Preparation, characterization and antibacterial properties of 6-deoxy-6-arginine modified chitosan

Journal Pre-proof Preparation, characterization and antibacterial properties of 6-deoxy-6-arginine modified chitosan Zhiwei Su, Qiming Han, Fang Zhang, Xianghong Meng, Bingjie Liu

PII:

S0144-8617(19)31303-7

DOI:

https://doi.org/10.1016/j.carbpol.2019.115635

Reference:

CARP 115635

To appear in:

Carbohydrate Polymers

Received Date:

3 June 2019

Revised Date:

25 September 2019

Accepted Date:

16 November 2019

Please cite this article as: Su Z, Han Q, Zhang F, Meng X, Liu B, Preparation, characterization and antibacterial properties of 6-deoxy-6-arginine modified chitosan, Carbohydrate Polymers (2019), doi: https://doi.org/10.1016/j.carbpol.2019.115635

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Preparation, characterization and antibacterial properties of 6-deoxy-6-arginine modified chitosan

Zhiwei Sua, Qiming Hana, Fang Zhanga, Xianghong Menga,b,*, Bingjie Liua,*

a

College of Food Science and Engineering, Ocean University of China, Qingdao 266003, China Pilot National Laboratory for Marine Science and Technology, Qingdao 266235, China

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b

Corresponding author. E-mail: [email protected] (B. Liu), [email protected] (X. Meng). Tel/Fax: +86-532-82032093

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*

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Graphical Abstract

Highlights 

The 6-deoxy-6-arginine chitosan (DAC) was prepared by chemical methods.



The DAC was characterized by FTIR, 1H and 13C NMR, DSC and elemental analysis.



The DAC have better antibacterial activity, water solubility than chitosan.

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

The DAC was non-toxic and might be used as a safe antibacterial agent.

Abstract In this study, 6-deoxy-6-arginine modified chitosan (DAC), was synthesized and characterized by Fourier Transform Infrared Spectroscopy (FTIR), 1H and

13

C nuclear magnetic resonance (NMR),

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differential scanning calorimetry (DSC) and elemental analysis. The arginine was grafted onto C6 groups of chitosan. Antibacterial activity of DAC against gram-negative bacteria Escherichia coli (E. coli) and gram-positive bacteria Staphylococcus aureus (S. aureus) were investigated at concentration

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between 0.02 mg/mL and 10 mg/mL. Cell viability assessment was estimated in vitro with Caco-2

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and L929 cells. Water solubility of DAC at different pH was also evaluated. The results showed that the minimum inhibitory concentration (MICs) of DAC against S. aureus and E. coli were 0.078

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mg/mL and 0.312 mg/mL, respectively. The minimum bactericidal concentration (MBC) against S.

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aureus and E. coli was 0.625 mg/mL. The cytotoxicity of chitosan and DAC was not significantly

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different. It demonstrated that DAC might be a potential safe antibacterial agent.

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Keywords: Chitosan; Arginine; Guanidyl; Modification; Water solubility; Antibacterial properties

1. Introduction

Chitosan, the second-most abundant biopolymer except for cellulose, is the N-deacetylation product of chitin. It is the only alkaline amino polysaccharide among natural polysaccharides. Because of its unique chemical structure, chitosan possesses a variety of biological properties,

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especially good antibacterial, antioxidation, immune regulation, and anti-tumour activity. Chitosan and its derivatives are widely used in environmental protection, food preservation, the chemical industry, agriculture, cosmetics and other fields (Yang, Jin, Xu, Fan, Wang & Xie, 2018; Yılmaz Atay & Çelik, 2017; Thaya et al., 2018). However, chitosan is difficult to be dissolved in neutral water solution, which limits its application. The C2 amino groups and C6 hydroxyl groups of chitosan are active functional groups, which can be modified to form various new derivatives (Hu et al., 2016;

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Tan, Li, Li, Dong & Guo, 2016; Luan et al., 2018). It is typically grafted with specific functional groups to enhance its antimicrobial and water-soluble properties (Sabaa, Elzanaty, Abdel-Gawad & Arafa, 2017; Jia, Duan, Fang, Wang & Huang, 2016; Wang et al., 2012).

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Although the chitosan’s antimicrobial activity has been well studied and several explanations of

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its antibacterial mechanism have been proposed (Alfaro, Chotiko, Chouljenko, Janes, King & Sathivel, 2018; Dragostin et al., 2016), the exact action mode of chitosan is still not fully understood.

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One of the mechanisms is that the amino groups at C2 in the glucose monomer form powerful groups

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of positively charged ions with free hydrogen ions, which can attract the negative charges carried by molecules on the surface of the bacterial cell membrane (Amato, Migneco, Martinelli, Pietrelli, Piozzi

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& Francolini, 2018; Zhong, Zhong, Xing, Li & Mo, 2010). This interaction can change the morphology of the bacterial cell membrane and increase the permeability of the cell membrane, which

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causes the outflow of cell contents and the death of bacteria (Zhong, Li, Xing & Liu, 2009; Alfaro, Chotiko, Chouljenko, Janes, King & Sathivel, 2018). Therefore, increasing the positive charges of chitosan and protonation are important modification methods to enhance its antibacterial activity (Dong et al., 2014). Grafting positively charged groups, such as quaternary ammonium (Jiao, Niu, Ma, Li, Tay & Chen, 2017; Oyervides-Muñoz, Pollet, Ulrich, de Jesús Sosa-Santillán & Avérous,

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2017), amino (Yan et al., 2016), or quaternary phosphonium (Tan et al., 2017; Wang, Xu, Guo, Peng & Tang, 2011), are the most direct and effective schemes. However, the introduction of these protonated groups has other unintended side effects, such as increased cytotoxicity (Rahmani et al., 2016; Wang, Xu, Guo, Peng & Tang, 2011). Guanidine can be viewed as a compound in which the oxygen atoms in the urea molecule are replaced by imine groups. Guanidyl groups are guanidine molecules in which hydrogen has been

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removed and the most bioactive organic base carrying a positive charge found in nature (Wu, Wang, Liu, Yin & Kuang, 2016; Tsetsekou, Brasinika, Vaou & Chatzitheodoridis, 2014). Arginine contains the guanidyl group. Several studies have reported that the carboxyl group of arginine can be grafted

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onto chitosan C2 amino group by chemical reaction to enhance the antibacterial activity (Tang et al.,

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2010; Lahmer, Williams, Townsend, Baker & Jones, 2012). However, previous studies mainly focused on the modification of C2 amino group and few focused on the C6 group modification of

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chitosan (Yang, Jin, Xu, Fan, Wang & Xie, 2018; Rahmani et al., 2016; Croce, Conti, Maake &

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Patzke, 2016). Our modified method was to graft an amino acid at C6 group of chitosan, which not only protects the C2 amino group with antibacterial activity from being destroyed, but also increases

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the positive charge of derivatives with the help of guanidine group of arginine. As a result, the introduction of C6 guanidyl could be produced to enhance the antibacterial activity. Therefore, the

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following measures were taken: C2 amino groups were protected by benzaldehyde and a substituted group was grafted on C6 groups of chitosan. The amino of arginine replaced the substituted group and the arginine-containing guanidyl was successfully grafted onto C6 groups. Finally, the C2 amino group was exposed by deprotection reaction. Based on the above hypothesis, chitosan was modified with arginine to produce an arginine-

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modified chitosan derivative, DAC. The DAC was characterized by FTIR, 1H NMR, 13C NMR, DSC and elemental analysis. The antimicrobial activity of DAC against E. coli and S. aureus was evaluated. 2. Materials and methods 2.1. Materials Commercial chitosan (average molecular weight 200 kDa, deacetylation 90%) was obtained from Qingdao Haihui Biochemical Corporation (Qingdao, China). L-arginine and 4-toluenesulfonyl

dichloromethane

were

purchased

from

J&K

Scientific

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chloride were purchased from Jinnan Fisher Chemical Co., Ltd. (Jinan, China). Benzaldehyde and Ltd.

(Shanghai,

China).

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dimethylaminopyridine (DMAp) and the other common chemical reagents were obtained from

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Sinopharm Group Chemical Reagent Co., Ltd. (Shanghai, China). All the chemical reagents were of

2.2. Preparation of DAC

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2.2.1. N-Benzaldehyde chitosan (b)

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analytical grade or better and used without further purification.

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As shown in Fig. 1, the synthetic method of N-benzaldehyde chitosan (b) was as previously described with modifications (Fan et al., 2018). Briefly, 1.50 g chitosan (a) was dispersed in 100 mL

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of 1% acetic acid aqueous solution and stirred until dissolution. NaOH aqueous solution (1 M) was added dropwise, and the purified chitosan was obtained after extraction and filtration. Purified

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chitosan was dispersed in 50 mL ethanol, and the mixture pH was adjusted to 6.5 with 1 M HCl aqueous solution. 20 mL ethanol containing 2.7 mL benzaldehyde was added to the mixture and stirred at 70 °C for 8 h. The mixture was cooled to room temperature and N-benzaldehyde chitosan was obtained by filtration. The solid was washed with ethanol and deionized water to remove the unreacted benzaldehyde.

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2.2.2. 6-Deoxy-6-p-toluenesulfonyl-N-benzaldehyde chitosan (c) One gram N-Benzaldehyde chitosan (b) was suspended in 30 mL dichloromethane, followed by the addition of 20 mL dichloromethane containing 5 g toluenesulfonyl chloride. Triethylamine and 4dimethylaminopyridine (DMAp) were added to the mixture as the acid trapping agent and catalyst, respectively. The reaction needed to be carried out in an ice bath for 2 h and at room temperature for another 3 h. The precipitate was collected by filtration, and washed with ethanol and ice water. 6-

in a vacuum freeze dryer. 2.2.3. 6-Deoxy-6-arginine-N-benzaldehyde chitosan (d) and DAC

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Deoxy-6-p-toluenesulfonyl-N-benzaldehyde chitosan (c) as brown solid was dried to a constant mass

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One gram 6-deoxy-6-p-toluenesulfonyl-N-benzaldehyde chitosan and 5 g arginine were dissolved

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in 30 mL deionized water. Na2CO3 (1.5 g) was added to the mixture as an acid trapping agent and then refluxed at 110 °C for 24 h. DAC was obtained by a one-pot reaction. The mixture was cooled

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to room temperature and 1 M HCl aqueous solution was added. The mixture was reacted for 24 h

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under stirring. The mixture pH was adjusted to 7 and the supernatant was collected by centrifugation. Finally, DAC was obtained through dialysis of the supernatant against distilled water for 3 days and

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lyophilization. 2.3. Characterization

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FTIR spectra of chitosan and DAC were carried out using the potassium bromide tablet method

on a Nicolet iS10 FTIR spectrometer (Shanghai, China). Spectra were performed ranging from 4000 to 500 cm−1 with 128 co-added scans and the resolution of 4 cm−1. 1H NMR and

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C NMR were

performed on a JEOL JNM-ECP600 spectrometer. The samples were dissolved in D2O. To measure the degree of substitution (DS) of DAC, the percentage content of H, C and N was estimated using

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Vario EL-III elemental analyser. Thermal stability analyses of chitosan and DAC were performed with a differential scanning calorimeter (DSC). The test was carried out in the temperature range of 25-500 °C at heating rate of 10 °C/min with 20 mL/min flow. 2.4. Water solubility test The solubility of DAC was measured as previously described with minor modifications (Dang et al., 2018). In brief, a vacuum freeze-dried sample (2 g) was weighed and dispersed in 10 mL pure

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water and the suspension was ultrasonicated for 30 min. When the solution was clear, the solute was added until precipitation occurred. After centrifugation, 5 mL supernatant was vacuum freeze dried at −65 °C. The freeze-dried sample was accurately weighed and expressed in g of DAC per 100 mL

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solution. The solubility of DAC was tested in the pH range of 3-11. In short, chitosan or DAC were

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dissolved in 1% acetic acid to prepare chitosan or DAC solution (1%, w/v), and stirred until the solution was clear. The transmittance of each solution was measured using a spectrophotometer at

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610 nm. Each experiment was carried out in triplicate, and the mean values and standard deviations

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were calculated.

2.5. Evaluation of DAC antibacterial activity

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2.5.1. Inhibition zone assay

The bacteria were incubated in LB broth for 12 h at 37 °C and diluted 1×107 cells/mL.

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Subsequently, 50 μL bacterial suspension was coated on a LB agar plate. Sterilized filter papers containing bacteriostatic agents were placed and incubated for 24 h at 37 °C. The diameter of the inhibition zone was measured three times and the mean values were calculated. 2.5.2. Determination of MICs and MBCs MICs were determined by classical broth dilution method as previously described [36]. In brief,

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the experimental strains were prepared by culture for 12 h and diluted at 1×107 cells/mL in LB broth. Antibacterial solution (20 mg/mL) was prepared by dissolving chitosan and DAC in the aqueous solution of 1% acetic acid and distilled water. A volume of 100 μL broth was added in the wells of the 96-well plate. The 100 μL antibacterial solution was serially diluted two-fold with nutrient broth in a 96−well microplate. The 100 μL mixture of well 11 was not further diluted, and only 200 μL LB broth was added in well 12. Thereafter, 100 μL bacterial suspension was added to wells 1 to 11. The

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plate was incubated at 37 °C for 24 h. The bacterial viability in the MIC assays was determined by OD values of the sample using microplate reader and set a blank control to remove the influence of background. MIC was defined as the lowest concentration of DAC where no growth was observed

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with a microplate reader. Additionally, 20 μL mixtures from wells with no growth were spread on

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agar plates for MBC determination. MBC was the lowest concentration of antibacterial agent where no colony growth was observed on LB agar plates after incubation at 37 °C for 48 h. The MIC and

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2.6. Cell cytotoxicity assay

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MBC determinations were carried out in triplicate.

Cell viability assessment was performed on Caco-2 and L929 fibroblast cells by MTT method as

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previously described (Li, Guan, Zhu, Wu & Sun, 2019; Rahmani et al., 2016; Rekha & Anila, 2019) with some modifications. Logarithmic growth phase cells were adjusted to the concentration of 2×104

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cells/mL and seeded in 96-well cell culture plates incubated at 37 °C in a 5% CO2 incubator for 24 h. Each well contained 100 μL of different concentration (0.2, 0.4, 0.6, 0.8, 1 mg/mL) of chitosan and DAC; the control group was incubated with culture medium. After the cells were cultured for 24 h and the medium was removed, and 200 μL of 0.5 mg /mL MTT solution was added to each well in the 96−well plate. The cells were cultured for 4 h; after removal of the culture medium, 150 μL DMSO

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was added to each well. The formazan was completely dissolved after mixing on an oscillator for 10 min. The absorbance of the solution at 570 nm was measured on a microplate ELISA reader. The viability was calculated by the following equation: Viability (%) =Abs sample/Abs sample×100% 2.7. Stability analysis The DAC stability in solution was measured by Zeta potential. 20 mg DAC was dissolved in 20

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mL aqueous solutions with pH of 3, 5, 7, 9, 11, respectively. The zeta potential at 40 °C, 60 °C, 80 °C and heating for 3 h, 6 h, 9 h was detected. The Zeta potential of DAC solution was detected within 15 d at room temperature.

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2.8. Statistical analysis

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All experiments were performed in triplicate. The results were analysed by one-way analysis of variance (ANOVA). P<0.05 was deemed to indicate statistical significance. The statistical analyses

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3. Results and discussion

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were performed using SPSS 17.0 software (IBM, New York, USA).

3.1. Synthesis and characterization

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The purpose of modifying chitosan was to increase its antibacterial properties and water solubility. The C2 amino group is the main group that has antibacterial activity. However, the C2 amino group

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was more active than the C6 hydroxyl group; therefore, the C2 amino group needed to be protected first to ensure that the C6 hydroxyl groups were involved in the reaction. Considering that the antibacterial activity of chitosan was related to the positive charge, that guanidine was the most bioactive organic base carrying a positive charge found in nature and that arginine is one of the most common guanidine-containing compounds, arginine was selected as the graft target. The active group

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on arginine was difficult to react with the C6 hydroxyl group on chitosan. Based on previous studies, the introduction of p-toluenesulfonyl chloride can resolve this problem (Cai, Wu, Liu, Wang, & Wang, 2019). As shown in Fig. 1, after these steps, target compounds were obtained and characterized by

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FTIR, 13C NMR, DSC and elemental analysis

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Fig. 1. Synthetic route of 6-deoxy-6-arginine chitosan (DAC). 3.1.1. FTIR spectrum analysis

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The spectra of arginine, intermediate products and the target product were shown in Fig. 2. For chitosan (a), the broad peak from 3200 cm−1 to 3500 cm−1 was assigned to the absorbance of -OH and

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-NH stretching vibration and the weak peak from 2850 cm−1 to 2950 cm−1 showed the stretching vibration of -CH. The peak at 1658 cm−1 was associated with the amide I stretching vibration and the -NH2 absorption peak is at 1595 cm−1. The peak at 1155 cm−1 was assigned to the characteristics of its polysaccharide structure. The peak from 1028 cm−1 to 1073 cm−1 is assigned to the absorbance peak of C6 and C3 hydroxyl groups, respectively (Sayed, Millard & Jardine, 2018). For arginine, the

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peaks at 1646 cm−1 and 1420 cm−1 were assigned to the characteristics of guanidine and the carboxyl of arginine, respectively (Xiao, Wan, Zhao, Liu & Zhang, 2011). Compared with chitosan, several new peaks appeared. The peak near 1645 cm−1 was attributed to N=C of N-benzaldehyde chitosan (b); two sharp peaks at 1581 cm−1 and 1452 cm−1 were the bending vibration absorption peaks of benzene rings. The peaks at 755 cm−1 and 692 cm−1 were caused by the deformation vibration of benzene rings. The absorption peak of –NH2 at 1600 cm−1 was

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weakened (Guo et al., 2007). All of the above changes demonstrated that the Schiff bases of chitosan have been synthesized.

For the intermediate products 6-deoxy-6-p-toluenesulfonyl-N-benzaldehyde chitosan (c), the

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peaks at 1645 cm−1 still exist because of the absorbance of -N=C-. In addition, (c) showed two

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characteristic absorption bands, one at 1175 cm−1 (S—O stretching vibration) and another at 814 cm−1 (symmetrical C—O—S vibration). There was no significant change in the C—O stretching vibration

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absorption peak (1068 cm−1) of C3 hydroxyl (Liu, Liu, Yue, Jiang & Xia, 2016), which indicated that

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the reaction occurred on the C6 hydroxyl group under the combined action of steric hindrance effect and reaction conditions. All of the above changes demonstrated that the 6-deoxy-6-p-toluenesulfonyl-

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N-benzaldehyde chitosan has been synthesized. DAC was obtained from (c) via a one-pot chemical reaction. Compared with the intermediate

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products (c), several new and obvious changes appeared in the infrared spectrum. The peaks at 1646 cm−1 were the characteristics of guanidine in arginine, in addition to the presence of amide bonds in chitosan, which were deformed. Dissociated symmetric stretching vibration peaks at approximately 1400 cm−1 were assigned to the characteristics of carboxylate in arginine. The carboxyl group on arginine may form the carboxylate in the final acid-base neutralization process (Hui, Guan & Hou,

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2001). Compared with the infrared absorption peak of chitosan, the peaks at 1028 cm−1 disappeared, which indicates that the hydroxyl group of chitosan C6 disappeared and the amino group of arginine was grafted to the C6 group. All of the above changes proved that the target product DAC has been

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synthesized.

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Fig. 2. FTIR spectra of arginine (Arg), chitosan (a), N-benzaldehyde chitosan (b), 6-deoxy-6-ptoluenesulfonyl-N-benzaldehyde chitosan (c), 6-deoxy-6-arginine-N-benzaldehyde chitosan (d) and

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DAC.

3.1.2. 1H NMR and 13C NMR analysis

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In 1H NMR spectra of DAC, as shown in Fig. 3, the peak at 1.91 ppm was corresponded to the

protons of N-acetyl glucosamine, and the peak at 2.80 ppm was assigned to the proton of glucosamine residues. The peak from 3.3 to 5.0 ppm were attributed to the non-anomeric protons of glucosamine (Sun, Shi, Wang, Fang, & Huang, 2017). It appeared that three peaks at 1.81 ppm, 3.28 ppm and 3.80 ppm belong to the proton of DAC at a, b, c position, respectively (Morris & Sharma, 2011). In the

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13

C NMR spectra, the main peaks of C1-C6 are basically consistent with previous results (Xiao, Wan,

Zhao, Liu & Zhang, 2011). Some non-common peaks in the spectrum may be caused by the effect of depolymerization, which was also previously reported (Kumirska, Weinhold, Steudte, Thöming, Brzozowski & Stepnowski, 2009). Carboxyl (176.05 ppm) and guanido (172.6 ppm) bands were present in the spectrum. In addition, methylene (40.4 ppm) and other distinct peaks of arginine

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confirmed that DAC was successfully prepared.

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Fig. 3. 1H NMR (A) and 13C NMR (B) spectra of DAC. 3.1.3. Thermal stability analysis

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The thermal stability of DAC was analysed by differential scanning calorimetry (DSC). The DSC

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curves of DAC and chitosan were shown in Fig. 4 (a). An endothermic peak appeared at approximately 100 °C, which was the peak of water. Compared with chitosan, DAC had a stronger endothermic peak (-0.67 vs. -0.60 mW/mg) and lower temperature (97.8 vs. 111.4 °C). It can be inferred that DAC may absorb more water (Li & Li, 2017), which can be referred to the greater hydrophilicity of DAC due to its guanidine and carboxylate. Compared with chitosan, the degradation temperature (227.8 vs. 271.4 °C ) of DAC was lower, which indicated that the introduction of arginine 13

altered the structure of chitosan and lead to the decrease of its thermal stability (Boggione, Mahl,

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Beppu & Farruggia, 2017).

Fig. 4. The DSC curves (a) and pH-dependent relative solubility (b) of chitosan and DAC.

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3.1.4. Elemental analysis

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The elemental analysis of DAC was used to quantitatively evaluate the extent of functionalization. The results of the elemental analysis were shown in Table 1. The DS of DAC was calculated based

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on the percentages of carbon and nitrogen according to the following equation: C% N%

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MC [12DS+6(DA-DS)+8(1-DA)]/MN[5DS+DA-DS+(1-DA)] = Mc: The molecular weight of C.

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MN: The molecular weight of N.

The C/N ratio of DAC was clearly lower than that of chitosan. This change was due to the introduction

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of arginine, which increases the content of nitrogen. That finding demonstrated that the guanidine groups were successfully grafted on chitosan. Table 1 The results of elemental analysis and DS. Sample

C%

N%

H%

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C/N

DS%

Chitosan

49.86

5.31

7.5

9.39

-

DAC

41.80

13.2

6.67

3.16

28.57

3.2. Solubility test The solubility of CS and DAC in distilled water and 1% acetic acid solution was shown in Table 2. As we all know, chitosan is almost insoluble in distilled water. However, DAC could be dissolved in distilled water, with a solubility of 5.78 g/100 mL. In acetic acid solution (1%, w/v), the solubility

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of DAC was higher than that of chitosan. After grafting arginine to the C6 groups on chitosan molecular chains, the guanidine and carboxylate moieties introduced endowed DAC with improved

Table 2

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The solubility of CS and DAC in different solutions.

Solubility (g/100 mL)

distilled water

1% acetic acid solution

insoluble

5.82±0.31

5.78±0.37

6.93±0.55

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Chitosan

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Sample

DAC

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solubility in acetic acid solution, and even in distilled water.

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The solubility of CS and DAC was measured over a pH range of 3 to 11. The results were shown in Fig. 4 (b). At low pH (≤6), chitosan and DAC had good solubility, and the transmittance of chitosan

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solution was close to 95%. With increased pH value, the transmittance of chitosan solution decreased rapidly, down to 5% in the range of pH 8 to 11. This was because the amino group on chitosan does not easily undergo protonation under alkaline conditions, resulting in poor water solubility (Dang et al., 2018). After the guanidine modification of chitosan, the transmittance of DAC solution was also greater than 75% in the range of pH 8 to 11. The results demonstrated that DAC had better solubility 15

than chitosan in neutral and alkaline solutions. In addition, after grafting arginine, the introduction of carboxylate also improved the solubility in neutral and alkaline solutions. 3.3. Evaluation of antibacterial activity 3.3.1. MICs and MBCs The antimicrobial activities of DAC against E. coli and S. aureus were carried out by the classical broth dilution method. The same method was performed on chitosan for control. As shown in Fig. 5,

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chitosan and target product DAC showed obvious robust antimicrobial activity against E. coli and S. aureus, which are some of the most common pathogenic strains responsible for contamination in food storage. DAC showed a more effective antibacterial activity compared with the same concentration

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of chitosan, in view of the OD values from 0.039 mg/mL to 0.625 mg/mL of antiseptics. The MIC

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and MBC values of chitosan and DAC were estimated, and the results were shown in Table 3. The MIC values of chitosan and DAC were 0.625 and 0.312 mg/mL against E. coli and 0.625 and 0.078

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mg/mL against S. aureus, respectively. The MBC values of chitosan and DAC were 2.5 and 0.625

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mg/mL against E. coli and 1.25 and 0.625 mg/mL against S. aureus, respectively. The results showed that the grafting of guanidyl groups to chitosan could improve its antibacterial activities. The

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antibacterial activity of chitosan against gram-positive and gram-negative bacteria did not show obvious differences; however, DAC showed significantly stronger antibacterial activity against gram-

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positive than gram-negative bacteria, which was consistent with previous studies (Tang et al., 2010; Lahmer, Williams, Townsend, Baker & Jones, 2012; Xiao, Wan, Zhao, Liu & Zhang, 2011). Table 3 The MIC and MBC of chitosan and DAC against E. coli and S. aureus. Bacteria

Chitosan

DAC

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MIC (mg/mL)

MBC (mg/mL)

MIC (mg/mL)

MBC (mg/mL)

E. coli

0.625

2.5

0.312

0.625

S. aureus

0.625

1.25

0.078

0. 625

3.3.2. Inhibition zones The diameters of the inhibition zones were measured for chitosan and DAC at a concentration of 1 mg/mL against E. coli and S. aureus (Fig. 5). The inhibition zone diameters of chitosan and DAC

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against E. coli and S. aureus were 9.4 and 10.2 mm and 11.3 and 12.4 mm, respectively. The DAC showed a larger inhibition zone diameter than that of chitosan, indicating that the antibacterial effect of chitosan grafted with arginine guanidine was significantly enhanced compared to chitosan alone.

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No antibacterial activity was found for an aqueous solution with the same concentration of arginine.

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Thus, the antibacterial activity of chitosan can be enhanced by grafting positive charge groups onto

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chitosan.

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b

a

a

a

8.9 mm

b

10.2 mm

c

c

a

12.4 mm

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11.3 mm

Fig. 5. MICs and diameters of the inhibition zones of chitosan and DAC against E. coli (left) and S.

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aureus (right). a: acetic acid solution (1%, v/v), b: chitosan solution (1 mg/mL), c: DAC solution (1

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mg/mL).

The possible mechanism of the higher antibacterial effect of DAC is the introduction of a

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positively charged guanidine group. The polymer has a stronger positive charge and combined with the negatively charged molecules on the surface of the bacterial cell membrane, this charge

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interaction can alter bacterial surface morphology, which either increases membrane permeability, causing leakage of intracellular substance or decreases membrane permeability, preventing nutrient transport (Tang et al., 2010 ; Xiao, Wan, Zhao, Liu & Zhang, 2011). DAC, contained not only the C2 amino group of chitosan itself, but also the cationic guanidine group, is introduced by arginine on C6 groups to make it have a strong positive charge and antibacterial activity. The results were

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consistent with previous research (Rahmani et al., 2016). 3.4. Cytotoxicity analysis To evaluate the safety of DAC, the cytotoxicity of DAC was tested against Caco-2 and L929 cells by the MTT assay. As shown in Fig. 6, the viability of Caco-2 and L929 cells in the presence of chitosan and DAC was nearly 100% at a low concentration (≤0.2 mg/mL), indicating that the cytotoxicity of DAC was nearly absent. Under the maximum concentration (1 mg/mL), the viability

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of Caco-2 cells and L929 cells in the presence of DAC or chitosan was still greater than 75.1% and 83.6%. There was no significant difference between DAC and chitosan in decreasing cell viability at increasing concentration. The result revealed that DAC with the concentration below 1 mg/mL

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was non-toxic. Therefore, the cytotoxicity of chitosan modified by guanidine was not increased,

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which might allow it to be used as a potential safe antibacterial agent.

Fig. 6. The cytotoxicity of chitosan and DAC against Caco-2 (a) and L929 (b) cells.

3.5. Stability analysis The result was shown in Fig. 7, DAC solution has no obvious regular distribution at different pH values, although the Zeta potential at 80 °C was slightly lower than that of 40 °C and 60 °C, it was still higher than 32 mV. There was no significant difference in potential change within 15 d at room 19

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temperature, we can believe that DAC solution has good stability.

Fig. 7 The stability of DAC solution

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4. Conclusion

In this paper, a novel bacteriostatic chitosan derivative, DAC, was synthesized through a series of

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reaction between chitosan and arginine. Not only C2 amino groups of chitosan with antibacterial

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activity was effectively protected, but the introduction of a guanidine group at C6 enhanced the positive charge of the polymer to enhance its antibacterial activity. DAC showed improved

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antibacterial activity against E. coli and S. aureus compared with chitosan. The cytotoxicity of DAC showed no obvious difference compared with chitosan. Overall, DAC, characterized by high water

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agent.

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solubility, strong antibacterial activity and biocompatibility, might be a potential safe antibacterial

Acknowledgements

This work is supported by National Key R&D Program of China (2018YFD0901106).

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