700 600 500 400 300 200

200

100

0 0

10

20

30

40

50

60

360

480

600

0

720

CTS11

Time (min)

CTS12

CTS13

CTS14

CTS15

10

Storage Modulus G' (Pa)

5

E)

10

3

10

2

10

Storage Modulus CTS21 CTS22 CTS23 CTS24 CTS25

1

10

-1

10

0

10

1

10

Loss Modulus CTS21 CTS22 CTS23 CTS24 CTS25

Loss Modulus G'' (Pa)

4

2

10

Angular Frequency(rad/s) Fig. 3. Rheological properties and swelling behaviors of CTS/NH3 and CTS-Ag+ /NH3 hydrogels. (A) Storage modulus (G , filled) and loss modulus (G , empty) as a function of angular frequency at strain = 0.5% for CTS/NH3 hydrogel (CTS0) and CTS-Ag+ /NH3 hydrogels (from CTS11 to CTS15). (B) Storage modulus (G , filled) and loss modulus (G , empty) as a function of temperature at strain = 0.5% and angular frequency = 1 rad/s for different hydrogels. (C) Water absorption kinetics of CTS-Ag+ /NH3 hydrogels (from CTS11 to CTS15). (D) Saturated water absorption of CTS-Ag+ /NH3 hydrogels (from CTS11 to CTS15) in pH = 7.4 PBS and deionized water. (E) Storage modulus (G , filled) and loss modulus (G , empty) as a function of angular frequency at strain = 0.5% for CTS-Ag+ /NH3 hydrogels (from CTS21 to CTS25).

until the sample weight no longer changed or the sample dissolved. The swelling ratio was calculated by the equation below: Swelling ratio =

Ws − Wd × 100% Wd

Where Ws and Wd are the weights of the swollen and dried sample, respectively. 2.3.7. Scanning electron microscopy (SEM) Hydrogel samples prepared from different concentrations of AgNO3 were lyophilized, mounted on a metal stub with a conductive tape and sputter-coated with a thin gold layer. The

morphologies of the hydrogels were imaged under a scanning electron microscopy (SEM, Hitachi S-4800, Japan) at an acceleration voltage of 5 kV. The porous structure parameters including porosity and average pore size of the freeze-dried hydrogels were analyzed by the method of Chhen et al. (2012). 2.3.8. Mechanical studies The mechanical properties of CTS-Ag+ /NH3 hydrogels were characterized by measuring their tensile strength () on a Universal Materials Testing Machine (Instron 6022, Instron Corporation, USA). Samples were cut into dumbbell shapes and stretched at a constant strain rate of 1 mm/min to the complete tensile fail-

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ure. Each test was repeated three times and the mean value was reported. 2.4. Tests on the antibacterial properties of CTS-Ag+ /NH3 hydrogels The antibacterial properties of chitosan/AgNO3 hydrogels were assessed via an ager diffusion test by the method described by Yan, Abdelgawad, El-Naggar and Rojas (2016). Both CTS/NH3 (CTS0) and CTS-Ag+ /NH3 hydrogels (CTS13) were cut into circular discs, placed on nutrient agar in Petri dishes that had been inoculated with bacteria, incubated at 35 ◦ C for 24 h and imaged for further evaluation. The experiments were conducted against five different kinds of bacteria including gram negative bacteria Escherichia coli (ATCC 25922 and ATCC 35218) and Pseudomonas aeruginosa (ATCC 27853), and gram positive bacteria Enterococcus faecalis (ATCC 29212), Staphylococcus aureus (ATCC 25923) and Staphylococcus aureus subsp. aureus (ATCC 29213). 3. Results and discussion 3.1. Formation of CTS-Ag+ /NH3 hydrogels The chitosan-Ag+ mixture was a transparent and stable solution. When it was exposed to the ammonia gas, a transparent hydrogel immediately appeared at the interface between the solution and the ammonia gas and extended to the bottom of the Petri dish. Eventually, a transparent CTS-Ag+ /NH3 hydrogel was formed. The color of the hydrogel changed from colorless to light brown with the increasing of Ag+ concentration. The higher Ag+ concentration was, the darker the color of the hydrogel was (Fig. 1A). The formation mechanism of CTS-Ag+ /NH3 hydrogels is shown in Fig. 1B. In the chitosan-AgNO3 solution, H+ combined with the unshared pair electron of the −NH2 of chitosan to form −NH3 + that could not coordinate with Ag+ . Instead, −NH3 + and Ag+ repulsed each other, which promoted the solubility of chitosan. When the solution was exposed to alkaline ammonia gas, the acidic −NH3 + was deprotonated and then coordinated with the Ag+ to form a cross-linked chitosan network. The ammonia gas was slowly infiltrated into the chitosan network, eventually resulting in a complete hydrogel. The ammonia gas was converted into ammonium acetate after the deprotonation of −NH3 + and washed out from the gel. As can be seen, the CTS-Ag+ /NH3 hydrogel formation was a self-assembly process and Ag+ functioned as a physical cross-linker to form noncovalent coordinate bonds with chitosan in the physical hydrogels. Ammonia gas worked as a pH regulator to neutralize the acidic chitosan solutions to initiate the gelation. This formation results in a homogeneous hydrogel. Alkaline ammonia gas instead of alkaline aqueous solutions, such as NaOH solution (Nie et al., 2015), was chosen in the present work as the pH regulator to avoid the formation of Ag2 O from the interaction between Ag+ and alkaline. The Ag2 O would cause dark brown color and rigidity of the hydrogel, which is undesired to wound dressings. 3.2. Characterization of CTS-Ag+ /NH3 hydrogels 3.2.1. FT-IR spectra The FT-IR spectra of CTS/NH3 and CTS-Ag+ /NH3 hydrogels formed at different Ag+ concentrations are shown in Fig. 2A. A broad absorption band at ∼3500–3200 cm−1 that was attributed to the stretching vibrations of O H and N H was observed on the spectra of all hydrogels. The absorption bands at 1640 cm−1 and 1570 cm−1 were ascribed to the C O stretching in the amide I and N H bending vibration in the NH2 groups in chitosan, respectively. The absorption band at 1570 cm−1 is much stronger than that at 1640 cm−1

for the CTS/NH3 physical hydrogel (CTS0) prepared with chitosan with a deacetylation degree of 90% (Mukhopadhyay, Mishra, Rana, & Kundu, 2012; Nawrotek, Tylman, Rudnicka, Balcerzak, ´ 2016; Vakili et al., 2016). The absorptions at 1410, & Kaminski, 1151, 1092 and 1027 cm−1 are associated with C H symmetrical deformation, the asymmetric stretching of C O C bridge, the C O stretching in CH OH and the C O stretching in CH2 OH of chitosan, respectively (Lejardi et al., 2014; Ostrowska-Czubenko & ˙ nska, ´ Gierszewska-Druzy 2009). As the Ag+ content in CTS-Ag+ /NH3 hydrogels gradually increased, the ratio between the band intensities at 1570 cm−1 and 1640 cm−1 decreased. As shown in Fig. 2B, the ratio of peak intensity of the peak at 1570 cm−1 and 1640 cm−1 indicated that the interaction between −NH2 and Ag+ gradually increased. The coordinate bonds between NH2 and Ag+ weakened the N H bending vibration in the NH2 groups. The absorption bands at ∼3500–3200 cm−1 that were assigned to the stretching vibrations of O H and N H shifted to a higher wavenumber, which indicated the enhanced intermolecular interactions in chitosan ´ by hydrogen bonds (Marsano, Vicini, Skopinska, Wisniewski, & Sionkowska, 2004). We conjectured that the molecular chains of chitosan became more regular due to the directivity of coordinate bonds, which was favorable to the hydrogen bonds between chitosan molecular chains. Bands at 1200–1000 cm−1 were ascribed to the skeleton vibration of C O C and stretching vibration of C O, and became weaker with the increase of the Ag+ concentration, indicating that some Ag+ also interacted with the lone pair electrons of O (Higazy, Hashem, ElShafei, Shaker, & Hady, 2010). However, the complexation activity of −NH2 is higher than C O C. Therefore, the interaction between NH2 and Ag+ was still the main factor affecting the crosslinking. Based on these results, it can be concluded that the CTS-Ag+ /NH3 hydrogel network was constructed by the hydrogen bonds between chitosan molecules and the coordinate bonds between the −NH2 of chitosan and Ag+ . 3.2.2. XPS spectroscopy Fig. 2C–G shows the XPS wide-scan spectra and high resolution N1s spectra. The characteristic peaks of carbon (C 1s), nitrogen (N 1s), and oxygen (O 1s) originated from chitosan revealed the similarity of the two samples in the wide-scan spectra. A peak in the XPS wide-scan spectrum of CTS12 assigned to Ag (Ag 3d) was observed (Alshehri et al., 2016; Nawrotek et al., 2016; Oliveira, Martins, Mafra, & Gomes, 2012). The resolved N 1s spectrum of CTS/NH3 hydrogel (CTS0, Fig. 2E) revealed two peaks at 400.3 eV and 398.1 eV that were assigned to N in NH-acetyl and free NH2 groups (Lawrie et al., 2007; Liu, Xu, Zhuang, & Cheng, 2014). Fig. 2F and G shows the resolved N 1s spectra of CTS-Ag+ /NH3 hydrogels, CTS12 and CTS14. The relative contents of the N atom interacted with Ag+ in the hydrogels are presented in Table 2. It is clear that the relative content of free NH2 decreased with the increase of Ag+ concentration due to their interactions. However, the relative content of NH-acetyl barely changed. In addition, a new peak appeared at ∼398.6 eV that was assigned to the coordinate bond between N and Ag+ and its intensity increased with the increase of Ag+ concentration. These results further confirmed that coordinate bonds were formed between Ag+ and the free NH2 groups of chitosan during the formation of CTS-Ag+ /NH3 hydrogels. 3.2.3. XRD analysis The XRD patterns of CTS, CTS/NH3 and CTS-Ag+ /NH3 hydrogels are shown in Fig. 2H. The diffraction peaks of CTS at 2␪ = ∼13◦ and 20◦ could be assigned to the strong intermolecular and intramolecular hydrogen bonds of the polymer chains, respectively. The peaks became stronger as the CTS/NH3 physical hydrogel formed, indicating that the crystallinity was enhanced by the chain entanglement

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Table 2 Relative contents of N and Ag in CTS/NH3 hydrogel and CTS-Ag+ /NH3 hydrogels determined by high resolution XPS of N1s. Samples

CTS0 CTS12 CTS14

NH-acetyl

–N-Ag+

–NH2

binding energy (eV)

Relative content

binding energy (eV)

Relative content

binding energy (eV)

Relative content

400.3 399.9 400.3

11.04% 10.36% 11.78%

398.1 397.9 397.7

88.96% 66.89% 41.71%

– 398.6 398.3

– 22.75% 46.51%

and stronger hydrogen bonds. The addition of Ag+ further increased the crystallinity of CTS-Ag+ /NH3 hydrogels due to the coordination interaction between Ag+ and −NH2 groups, promoting the dense of the hydrogel networks and enhancing the hydrogen bond interaction between the molecular chains. The new peaks at 29.6◦ , 36.2◦ , 39.8◦ , and 43.6◦ were attributed to the Ag+ .

the G and G values declined with the increase of angular frequency. Although the ratio of coordinate bonds remained constant, the higher chitosan concentrations provided more chain entanglements, resulting in higher G and G . The tests indicated that the CTS-Ag+ /NH3 physical hydrogels remained elastic at angular frequency lower than 400 rad/s.

3.2.4. Solid state 13 C NMR analysis The 13 C NMR of freeze-dried CTS-Ag+ /NH3 was determined. However, the peaks of carbon of CTS-Ag+ /NH3 are the same with pure chitosan (Liu et al., 2016), which indicated that the addition of Ag+ has no influence on the chemical structure of the carbon atoms on the chain of chitosan.

3.2.6. Swelling behaviors of freeze-dried CTS-Ag+ /NH3 hydrogels Fig. 3C shows the swelling behaviors of different hydrogels in distilled deionized water. The freeze-dried CTS/NH3 hydrogels (CTS0) rapidly dissolved in water and no water absorption was achieved. The CTS/NH3 hydrogel network was formed mainly by the weak intermolecular forces, chain entanglements, hydrophobic interactions and hydrogen bonds (Montembault et al., 2005b), which could be easily broken upon contacting with water. The swelling ratio of CTS-Ag+ /NH3 hydrogels during the initial period (t < 20 min) increased first and then decreased with the increasing of Ag+ concentration. A maximum swelling ratio of 1912.5% was achieve with mAg+ /mCTS = 0.085 (CTS12). The swelling ratio of CTS-Ag+ /NH3 hydrogels prepared at mAg+ /mCTS ratios less than 0.085 during the initial swelling stage (t < 20 min) increased with the Ag+ concentration and decreased as the prolong of swelling time, indicating that the networks in these CTS-Ag+ /NH3 hydrogels were unstable and could be broken as exposed to water for a long period of time. However, the swelling ratio of CTS-Ag+ /NH3 hydrogels prepared at mAg+ /mCTS ratios higher than 0.085 during the initial period (t < 20 min) decreased with the increase of Ag+ concentration. The swelling behavior of the CTS-Ag+ /NH3 hydrogel was affected by its degree of cross-linking (Liu et al., 2014). Higher Ag+ concentrations resulted in higher degrees of cross-linking and consequently lower swelling ratios and higher resistance of the hydrogels to the network destruction by water. The cross-linking coordinate bonds in the hydrogels prepared at mAg+ /mCTS ratio over 0.170 were sufficient enough to afford hydrogels with stable swelling ratios in water for over 10 h. Fig. 3D shows the saturated water adsorption of freeze-dried CTS-Ag+ /NH3 hydrogels in PBS (pH = 7.4) and deionized water. No significant difference was observed for the changing of the swelling tendency of hydrogels in PBS and deionized water except CTS11. Freeze-dried CTS11 swollen in PBS, however dissolved in deionized water, indicating that these hydrogels were stable in PBS. The saturated water adsorption of hydrogels in PBS increased initially and then decreased from CTS11 to CTS15 and was slightly lower than that in deionized water because of the higher ionic concentration and osmotic pressure of PBS. In all, the swelling behaviors of CTS-Ag+ /NH3 hydrogels indicated that addition of Ag+ stabilized their cross-linking networks due to the coordinate bonds formed between the NH2 groups of chitosan and Ag+ . The CTS-Ag+ /NH3 hydrogels were durable and remained stable upon exposing to water. Ag+ could improve the swelling capacity of chitosan hydrogels, which was an important parameter for evaluating their application in biomaterials (Cui, Jia, Guo, Liu, & Zhu, 2014).

3.2.5. Rheological properties of CTS-Ag+ /NH3 hydrogels Rheological measurements were performed to characterize the coordinate bonds between Ag+ and the NH2 groups of chitosan in CTS-Ag+ /NH3 hydrogels. Fig. 3A, B and E shows the rheological properties of CTS/NH3 and CTS-Ag+ /NH3 hydrogels. The storage modulus (G ) and loss modulus (G ) as the function of angular frequency at strain = 0.5% for CTS/NH3 hydrogel (CTS0) and CTS-Ag+ /NH3 hydrogels (CTS11-CTS15) are shown in Fig. 3A. The G value of CTS0 (black hollow square) was less than the G at the angular frequency below ∼200 rad/s (black solid square) and became equal at ∼200 rad/s. The G value of CTS0 was greater than the G value at the angular frequency above 200 rad/s, implying that the interactions in CTS/NH3 hydrogel could be easily broken. In contrast, for all of the CTS-Ag+ /NH3 hydrogels, while the angular frequency was lower than 400 rad/s, the G values of them were always greater than their G values, indicating that CTS-Ag+ /NH3 hydrogels were elastic dominantly hydrogels. Both G and G increased with the increase of Ag+ concentration because more coordinate bonds formed between Ag+ and NH2 groups at high Ag+ concentrations, indicating that the intermolecular interactions and chain entanglements were enhanced to make the hydrogels more elastic. However, the G value decreased as the angular frequency increased over 400 rad/s and became equal to the G value, implying that the physical interactions, mainly coordinate bonds between Ag+ and the NH2 groups, were broken at angular frequency higher than 400 rad/s. The networks in CTS-Ag+ /NH3 hydrogel formed by the complexing interaction could be broken at the higher angular frequency, which transformed the hydrogels from an elastic solid to a viscous liquid. This is typical characteristics of physical hydrogels and the intersection of G and G is an important marker of sol-gel transition (Cai & Zang, 2006; Liu, Chen, Zhao, Tong, & Chen, 2015). The effects of temperature on G and G of CTS/NH3 hydrogel (CTS0) and CTS-Ag+ /NH3 hydrogels (CTS11-CTS15) are showed in Fig. 3B. The G values of all tested hydrogels were greater than their G values in the testing temperature range, implying that the hydrogels had high thermal stability and their network structure could not be broken by heating. Fig. 3E shows the rheological properties of CTS-Ag+ /NH3 hydrogels in group 2 (CTS21-CTS26) with mAg+ /mCTS (mass ratio of AgNO3 to chitosan) = 0.128 as the function of angular frequency at strain = 0.5%. The G values of the hydrogels were greater than their G values at low angular frequencies, and the difference between

3.2.7. Morphology of freeze-dried hydrogels The surface morphologies of freeze-dried CTS/NH3 and CTSAg+ /NH3 hydrogels are presented in Fig. 4.

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Fig. 4. SEM images of freeze-dried CTS/NH3 and CTS-Ag+ /NH3 hydrogels.

A)

(A) CTS0, (B) CTS11, (C) CTS12, (D) CTS13, (E) CTS14.

B)

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CTS12

CTS13

CTS14

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CTS21

CTS22

CTS23

CTS24

CTS25

CTS26

Fig. 5. Mechanical propertiesof CTS-Ag+ /NH3 hydrogels. (A) Tensile strength of CTS-Ag+ /NH3 hydrogels in group 1 (from CTS11 to CTS15). (B) Tensile strength of CTS-Ag+ /NH3 hydrogels in group 2 (from CTS21 to CTS26).

The SEM images of CTS/NH3 hydrogel (CTS0) revealed a highly porous structure. The porosity of the freeze-dried hydrogel was 34.6% and the average pore size was 73.63 ␮m, indicating that its network was loose due to the weak interactions between chitosan molecular chains. The addition of Ag+ reduced the porosity of the network and resulted in compact surfaces of on CTS-Ag+ /NH3 hydrogels. The porosity of the freeze-dried CTS11 hydrogel was decreased to 19.3%. The porous architectures gradually turned into a compact and smooth surface as the Ag+ concentration further increased and the porosity became less than 11.0%. These results demonstrated that the coordinate bonds between the NH2 groups and Ag+ were much stronger than chain entanglements, hydrophobic interactions and hydrogen bonds. 3.2.8. Mechanical properties of CTS-Ag+ /NH3 hydrogels The CTS/NH3 hydrogel was soft and fragile and could not be cut into dumbbell shapes for the tensile test. CTS-Ag+ /NH3 hydrogels (CTS11-CTS15) were tested to investigate the effects of Ag+ on the mechanical properties of CTS-Ag+ /NH3 hydrogel. As shown in Fig. 5A, tensile strength increased with the increase of Ag+ concentrations and the ultimate stress was 0.17 MPa. As the Ag+ concentration increased, more coordinate bonds were formed and the porous architectures of the hydrogels gradually turned into a

compact structure (Fig. 4). Therefore, the mechanical strength of CTS-Ag+ /NH3 hydrogels increased. For the CTS-Ag+ /NH3 hydrogels prepared at different concentrations of chitosan with a constant mass ratio of AgNO3 to chitosan (CTS21-CTS26), their tensile strengths increased first and then decreased with the increase of chitosan concentration (Fig. 5B). The highest ultimate stress of 0.33 MPa was obtained at the chitosan concentration of 1.5 wt.% (CTS23). The enhanced tensile strengths of the hydrogels prepared at chitosan concentrations lower than 1.5 wt.% was mainly due to the increased chain entanglements. The viscosity of chitosan solutions significantly increased at concentrations higher than 1.5 wt.%, which made the diffusion of ammonia in the chitosan solution very difficult and thus interfered the neutralization of NH3 + . Therefore, the formation of the coordinate bonds and cross-linking network in the hydrogels were affected, leading to low tensile strengths of the hydrogels. The tensile test results further confirmed that the addition of Ag+ indeed improved the mechanical properties of CTS-Ag+ /NH3 hydrogels. The tensile strength of CTS23 is close to the highest tensile strength of chitosan/␤-glycerophosphate disodium salt/attapulgite (CS/GP/ATP) hydrogels reported by other authors (Wang and Chen, 2016).

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Fig. 6. Antibacterial activities of CTS/NH3 and CTS-Ag+ /NH3 hydrogels. Antibacterial activity of CTS/NH3 hydrogels (CTS0) and CTS-Ag+ /NH3 hydrogels (CTS13) against (A) Escherichia coli (ATCC 25922), (B) Escherichia coli (ATCC 35218), (C) Pseudomonas aeruginosa (ATCC 27853), (D) Enterococcus faecalis (ATCC 29212), (E) Staphylococcus aureus (ATCC 25923) and (F) Staphylococcus aureus subsp. aureus (ATCC 29213).

3.3. Antibacterial properties of CTS-Ag+ /NH3 hydrogels Fig. 6 shows the antibacterial activities of CTS/NH3 and CTSAg+ /NH3 hydrogels against gram negative and gram positive bacteria. No inhibition zone was observed for the activity tests of CTS0 against the bacteria, which indicated the poor antibacterial activity of the CTS/NH3 hydrogel. In contrast, the CTS-Ag+ /NH3 hydrogel, CTS13, showed obvious inhibition zones against all bacteria, indicating that the antibacterial activity of CTS-Ag+ /NH3 hydrogels was improved due to the surface Ag+ on the hydrogels that could destruct the cell walls of bacteria (Li, He, Li, & Zhang, 2015). The excellent and broad antibacterial activities of the CTSAg+ /NH3 hydrogels against both gram negative and gram positive bacteria make them a promising candidate for wound dressing materials. 4. Conclusion A novel method was developed for the preparation of transparent CTS-Ag+ /NH3 hydrogels with improved mechanical properties and antibacterial activities. CTS-Ag+ /NH3 hydrogels were prepared by the gelation of chitosan solutions in the presence of AgNO3 under a gaseous ammonia atmosphere. The formation mechanism of the hydrogels was deduced and demonstrated. The enhanced mechani-

cal properties of the CTS-Ag+ /NH3 hydrogels were attributed to the entanglement of chitosan chains and coordinate bonds between Ag+ and the NH2 groups of chitosan. The AgNO3 content in the hydrogels ranged from 0.064 wt.% to 0.424 wt.%, which was much lower than that reported in the previous reports, but was able to endow the hydrogels great antibacterial properties. The morphology and swelling behavior of CTS-Ag+ /NH3 hydrogels were also significantly improved by the addition of Ag+ . These results suggest that CTS-Ag+ /NH3 hydrogels are promising biomaterials for wound dressings. Acknowledgments This work was supported by the Youth Talent Plan of Beijing City, the Combination Project of Guangdong Province and the “Yangfan” Innovative Research Team Project of Guangdong Province. References Alshehri, S. M., Almuqati, T., Almuqati, N., Al-farraj, E., Alhokbany, N., & Ahamad, T. (2016). Chitosan based polymer matrix with silver nanoparticles decorated multiwalled carbon nanotubes for catalytic reduction of 4-nitrophenol. Carbohydrate Polymers, 151, 135–143. Boucard, N., Viton, C., & Domard, A. (2005). New aspects of the formation of physical hydrogels of chitosan in a hydroalcoholic medium. Biomacromolecules, 6(6), 3227–3237.

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