Decanoic acid functionalized chitosan: Synthesis, characterization, and evaluation as potential wound dressing material

International Journal of Biological Macromolecules 139 (2019) 1046–1053

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International Journal of Biological Macromolecules journal homepage: http://www.elsevier.com/locate/ijbiomac

Decanoic acid functionalized chitosan: Synthesis, characterization, and evaluation as potential wound dressing material Qifeng Dang a,1, Qianqian Zhang a,1, Chengsheng Liu a,⁎, Jingquan Yan b, Guozhu Chang a, Ying Xin c, Xiaoyu Cheng c, Yachan Cao a, Hong Gao a, Yan Liu a a b c

College of Marine Life Sciences, Ocean University of China, 5 Yushan Road, Qingdao 266003, PR China School of Medicine and Pharmacy, Ocean University of China, 5 Yushan Road, Qingdao 266003, PR China The Affiliated Hospital of Qingdao University, Qingdao University, 308 Ningxia Road, Qingdao 266071, PR China

a r t i c l e

i n f o

Article history: Received 9 April 2019 Received in revised form 20 July 2019 Accepted 8 August 2019 Available online 09 August 2019 Keywords: Decanoic acid functionalized chitosan Characterization Biocompatibility Wound healing

a b s t r a c t Skin wound dressing materials, which can accelerate wound healing and have the synthetic advantages of simplicity, environmental safety, and resource abundance, are becoming a hot topic of research now. Following such a research trend, we prepared novel decanoic acid functionalized chitosan (CSDA) with good solubility by acylation via a facile one-step method. FTIR, 1H NMR, and UV–Vis results demonstrated that alkyl chains were successfully grafted onto C2 positions of chitosan (CS) skeleton through acylation. XRD patterns implied that the crystallinity of CSDA greatly declined due to the introduction of alkyl moieties, favorable for improving water solubility. Conductometric titration results showed that the degrees of substitution of CSDA, CSDA1, and CSDA2 were 41.42, 26.12, and 23.17%, respectively. MTT assay and hemolysis experiments illustrated that all the CSDA samples tested in this work possessed good hemocompatibility (hemolysis rate b 2%) and excellent cytocompatibility (relative cell viability N75%) toward L929 cells. Moreover, CSDA-soaked gauze dressings and full-thickness excisional wound models were employed to estimate the feasibility of CSDA as wound dressing material, and the results displayed that CSDA with the degree of substitution of 41.42% could enhance the wound healing rate to 100% on day 16. Altogether, CSDA might be potential material used as wound dressing. © 2019 Elsevier B.V. All rights reserved.

1. Introduction Skin, as the first line of defense of human body, plays an important role in protecting the internal environment and maintaining the homeostasis [1,2]. Skin wounds caused by physical, chemical, and mechanical injuries often make people suffer pains mentally and physically. Although a large number of strategies and materials have been widely proposed and investigated for skin wound healing, biomaterials that help promote wound healing have increasingly attracted much attention because the current therapeutic materials are still limited, costly, and inefficient [3]. Satisfactory materials for wound healing are expected to be hemostatic, antibacterial, nontoxic, biodegradable, and biocompatible [4]. Chitosan (CS) is just one of the suitable candidates that comply with the requirements mentioned above [5–7]. It is reported that CS has a similar chemical structure to hyaluronic acid in extracellular matrixes, which exerts great efficiency in promoting wound healing [8]. Especially N-acetyl-D-glucosamine, as a major component in both hyaluronic ⁎ Corresponding author. E-mail address: [email protected] (C. Liu). 1 These authors contributed equally to this work.

https://doi.org/10.1016/j.ijbiomac.2019.08.083 0141-8130/© 2019 Elsevier B.V. All rights reserved.

acid and CS, is necessary for repairing skin wound [9]. Except for Nacetyl-D-glucosamine, glucosamine is also an important unit in CS molecules. Previous studies have shown that the moderate amount of glucosamine can promote wound healing, owing to its haemostatic activity [10], and to adsorption toward growth factors and cell surface receptors [11]. However, too much glucosamine has ample free amino groups, which can rupture cell membranes and further lead to high cell mortality [12]. In addition, strong intra- and inter-molecular hydrogen bonds substantially affect the solubility of CS, and are also disadvantageous to applications in skin wound repairing [6]. Hence, reducing the number of free amino groups in CS and improving water solubility are of considerable interest in wound repairing area. It is reported that long alkyl chains can be anchored onto blood cell membranes to connect cells to form 3D network structures, thus promoting blood clotting [13]. Furthermore, the introduction of alkyl chains to C2−NH2 can considerably weaken intra- and/or inter-molecular hydrogen bonds in CS molecules [6], likely improving the water solubility of CS. Therefore, we here proposed a hypothesis on the reduction in free amino percentage in CS by introducing long alkyl chains to C2−NH2, which should promote wound healing in theory. Modifications to CS are supposed to be one of the most up-and-coming investigation domains ever since the last century. Several CS

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derivatives such as peptide-decorated CS [14], CS-sulfonamide derivatives [15], 3,6-O-N-acetylethylenediamine modified CS [16] and Nsuccinyl-CS [17] have been prepared in order to improve the repairing effect of wounded skin. Compared with CS, the above CS derivatives largely promote skin wound repairing and are easy to be used to a certain extent. However, the modification processes of most CS derivatives are multi-step and time-consuming, leading to the following issues: (i) the waste of manpower and resources; (ii) environmental pollution caused by excessive reagents used and by-products produced in the reaction processes; and (iii) inevitable harm to human body caused by large amounts of chemicals used during the experimental operation. Consequently, it is necessary to explore a simple and short timeconsuming method for CS derivative preparation. Although numerous papers have been published on CS modifications, however, decanoic acid (DA) functionalized CS (CSDA) synthesized via directly grafting DA to pristine CS chains using a facile onestep method has not been found so far. In the present work, novel CSDA was successfully prepared using a simple and short timeconsuming protocol through introducing long alkyl chains of DA onto amino groups in CS via acylation. Theoretically, newly synthetized CSDA should possess the high possibility of low cytotoxicity due to the introduction of alkyl moieties, beneficial to skin wound repairing. Nevertheless, using CSDA as wound dressing has not yet been reported. The chemical structure and physical properties of CSDA were characterized by FTIR, 1H NMR, XRD, UV–Vis, and conductometric titration techniques. The solubility of CS and CSDA in different solvents was measured. Hemolysis and MTT tests were used to evaluate the biocompatibility of CSDA. Moreover, CSDA-soaked gauze dressings were employed in fullthickness excisional wound models to assess the wound healing property of CSDA. 2. Materials and methods 2.1. Materials, chemicals, and animals CS with the degree of deacetylation of 93% and mean molecular weight of 410 kDa was purchased from Qingdao Baicheng Marine Biological Resources Development Co., Ltd. (Qingdao, China). MTT, Dulbecco's modified Eagle's medium (DMEM), and fetal bovine serum (FBS) were commercially obtained from Sigma–Aldrich Co. (St. Louis, USA). 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), N-hydroxysulfosuccinimide sodium salt (NHS), and all the other chemical reagents were of analytical grade and purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). L929 cells were donated by Lab of Biochemistry, College of Marine Life Sciences, Ocean University of China. Male SD rats (body weight of 180–210 g each) and New Zealand white rabbits were purchased from Qingdao Food and Drug Administration (Qingdao, China). The procedure for animal experiments was conducted in accordance with the UK Animal (Scientific procedure) Act, 1986 and the related guidelines.

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low), the products were named as CSDA, CSDA1, and CSDA2, respectively. 2.3. Characterizations of CSDA 2.3.1. FTIR spectroscopy FTIR spectra were recorded on an FTIR spectrometer (AVATAR 360, Thermo Nicolet Corp., USA). Samples and KBr powders were dried for 4 h. Then, 2 mg of each sample was mixed with 100 mg of KBr, and the mixtures were ground to prepare discs. The discs were dried under a tungsten lamp for 5 min. All the samples were scanned against a blank KBr disc background in the range of 4000–400 cm−1 at a resolution of 4.0 cm−1 at room temperature. The results were analyzed by Omnic v8.0 software (Thermo Nicolet Corp., USA). 2.3.2. XRD crystallography XRD patterns of CS and CSDA were recorded on a diffractometer (D −8 ADVANCE, Bruker AXS Inc., Germany) with a detector operating at 40 kV and 40 mA using Cu Ka radiation. Scan angles from 5 to 60° were selected and the scanning speed was 4°/min. MDI Jade 6.5 software (Materials Data Inc., USA) was used to analyze the crystalline state of CS or CSDA. 2.3.3. 1H NMR analysis A total of 3 mg of CS or CSDA was accurately weighed and dried to constant weight. Subsequently, each sample was dissolved in 500 μL DCl solution (1%, D2O as solvent). 1H NMR spectra were procured from an NMR spectrometer (AV500 MHz, Bruker Optics Inc., Germany) at room temperature. 2.3.4. UV–vis scanning CSDA, CSDA1, and CSDA2 solutions were prepared at a concentration of 0.1 mg/mL. UV–Vis absorbance was measured on a spectrophotometer (UV2802S, Unico (Shanghai) Instrument Co., Ltd., China) over the wavelength range from 190 to 400 nm. HAc aqueous solution (0.1 mg/mL) was used as control. 2.3.5. Conductometric titration The degrees of substitution of CSDAs were determined by conductometric titration. Briefly, 0.1 g of each sample was dissolved in 15 mL of HCl solution (0.1 M) under stirring for 2 h to ensure fully dissolution. Then, 25 mL of NaOH solution (0.1 M) was added drop by drop, and the conductometric titration curves were recorded by using a conductivity meter (DDSJ−308A, INESA Scientific Instrument Co., Ltd., China). The mass percentage of NH2 was calculated by Eq. (1), and the degree of substitution (DS) of amino groups was determined according to Eq. (2). NH2 ð%Þ ¼ DS ¼

ðV 2 −V 1 Þ  10−3  C  16:0226  100% m

NH2 ðCSÞ−NH 2 ðSampleÞ  100% NH2 ðCSÞ

ð1Þ ð2Þ

2.2. Synthesis of CSDA The synthetic route of CSDA prepared via a one-step approach is shown in Scheme 1. Briefly, 1.0 g of CS was dissolved in 50 mL of acetic acid (HAc) solution (1%, v/v) under stirring for 24 h to ensure fully complete dissolution. DA ([1] 0.534, [2] 0.356, or [3] 0.134 g), EDC ([1] 1.785, [2] 1.190, or [3] 0.448 g), and NHS ([1] 0.357, [2] 0.238, or [3] 0.089 g) were in turn dissolved together in 20 mL of dimethyl sulphoxide (DMSO) and stirred for 2 h, and then slowly added dropwise to CS solution and allowed to react for 24 h. All the above reactions were carried out at room temperature. The resultant product was obtained by precipitating, washing with ethanol, and drying in a vacuum desiccator. According to the substitution degrees of amino groups (from high to

where V1 (mL) is the volume of NaOH solution to neutralize free H+, V2 + (mL) is the total volume of NaOH solution to neutralize NH+ 3 and H , C (M) is the molar concentration of NaOH solution, m (g) is the mass of sample, and 16.0226 (g/mol) is the molar mass of the amino group. 2.3.6. Solubility test The solubility of CSDA in different solvents was determined at room temperature according to the method described previously [18], with some modifications. Specifically, 0.1 g of the accurately weighed CSDA powders was added to 10 mL of a certain solvent under stirring at room temperature for 2 h. CS powders was used as control. The solvents used include ethanol (EtOH), isopropyl alcohol (IPA), dimethyl

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Scheme 1. Schematic synthesis route of CSDA. (CSDA, decanoic acid functionalized CS; CS, chitosan; DA, decanoic acid; EDC, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; NHS, N-hydroxysulfosuccinimide sodium salt.)

formamide (DMF), DMSO, HAc solution (1%, v/v), NaOH solution (0.1 M), and deionized water (DW).

expressed as the means ± standard deviations (SDs). The relative cell viability (RCV) was calculated by Eq. (3).

2.4. Biocompatibility of CSDA

RCV ð%Þ ¼

2.4.1. MTT assay In order to detect the biocompatibility of CSDA, we used CSDA solutions with different concentrations (0.5, 1, 2, and 4 mg/mL) to culture L929 cells by MTT assay. In brief, CSDA solutions were filtered through 0.22 μm membrane filters to remove bacteria. Cell suspensions were prepared using L929 cells grown to logarithmic phase and were incubated on 96 well cell culture plates with a density of 1 × 104 cells/well at 37 °C. After 12 h of cultivation, the culture media were replaced by CSDA solutions with different concentrations, and the blank complete medium was used as control. After each given incubation time, 5 mg/mL of MTT solution was added (20 μL per well) and incubated for 4 h. Then, the supernatant in each well was removed, and DMSO was added (150 μL per well) and incubated at 37 °C for 10 min. Finally, cell viability was assessed by measuring the absorbance at 490 nm on a microplate reader (Sunrise–basic, Tecan Group Ltd., Austria). Each sample was divided into five parallels. All the recorded values were estimated from the data of five individual experiments and were

OD490e  100% OD490c

ð3Þ

where OD490e and OD490c represent the absorbance values of the experimental and control groups, respectively. 2.4.2. Hemolysis test The hemolysis of CSDA was estimated according to the method described in American Society for Testing and Materials (ASTM F756 −17) [19]. Briefly, a certain amount of CSDA powders was dissolved in normal saline (NS; 1%, w/v) to prepare solutions with different concentrations (0.5, 1, 2, 4, and 10 mg/mL) and incubated at 37 °C for 1 h. Fresh New Zealand white rabbit blood (4 mL) was quickly injected into the EDTA-K2 vacuum blood collection tube, and diluted with 5 mL of NS. Diluted blood (0.06 mL) was added to each sample and incubated gently at 37 °C for another 1 h and 2 h. Then, the erythrocyte fragments were removed by centrifuging (1200 g) for 10 min, and the absorbance of the supernatants was measured at 545 nm using a UV– Vis spectrophotometer. Distilled water and NS were used as positive

Fig. 1. (A) FTIR spectra and (B) XRD patterns of CS and CSDA, and 1H NMR spectra of (C) CS and (D) CSDA.

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Fig. 2. (A) UV–Vis absorption spectra and (B) conductometric titration curves of CSDAs with different degrees of substitution.

(pc, 100% lysis) and negative (nc, 0% lysis) controls, respectively. The hemolysis rate (HR %) was calculated according to Eq. (4).

3. Results and discussion 3.1. Characterization of CSDA

HRð%Þ ¼

ODsample −ODnc  100% ODpc −ODnc

ð4Þ

2.5. In vivo wound healing study Based on MTT assay and hemolytic results, CSDA was selected for the following experiments. To preliminarily evaluate the feasibility of CSDA as wound dressing, the humanized full-thickness excisional round wound (d = 1 cm) was established on the backsides of healthy male SD rats [20]. The male rats (around 210 g each) were randomly divided into two groups (NS and CSDA groups) and each group included 12 animals. Sterile gauze (6-layer, 1.5 × 1.5 cm2) was soaked with 1 mL of NS or CSDA solution (1%, w/v). The wounds in NS and CSDA groups were covered with NS-soaked and CSDA-soaked gauze dressings, respectively. Then, all the dressings were fixed with dry sterile gauzes. During the experimental process, the dressings of all groups were changed every other day. Clinical signs, mortality, and mean body weight of all the rats in NS and CSDA groups were recorded every day during the experimental period. In addition, blood was collected for hematologic and plasma biochemical measurements, including white blood cell (WBC), red blood cell (RBC), hemoglobin (HGB), platelet (PLT), alanine aminotransferase (ALT), aspartate aminotransferase (AST), uric acid (Ua), urea, and serum creatinine (CR), at the scheduled time (day 4, 8, 12, and 16). Wound areas (WA) were measured at every four days and calculated by ImageJ software (National Institutes of Health, USA). The wound healing rate (%) was defined as Eq. (5).

Wound healing rateð%Þ ¼

WA0 −WAt  100% WA0

3.1.1. FTIR analysis FTIR spectra of CS and CSDA are displayed in Fig. 1A. Two absorption bands at 1653 cm−1 (C_O stretching vibration, amide I) and 1558 cm−1 (N−H bending vibration, amide II) of CSDA were strengthened in intensity, compared to those at 1655 and 1593 cm−1 in CS spectrum [21]. Besides, the signals at 2855 and 2925 cm−1 (asymmetric and symmetric stretching vibration of C−H) in CSDA spectrum largely increased, compared to the counterparts at 2855 and 2925 cm−1 in CS spectrum [22]. Such findings above indicated that the alkyl chains (DA moieties) were introduced into −NH2 on CS backbone. There were no obvious differences between the absorption bands at 1030 and 1076 cm−1 in CS and CSDA spectra, implying that the hydroxyl groups on CS skeleton were not involved in the grafting reaction. Overall, these results illustrated that the alkyl chains of DA were successfully grafted to C2 position in CS molecular skeleton.

3.1.2. XRD study The crystallinity of CS and CSDA was determined by XRD crystallography and the results are shown in Fig. 1B. For CS, there were two peaks at around 20.2° (an intense diffraction peak, attributed to (100)) and 10.7° (a weak diffraction peak, attributed to (020)). These two peaks indicated the high crystallinity of CS, which was mainly caused by the formation of strong intra- and/or inter-molecular hydrogen bonds between amino and hydroxyl groups of CS [23]. Compared with that of CS, the XRD pattern of CSDA displayed an extremely weak diffraction peak at 20.2° only, demonstrating generous decrystallization. The introduction of long alkyl chains weakened the intra- and inter-molecular hydrogen bonds in CS, which led to the damage of symmetry and stereoregularity, and then led to the formation of amorphous structure in CSDA. The decrystallization due to damage of symmetry and

ð5Þ

where WA0 (cm2) is the original wound area, and WAt (cm2) is the wound area at time t.

2.6. Statistical analysis Experiments were conducted at least three times and all data were expressed as the means ± SDs. Statistical analyses were conducted by using a one-way ANOVA test to deal with the data via SPSS 16.0 software (IBM Corp., USA), and p b 0.05 and p b 0.01 were considered to be significant and highly significant, respectively.

Table 1 Solubility of CS and CSDAs in different solvents at 25 °C. Samplea

CS CSDA CSDA1 CSDA2 a

Solventb EtOH

IPA

DMF

DMSO

HAc

NaOH

DW

− − − −

− − − −

− − − −

− − − −

+ + + +

− − − −

− + + +

CS, chitosan; CSDA, decanoic acid functionalized CS. EtOH, ethanol; IPA, isopropyl alcohol; DMF, dimethyl formamide; DMSO, dimethyl sulphoxide; HAc, acetic acid solution (1%, v/v); NaOH, sodium hydroxide solution (0.1 M); DW, deionized water; “+” and “−” denote soluble and insoluble, respectively. b

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stereoregularity was one of the reasons for the improvement of water solubility of CSDA. 3.1.3. 1H NMR spectra 1 H NMR was used to further detect the chemical structures of CS and CSDA, and the spectra are shown in Fig. 1C and D, respectively. In CS spectrum (Fig. 1C), the chemical shift of protons from N-acetyl was appeared at 1.91 ppm, and the signals at 3.06 and 4.76 ppm were attributed to H2 proton in glucosamine (GluN) and H1 proton in glucosamine (GluN), respectively [24]. In addition, the multiplet at 3.50–3.70 ppm belonged to the chemical shifts of H3–H6 on CS backbone [25], and the singlet at 4.96 ppm was due to the proton signal from solvent (HOD). In CSDA spectrum (Fig. 1D), compared to CS spectrum, all the above signals were present. Notably, new signals at 0.75 and 1.24 ppm were attributed to the protons of methyl (−CH3) at the end of the alkyl chains and methylene (−CH2) connected to the end methyl, respectively. Besides, the signal at 1.91 ppm was strengthened compared to that of CS, which was corresponded to methylene (−CH2) directly linking to amide bond overlapped with the N-acetyl group. The signal at 4.49 ppm was the chemical shift of H1 in Nacetylglucosamine (GluNAc) [24]. The above results indicated that CSDA had a complete glycol cyclic structure, and that DA was successfully grafted to free amino groups via acylation.

NH2 destroyed intra- and inter-molecular hydrogen bonds, and further compromised the regularity of the molecular structures and reduced the internal crystal region of CSDA. Hence, the modification to CS with DA obviously improved the solubility of CSDA in DW. 3.2. Biocompatibility of CSDA 3.2.1. MTT assay In order to evaluate the biocompatibility of CSDA, the effect of CSDA solutions on the viability of L929 cells was detected by using the MTT

3.1.4. UV absorption analysis UV–Vis absorption is caused by the transition of electrons that occurs after a molecule or ion absorbs ultraviolet or visible light. The structural units that absorb ultraviolet or visible light are known by chromophores, such as C_C, C_O, and C=CR−O−. Therefore, UV spectra are usually employed to analyze the structures of organic compounds. In this study, UV absorption spectra at a wavelength range from 190 to 400 nm were used as an assistant method to analyze the structures of CSDA, CSDA1, and CSDA2, and the results are displayed in Fig. 2A. Generally, there are no chromophores in the polysaccharide molecules, whereas chitin and CS have weak and broad absorption peaks at around 230 nm because of C_O of acetyl groups at the C2 position of each sugar residue [26]. In each sample spectrum, an obvious absorption peak at 230 nm was observed, and increased in intensity as the substitution degrees of amino groups increased. Accordingly, it could be inferred that CSDA contained much more C_O (chromophore) than CSDA1 and CSDA2, and that DA was successfully grafted to amino groups via acylation. 3.1.5. Conductometric titration The conductometric titration curves of CS and CSDA are shown in Fig. 2B and could be divided into three regions. In the first region, or at the initial titration stage, H+ in HCl solution was neutralized by OH− in NaOH solution and thus, the titration curves decreased rapidly until H+ was completely neutralized. In the second region, the residual amino groups on CSDA were protonated to form NH+ 3 and combined with OH−, so the decrease in NH+ 3 in this region maintained equilibrium with the increase in Na+, and therefore the conductivity remained basically stable. In the third region, the conductivity of the solution increased with the continuous addition of Na+ on account of the fact that NH+ 3 was completely neutralized. In this region, the conductivity increased slowly due to the buffering effect of salts that were formed in the aforementioned two stages. Compared with CS, the amino percentage in CSDA decreased due to the introduction of DA. Based on the calculation results, the substitution degrees of amino groups of CSDA, CSDA1, and CSDA2 were 41.42, 26.12, and 23.17%, respectively. 3.1.6. Solubility determination The solubility of CSDA and CS in different solvents is shown in Table 1. CS was not able to dissolve in the tested solvents except HAc solution (1%, v/v), as described previously [18]. Unlike CS, CSDA was soluble in both DW and HAc solution. The introduction of alkyl chains to

Fig. 3. In vitro cytotoxicity evaluation of different concentrations (0.5, 1, 2, and 4 mg/mL) of (A) CSDA, (B) CSDA1, and (C) CSDA2 solutions. (Data are obtained by the MTT assay, and error bars indicate standard deviations calculated from five parallel tests; *, p b 0.05; **, p b 0.01.)

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Fig. 4. Hemolysis rates of samples with different concentrations (0.5, 1, 2, 4, and 10 mg/mL) for (A) 1 h and (B) 2 h. (Error bars indicate standard deviations calculated from five parallel tests; *, p b 0.05; **, p b 0.01.)

assay and the results are shown in Fig. 3. All the tested CSDA solutions showed low toxicity to L929 cells. Cells cultured in CSDA solutions for 24, 48, and 72 h had RCV% values of above 75%, within the acceptable limitation [27]. In addition, the RCV% of all samples increased with the increase in culture time. It is well known that the animal cytomembrane has a flexible structure composed of phospholipid bilayer and glycoprotein. The phospholipid molecules are shaped with hydrophilic heads and hydrophobic tails. The tails tend to stick to each other to avoid water and let the heads face the aqueous areas inside and outside the cell. The two layers of phospholipid molecules create the lipid bilayer. The alkyl chains on CSDA could insert into the lipid bilayer without destroying the cell membrane, which could promote the cell adhesion of CSDA. Moreover, free amino groups on CSDA could electrostatically attract the negative charge on cellular membranes [28], and thus

promoting cells adhesion. Hence, there were no significant differences between the tested CSDA samples with different alkyl content. 3.2.2. Hemolysis test Hemolysis test is a simple and credible method to evaluate the hemocompatibility of materials for tissue engineering. According to ASTM F756–17 [19], biomedical materials with hemolysis rates of b2% are considered to be non-hemolytic, and to be slightly hemolytic for the range of 2 to 5%. The hemolysis rates of CSDA samples are shown in Fig. 4. The hemolysis rates induced by all samples were b2%, indicating that all samples were non-hemolytic. Also, Fig. 4A and B show that the hemolysis rates of all samples increased with the increase in sample concentrations, as well as in contact time. Additionally, at the same time, the hemolysis rates of CSDA1 and CSDA2 with the same

Fig. 5. (A) Wound healing photographs of SD rats, and (B) average body weight of rats over time and (C) wound healing rates. (Error bars indicate standard deviations calculated from three parallel tests; *, p b 0.05; **, p b 0.01; NS, normal saline.)

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Fig. 6. (A) Hematologic and (B) plasma biochemical parameters of SD rats during the wound healing process. (WBC, white blood cell; RBC, red blood cell; HGB, hemoglobin; PLT, platelet; ALT, alanine aminotransferase; AST, aspartate aminotransferase; Ua, uric acid; CR, serum creatinine; error bars indicate standard deviations calculated from three parallel tests; NS, normal saline.)

concentration were higher than CSDA. Such results might be due to the high residual free amino groups on CSDA1 and CSDA2 molecules. High residual free amino groups had stronger electrostatic interaction with red blood cells, resulting in cell disruption and further increasing the hemolysis rates. Such findings implied that non-hemolytic CSDA could be obtained by increasing the content of alkyl chains. 3.3. In vivo wound healing study In the present work, the effects of CSDA on wound healing were preliminarily evaluated in rat models with full-thickness excisional wounds. Fig. 5A shows digital photographs of wounds in each group on day 0, 4, 8, 12, and 16. The wounds in CSDA group healed up faster than those in NS group. The changes of wound healing rate (%) in both CSDA and NS groups were calculated and the results are presented in Fig. 5C. Compared to the control group, the rats in CSDA group had higher wound healing rates on day 0, 4, 8, 12, and 16. Besides, the rats in both CSDA (CSDA-soaked gauze dressings) and NS groups (NSsoaked gauze dressings) survived and had monotonical increases in body weight (Fig. 5B) during the observation period. Therefore, CSDA had the function of promoting wound healing. Skin wound healing is a complex process that requires the participation of multiple growth factors, repair cells, as well as complete hemostatic and inflammatory mechanisms [29,30]. At the different stages of wound healing, each kind of cell has its unique function, such as fibroblasts, neutrophils, and macrophages. As soon as the wound comes into being, it will have different degrees of tissue necrosis and blood vessel rupture bleeding, and then there will be inflammatory reaction in a few hours, which is characterized by wound congestion, oozing, and local swelling. Therefore, hemostasis is the primary body reaction after injury. In the present work, long alkyl chains on CSDA might promote blood clotting in such a way that the long alkyl chains were anchored onto blood cells to form 3D network structures between cells [31]. Wound scars were formed in both CSDA and NS groups on day 4. The wound scars formed by the coagulation of proteins in wound exudates could protect the wound temporarily. Moreover, the positive charge of the remaining amino groups on CSDA backbone could react with the negative charge on the surface of red blood cells, which could stop bleeding quickly. Furthermore, the moderate amino groups in CSDA could bind with chondroitin sulfate proteoglycans through cell membranes, which would promote the migration of fibroblasts to the wound site [32]. Therefore, we speculated that the synergetic effect of the hemostasis and migration of fibroblasts might be the reasons for CSDA to promoting the wound healing. Hematology and plasma biochemical parameters, as two important indicators for physiological conditions of an animal, were handled to assess hematopoiesis and functions of liver and kidney. As shown in Fig. 6A, there were no significant differences (p N 0.05) in the levels of red blood cells (RBC), white blood cells (WBC), platelet (PLT),

hemoglobin (HGB) between the rats in NS and CSDA groups, indicating that CSDA did not cause a functional disorder of the blood system in rats. Notably, in the early stage of wound healing, the PLT level of rats in the CSDA group was slightly higher than that in the NS group, due to the effect of CSDA on coagulation. The effect of CSDA-soaked gauze dressing on plasma biochemical parameters are shown in Fig. 6B. The results showed that all the blood biochemical parameters in two groups were within the normal level, and did not show significant differences (p N 0.05) between CSDA and control groups. These results indicated that the treatment with CSDA-soaked gauze dressing did not cause abnormality of kidney and liver functions. In conclusion, CSDA could be used as potential wound dressing material.

4. Conclusions In this work, CSDA with good water solubility was successfully prepared by acylation via a one-step method. FTIR, 1H NMR, and UV–Vis analyses indicated that alkyl chains were directly introduced into the C2−NH2 on CS skeleton. CSDA had lower crystallinity than CS, as evidenced by XRD results. Conductometric titration results displayed that the substitution degrees of amino groups of CSDA, CSDA1, and CSDA2 were 41.42, 26.12, and 23.17%, respectively. Hemolysis and MTT results demonstrated that CSDA was non-hemolytic and had low cytotoxicity toward L929. Moreover, the wound healing tests showed that CSDA could accelerate the wound healing rates (p b 0.05). Overall, CSDA, with good solubility, low cytotoxicity, satisfactory hemocompatibility, and outstanding performance for promoting wound healing, could be used as potential skin repair material. Of course, further studies on wound healing properties, particularly the effects of CSDA on blood coagulation, angiogenesis, collagen deposition, and proliferation and differentiation of epidermal cells, are required for the evaluation of this new material. All the tests mentioned above are underway now.

Declaration of competing interest None. Acknowledgments This work was supported by National Nature Science Foundation of China (NSFC) [Grant number 31400812], and Science and Technology Development Funds of Qingdao Shinan [Grant numbers 2014-14-003SW, 2015-5-015-ZH]. References [1] J.S. Barbieri, K. Wanat, J. Seykora, Skin: basic structure and function, Pathobiol. Hum. Dis. (2014) 1134–1144, https://doi.org/10.1016/B978-0-12-386456-7.03501-2.

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