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Degradation and compatibility behaviors of poly(glycolic acid) grafted chitosan
Materials Science and Engineering C 33 (2013) 2626–2631
Contents lists available at SciVerse ScienceDirect
Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Degradation and compatibility behaviors of poly(glycolic acid) grafted chitosan Luzhong Zhang a, Sufeng Dou a, Yan Li b, Ying Yuan a, Yawei Ji a, Yaling Wang c, Yumin Yang a,⁎ a b c
Jiangsu Key Laboratory of Neuroregeneration, Nantong University, Nantong 226001, P. R. China College of Life Science and Pharmaceutical Engineering, Nanjing University of Technology, Nanjing 210009, P. R. China College of Chemistry and Chemical Engineering, Nantong University, Nantong 226001, P. R. China
a r t i c l e
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Article history: Received 26 April 2012 Received in revised form 20 January 2013 Accepted 15 February 2013 Available online 21 February 2013 Keywords: Chitosan Poly(glycolic acid) Grafting Degradation Biocompatibility
a b s t r a c t The films of poly(glycolic acid) grafted chitosan were prepared without using a catalyst to improve the degradable property of chitosan. The films were characterized by Fourier transform-infrared spectroscopy and X-ray photoelectron spectroscopy (XPS). The degradation of the poly(glycolic acid) grafted chitosan films were investigated in the lysozyme solution. In vitro degradation tests revealed that the degradation rate of poly(glycolic acid) grafted chitosan films increased dramatically compared with chitosan. The degradation rate of poly(glycolic acid) grafted chitosan films gradually increased with the increasing of the molar ratio of glycolic acid to chitosan. Additionally, the poly(glycolic acid) grafted chitosan films have good biocompatibility, as demonstrated by in vitro cytotoxicity of the extraction fluids. The biocompatible and biodegradable poly(glycolic acid) grafted chitosan would be an effective material with controllable degradation rate to meet the diverse needs in biomedical fields. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Chitosan is a linear natural polysaccharide containing β-(1,4)2-amino-D-glucose and β-(1,4)-2-acetamindo-D-glucose unit, and obtained by N-deacetylation of chitin extracted from crustacean shells [1]. Because of its good biocompatible, antibacterial and low toxic properties, chitosan has been widely used for biomedical applications, such as sustained drug and protein delivery [2–4], non-viral gene delivery [5] and tissue engineering [6,7]. However, the use of chitosan in the biomedical fields has been limited because of the slow degradation rate [8], poor mechanical properties and the fact that it is hard to handle [9]. Since grafting copolymerization can modify the structure and properties of natural polysaccharides, it is an important resource for developing advanced materials [10,11]. Chitosan has reactive amine and hydroxyl side groups occurring in the units of polymer chains, and it acts as a desirable backbone to graft synthetic polymer. For instance, chitosan-g-N-isopropylacrylamide copolymer with thermoresponsive, fully reversible property was synthesized [12]. In the view of the designing of biologically degradable tissue engineering biomaterials, it would be advantageous to prepare a grafting copolymer composed of chitosan and poly(glycolic acid). Poly(glycolic acid), which is a fascinating synthetic polymer, has exhibited good mechanical properties, biodegradability and biocompatibility [13]. It has been widely used in biomedical applications, such as tissue engineered scaffold [14,15], controlled drug delivery
⁎ Corresponding author. Tel./fax: +86 513 85511585. E-mail address: [email protected] (Y. Yang). 0928-4931/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.msec.2013.02.024
systems [16] and implants for orthopedic device [17]. However, poly(glycolic acid) generally produces acidic degradation products at the implanted site. The degraded glycolic acid decreases the pH in the surrounding of the scaffold, and then evokes inflammatory tissue reactions [18]. Since basic glucosamine released from degrading chitosan is expected to neutralize acidic products, the tissue reactions may be alleviated by chitosan grafting copolymer. Importantly, poly(glycolic acid) grafted chitosan could not only retain the good properties of chitosan, but also may bring the easy degradation. In the present work, the films of poly(glycolic acid) grafted chitosan were prepared without using a catalyst to improve the degradable properties of chitosan. The films were characterized by Fourier transforminfrared spectroscopy (FT-IR) and X-ray photoelectron spectroscopy (XPS) analysis. The degradation rates of the poly(glycolic acid) grafted chitosan films were investigated in the lysozyme solution. It is found that the poly(glycolic acid) grafted chitosan films are more easily degradable compared with chitosan film, and the degradation rate of poly(glycolic acid) grafted chitosan films could be regulated. The in vitro cytotoxicity of the extraction fluids revealed that the films of poly(glycolic acid) grafted chitosan have no obvious cytotoxic effect. 2. Experimental section 2.1. Materials and methods Chitosan (the average molecular weight was 2.8 × 10 4 Da, the degree of deacetylation was about 95.3%) was purchased from Nantong Xincheng Biochemical Company. The glycolic acid was obtained from Acros Organics Company. The lysozyme was bought from Shanghai
L. Zhang et al. / Materials Science and Engineering C 33 (2013) 2626–2631
Aladdin Reagent Company. Other reagents were used as received without further purification. FT-IR spectra of chitosan and poly(glycolic acid) grafted chitosan films were recorded with a spectrometer (Nicolet 5700, Madison, WI). The X-ray photoelectron spectroscopy (XPS) of chitosan and poly(glycolic acid) grafted chitosan films were recorded on a Thermo Scientific K-Alpha spectrometer (Thermo Scientific, USA) equipped with a monochromatic Al-Kα X-ray source. The morphology of the chitosan film and the poly(glycolic acid) grafted chitosan films before and after degradation were observed using an S-3400 NII scanning electron microscopy (SEM, Hitachi, Japan). The samples were coated with gold using a JFC-1600 unit (JEOL Inc., Japan) Ion Sputter before examination under the SEM. 2.2. Preparation of poly(glycolic acid) grafted chitosan films Chitosan was dissolved in glycolic acid solutions with different concentrations and the mixtures were then standing overnight at room temperature. The solutions were poured into frame molds and dried at 45 °C to a constant weight. Then, the films were treated at 80 °C at a reduced pressure (10–12 mm Hg) for another 24 h to promote dehydration of the copolymer salts and the polymerization of glycolic acid. At last, the samples were extracted with methanol to remove unreacted glycolic acid and oligo(glycolic acid). 2.3. Preparation of chitosan film Chitosan was dissolved in acetic acid solutions with different concentrations and the mixtures were then standing overnight at room temperature. The solution was poured into a frame mold and dried at 45 °C to a constant weight. The samples were then extracted with methanol to remove acetic acid, and then the films were immersed in 2 M sodium hydroxide solution for 4 h. All prepared films were sterilized with 70% alcohol and washed with sterilized phosphate buffered saline (PBS, 0.1 M, pH 7.4) prior to use. 2.4. In vitro degradation of poly(glycolic acid) grafted chitosan films and chitosan film The enzymatic degradation of poly(glycolic acid) grafted chitosan films in vitro was investigated in the lysozyme solution. The poly(glycolic acid) grafted chitosan films (0.1 g) were immersed in 10 mL of lysozyme solution (2 mg/mL) in phosphate buffered saline (PBS) (pH 7.4) at 37 °C. For the sake of comparison, chitosan film was treated in the same manner. All the solutions were changed weekly, and the films were taken out at the predetermined time points. The degraded samples were washed three times with distilled water, dried under vacuum at 40 °C and weighted. The measurements were performed in triplicate and the results were the average of the three times. The degradation ratio was determined according to the following equation:
Degradation ratio ¼
2.6. In vitro cytotoxicity According to ISO-10993, the MTT [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide] assay was performed to determine the in vitro cytotoxicity of the extracts of films. L929 cells were seeded into a 96-well plate at a density of 5000 cells per well and incubated with 100 μL culture medium containing either in the DMEM medium or the film extract fluid at 37 °C for various time periods. After the incubation, the culture medium in each well was removed and the cells were washed three times with PBS. 20 μL of MTT solution (5 mg/mL) was added to each well and cells were cultured for another 4 h. The supernatant was discarded and then 100 μL of DMSO was added to each well. The OD values of the plate were measured on an EIX-800 Microelisa reader at 570 nm (Bio-Tek Inc., USA). 2.7. Bright-field microscopy measurement of cells The cells were seeded onto the films, which had been placed into a 6-well plate at a density of 2.5 × 10 5 cells per well, and incubated for 12 h. Thereafter, the cells were observed by optical microscopy. 3. Results and discussion The poly(glycolic acid) grafting chitosan were obtained after the reaction between chitosan and glycolic acid without using a catalyst (Scheme 1). When chitosan was dissolved in a glycolic acid aqueous solution, the amino groups of chitosan were protonated and the chitosan amino glycolic salt was formed [19]. The dehydration of the salt was done to form amide groups between chitosan and glycolic acid by heating the solution, and the polycondensation of glycolic acid was carried out simultaneously. The different poly(glycolic acid) grafted chitosan films were prepared via the variation of the feeding molar ratios of glucosamine unit to glycolic acid (the molar ratios of glucosamine unit to glycolic acid was changed from 1/1 to 1/10). Fig. 1 shows the IR spectra of chitosan and the poly(glycolic acid) grafted chitosan copolymer films extracted with methanol. For the chitosan film (Fig. 1a), the absorption peaks at 1595 and 1658 cm−1 are attributed to the N\H bending vibrations of non-acylated NH2 group and the carbonyl stretching of secondary amide (amide I band), respectively. Compared with chitosan film, the film of poly(glycolic acid) grafted chitosan (CS:GA = 1:1) (Fig. 1b) has new peaks appearing at 1639 and 1087 cm−1 corresponding to the carbonyl stretching of secondary amide (the NH2 groups of chitosan unit are acylated by glycolic acid) and C\O stretching of glycolic acid (CH2–OH), respectively. With the molar ratios of glucosamine unit to glycolic acid increased to 1/5 and 1/10 (Fig. 1c and d), the peaks located at 1749 and 1184 cm−1 can be assigned to the ester carbonyl stretching and C\O stretching of oligo(glycolic acid), indicating the formation of oligo(glycolic acid) attached to the chitosan. Additionally, no peak corresponding to ether groups from the reaction between the hydroxyl groups was found in the spectrum of the poly(glycolic acid) grafted
Initial dry weightdry weight after degradation : Initial dry weight
2.5. Cell culture The mouse fibroblast cells (L-929) were grown at 37 °C in a humidified atmosphere of 5% CO2 in Dulbecco's modified Eagle's medium (DMEM). The culture medium was supplemented with 10% fetal calf serum, 100 μg/mL streptomycin, 100 U/mL penicillin and 4 × 10 −3 M L-glutamine.
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Scheme 1. Grafting copolymerization of chitosan and glycolic acid.
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Fig. 1. FT-IR spectra of chitosan (a) and the poly(glycolic acid) grafted chitosan (b, CS: GA = 1:1; c, CS:GA = 1:5; d, CS:GA = 1:10; the feeding molar ratio of glucosamine unit (CS) to glycolic acid(GA)).
chitosan, suggesting the successful synthesis of poly(glycolic acid) grafted chitosan. In order to confirm the chemical composition, XPS was used to obtain the information about different elements at the surface. Fig. 2 shows that the XPS N 1s region corresponding to the atomic orbital 1s of nitrogen occurred in the spectra of chitosan and poly(glycolic acid) grafted chitosan films. For the chitosan films after the NaOH treatment,
5600 5400
a
Pos. 399
the N 1s narrow scan (Fig. 2a) displayed only one peak at 399 eV approximately, corresponding to the amine and amide chemical binding [20]. In contrast to the chitosan film, the N 1 s narrow scan of the poly(glycolic acid) grafted chitosan (CS:GA= 1:1, Fig. 2b) displayed two peaks located at 399 eV and 401 eV approximately, corresponding to the amide and protonated amine [20]. The atomic ratios of these peaks were about 54% and 46%. With the molar ratios of glucosamine unit to glycolic acid increased to 1/5 and 1/10, the atomic ratios of the protonated amine decreased to 35% and 29% [Fig. 2c and d], respectively, indicating that the formation of amide groups between the chitosan and glycolic acid. The XPS C 1s spectrum of the chitosan film and chitosan graft copolymer films were also shown in Fig. 3. The C 1s narrow scan (Fig. 3a) at 284 eV approximately, was mainly assigned to the carbon surface contaminant \CH2- for the chitosan films [21]. The C 1s peak at 286 eV approximately was assigned to the C\N\C_O in the spectrum of the poly(glycolic acid) grafted chitosan (CS:GA = 1:1, Fig. 3b), suggesting that the glycolic acid has grafted the chitosan [22]. When the molar ratio of chitosan and glycolic acid in the poly(glycolic acid) grafted chitosan came to 1/5, a new peak located at 288 eV approximately emerged (Fig. 3c), demonstrating that more glycolic acid was grafted onto chitosan [20]. With the molar ratio decreasing to 1/10, the atomic ratio of the peak at 284 eV increased and can be reasoned that more glycolic acid were grafted onto the chitosan (Fig. 3d). The controllable biodegradability and biocompatibility are the important requirements for the development of any medical implant materials [23]. For example, the biomaterial degradation rate is regarded as an important design consideration for artificial nerve scaffolds, since non-degradable tubes could compress regenerated nerves in the long term. Proper degradation rate is an important factor for a tissue-engineered scaffold to resist rearing and stretching
10000
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%Area 54.37 45.63
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Fig. 2. XPS N 1s narrow scans with the curve fit of chitosan (a) and poly(glycolic acid) grafted chitosan (b, CS:GA = 1:1; c, CS:GA = 1:5; d, CS:GA = 1:10; the feeding molar ratio of glucosamine unit (CS) to glycolic acid(GA)).
L. Zhang et al. / Materials Science and Engineering C 33 (2013) 2626–2631
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Fig. 3. XPS C 1s narrow scans with the curve fit of chitosan (a) and poly(glycolic acid) grafted chitosan (b, CS:GA = 1:1; c, CS:GA = 1:5; d, CS:GA = 1:10; the feeding molar ratio of glucosamine unit (CS) to glycolic acid(GA)).
forces during nerve regeneration process [24]. To evaluate the performance of the poly(glycolic acid) grafted chitosan in biomedical application, the degradation behaviors of the chitosan or poly(glycolic acid) grafted chitosan films in vitro were investigated in the presence of lysozyme at 37 °C. Since hen egg white (HEW) lysozyme as well as human lysozyme could cleavage the β(1-4)-linked unit of chitosan
[25], the in vitro degradation behavior of chitosan-g-poly(glycolic acid) has been investigated by using HEW lysozyme in this study. Fig. 4 shows the degradation behavior of the poly(glycolic acid) grafted chitosan samples, including chitosan film, poly(glycolic acid) grafted chitosan films (the molar ratios of glycolic acid to glucosamine unit was changed from 1/1, 3/1, 5/1, 10/1). It can be observed that only 0.70% weight was lost in the group of chitosan film within
100
chitosan CS:GA=1:1 CS:GA=1:3 CS:GA=1:5 CS:GA=1:10
Weight loss (%)
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0 0
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Degradation Time (days) Fig. 4. Dry weight loss after in vitro degradation of poly(glycolic acid) grafted chitosan films and chitosan film in 4 mg/mL lysozyme solution in PBS (pH 7.4) incubated at 37 °C for various time periods (the feeding molar ratio of glucosamine unit to glycolic acid, CS:GA).
Fig. 5. SEM images of the chitosan film and the poly(glycolic acid) grafted chitosan film before (a, chitosan; c, poly(glycolic acid) grafted chitosan and after degradation (b, chitosan; d, poly(glycolic acid) grafted chitosan) for 4 weeks. The chitosan-g-poly(glycolic acid) molar ratio of glucosamine unit to glycolic acid is 1:10.
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1.5
OD value
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CS:GA=1:1 CS:GA=1:5 CS:GA=1:10 chitosan DMEM
0.9
0.6
0.3
0.0 1
3
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Time (Days) Fig. 6. The changes in cell viability of L929 cells after they were cultured in the DMEM medium or different poly(glycolic acid) grafted chitosan extraction fluids for 1 d, 3 d, 5 d, 7 d, respectively (CS:GA, the molar ratio of glucosamine unit to glycolic acid).
28 days [1,26]. Thus, the chitosan film has a very slow degradation rate. Compared to the chitosan film, the weight loss percentage of poly(glycolic acid) grafted chitosan significantly increased. The mean percentage of weight loss at the termination of the experiment for the groups of poly(glycolic acid) grafted chitosan (1/1) and poly(glycolic acid) grafted chitosan (10/1) are 4.67% and 85.60%. The percentage of weight loss gradually increased with the increasing of the molar ratio of glycolic acid to chitosan. This phenomenon can be attributed to the fact that the poly(glycolic acid) is easy to be degraded [27] and the amide of chitosan will make the degradation faster [26]. Additionally, the pH values did not change significantly during the degradation in the PBS. The various degradation rates would help to design poly(glycolic acid) grafted chitosan based biomedical materials with the predetermined degradation time. Therefore, the degradation rate of chitosan based materials could be regulated by varying the molar ratio of glycolic acid to chitosan. The SEM analysis further showed the morphological change of poly(glycolic acid) grafted chitosan after degradation over 4 weeks (Fig. 5). It is found that the surface of chitosan and poly(glycolic
acid) grafted chitosan films were smooth before degradation. The sample of chitosan film degraded in PBS containing lysozyme shows slightly rough surface, while the sample of poly(glycolic acid) grafted chitosan film shows obviously rough surface under the same condition. These results demonstrated that the poly(glycolic acid) grafted chitosan films have the faster degradation rate than chitosan film, which is consistent with the results of the mean weight loss of poly(glycolic acid) grafted chitosan after 4 weeks. To examine the compatibility of the poly(glycolic acid) grafted chitosan, the in vitro cytotoxicity tests of the film extracts against the mouse fibroblast cells (L929) were conducted according to ISO-10993. Since L929 cells are easy to prepare and culture, it is routinely used for cytotoxicity studies [28]. The L929 cells were cultivated in the DMEM culture medium and the poly(glycolic acid) grafted chitosan film extract fluid, for 1, 3, 5, and 7 days (Fig. 6), respectively. It is found that the viability of L929 cells cultured in the film extract fluid was not significantly different from that in DMEM supplemented with 10% fetal calf serum (blank control) after 1, 3, 5, and 7 days, indicating that the films of poly(glycolic acid) grafted chitosan have no obvious cytotoxic effect at all used conditions. Furthermore, the cells were observed by optical microscopy after being cultured for 12 h on the chitosan and different poly(glycolic acid) grafted chitosan films (Fig. 7). Bright-field images show that all cells proliferate very well and maintain their normal configuration on all the used films (The normal L929 cells should be mainly adhered to the surface of the culture plate, plump, spindle-shaped and glossy, usually accompanied by some round dividing cells.). Therefore, the poly(glycolic acid) grafted chitosan films showed no obvious cytotoxicity and presented a good substrate for the growth of L929 cells. 4. Conclusion The poly(glycolic acid) grafted chitosan films were prepared without using a catalyst via dehydration reaction, and characterized by FT-IR and XPS analysis. The poly(glycolic acid) grafted chitosan films degraded much faster than chitosan film, and the degradation rate of poly(glycolic acid) grafted chitosan films gradually increased with the increasing of the molar ratio of glycolic acid to chitosan. In vitro cytotoxicity tests exhibited that the poly(glycolic acid) grafted chitosan films have good biocompatibility. These results suggested that poly(glycolic acid) grafted chitosan could be a good biocompatible and biodegradable materials. The poly(glycolic acid) grafted
Fig. 7. Bright-field microscopic images of L929 cells after 12 h cultured in the DMEM medium (a), chitosan extraction film (b) and different extraction films of poly(glycolic acid) grafted chitosan (c, CS:GA = 1:1; d, CS:GA = 1:5; e, CS:GA = 1:10) (CS:GA, the molar ratio of glucosamine unit to glycolic acid). The scale bar represents 50 μm.
L. Zhang et al. / Materials Science and Engineering C 33 (2013) 2626–2631
chitosan would be an effective material with controllable degradation rate to meet the diverse needs in biomedical fields. Acknowledgments
[8] [9] [10] [11] [12]
This study was supported by the Hi-Tech Research and Development Program of China (863 Program, No. 2012AA020502), Natural Science Foundation of China (No. 81171457, 21242005, 30970996 and 30970713), Natural Science Foundation of Nantong City (No. BK2012089, AS2011016), the Priority of Academic Program Development of Jiangsu Higher Education Institutions (PAPD) and Natural Science Foundation of Nantong University (No. 03080470, 10Z014).
[13] [14]
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Contents lists available at SciVerse ScienceDirect
Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Degradation and compatibility behaviors of poly(glycolic acid) grafted chitosan Luzhong Zhang a, Sufeng Dou a, Yan Li b, Ying Yuan a, Yawei Ji a, Yaling Wang c, Yumin Yang a,⁎ a b c
Jiangsu Key Laboratory of Neuroregeneration, Nantong University, Nantong 226001, P. R. China College of Life Science and Pharmaceutical Engineering, Nanjing University of Technology, Nanjing 210009, P. R. China College of Chemistry and Chemical Engineering, Nantong University, Nantong 226001, P. R. China
a r t i c l e
i n f o
Article history: Received 26 April 2012 Received in revised form 20 January 2013 Accepted 15 February 2013 Available online 21 February 2013 Keywords: Chitosan Poly(glycolic acid) Grafting Degradation Biocompatibility
a b s t r a c t The films of poly(glycolic acid) grafted chitosan were prepared without using a catalyst to improve the degradable property of chitosan. The films were characterized by Fourier transform-infrared spectroscopy and X-ray photoelectron spectroscopy (XPS). The degradation of the poly(glycolic acid) grafted chitosan films were investigated in the lysozyme solution. In vitro degradation tests revealed that the degradation rate of poly(glycolic acid) grafted chitosan films increased dramatically compared with chitosan. The degradation rate of poly(glycolic acid) grafted chitosan films gradually increased with the increasing of the molar ratio of glycolic acid to chitosan. Additionally, the poly(glycolic acid) grafted chitosan films have good biocompatibility, as demonstrated by in vitro cytotoxicity of the extraction fluids. The biocompatible and biodegradable poly(glycolic acid) grafted chitosan would be an effective material with controllable degradation rate to meet the diverse needs in biomedical fields. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Chitosan is a linear natural polysaccharide containing β-(1,4)2-amino-D-glucose and β-(1,4)-2-acetamindo-D-glucose unit, and obtained by N-deacetylation of chitin extracted from crustacean shells [1]. Because of its good biocompatible, antibacterial and low toxic properties, chitosan has been widely used for biomedical applications, such as sustained drug and protein delivery [2–4], non-viral gene delivery [5] and tissue engineering [6,7]. However, the use of chitosan in the biomedical fields has been limited because of the slow degradation rate [8], poor mechanical properties and the fact that it is hard to handle [9]. Since grafting copolymerization can modify the structure and properties of natural polysaccharides, it is an important resource for developing advanced materials [10,11]. Chitosan has reactive amine and hydroxyl side groups occurring in the units of polymer chains, and it acts as a desirable backbone to graft synthetic polymer. For instance, chitosan-g-N-isopropylacrylamide copolymer with thermoresponsive, fully reversible property was synthesized [12]. In the view of the designing of biologically degradable tissue engineering biomaterials, it would be advantageous to prepare a grafting copolymer composed of chitosan and poly(glycolic acid). Poly(glycolic acid), which is a fascinating synthetic polymer, has exhibited good mechanical properties, biodegradability and biocompatibility [13]. It has been widely used in biomedical applications, such as tissue engineered scaffold [14,15], controlled drug delivery
⁎ Corresponding author. Tel./fax: +86 513 85511585. E-mail address: [email protected] (Y. Yang). 0928-4931/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.msec.2013.02.024
systems [16] and implants for orthopedic device [17]. However, poly(glycolic acid) generally produces acidic degradation products at the implanted site. The degraded glycolic acid decreases the pH in the surrounding of the scaffold, and then evokes inflammatory tissue reactions [18]. Since basic glucosamine released from degrading chitosan is expected to neutralize acidic products, the tissue reactions may be alleviated by chitosan grafting copolymer. Importantly, poly(glycolic acid) grafted chitosan could not only retain the good properties of chitosan, but also may bring the easy degradation. In the present work, the films of poly(glycolic acid) grafted chitosan were prepared without using a catalyst to improve the degradable properties of chitosan. The films were characterized by Fourier transforminfrared spectroscopy (FT-IR) and X-ray photoelectron spectroscopy (XPS) analysis. The degradation rates of the poly(glycolic acid) grafted chitosan films were investigated in the lysozyme solution. It is found that the poly(glycolic acid) grafted chitosan films are more easily degradable compared with chitosan film, and the degradation rate of poly(glycolic acid) grafted chitosan films could be regulated. The in vitro cytotoxicity of the extraction fluids revealed that the films of poly(glycolic acid) grafted chitosan have no obvious cytotoxic effect. 2. Experimental section 2.1. Materials and methods Chitosan (the average molecular weight was 2.8 × 10 4 Da, the degree of deacetylation was about 95.3%) was purchased from Nantong Xincheng Biochemical Company. The glycolic acid was obtained from Acros Organics Company. The lysozyme was bought from Shanghai
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Aladdin Reagent Company. Other reagents were used as received without further purification. FT-IR spectra of chitosan and poly(glycolic acid) grafted chitosan films were recorded with a spectrometer (Nicolet 5700, Madison, WI). The X-ray photoelectron spectroscopy (XPS) of chitosan and poly(glycolic acid) grafted chitosan films were recorded on a Thermo Scientific K-Alpha spectrometer (Thermo Scientific, USA) equipped with a monochromatic Al-Kα X-ray source. The morphology of the chitosan film and the poly(glycolic acid) grafted chitosan films before and after degradation were observed using an S-3400 NII scanning electron microscopy (SEM, Hitachi, Japan). The samples were coated with gold using a JFC-1600 unit (JEOL Inc., Japan) Ion Sputter before examination under the SEM. 2.2. Preparation of poly(glycolic acid) grafted chitosan films Chitosan was dissolved in glycolic acid solutions with different concentrations and the mixtures were then standing overnight at room temperature. The solutions were poured into frame molds and dried at 45 °C to a constant weight. Then, the films were treated at 80 °C at a reduced pressure (10–12 mm Hg) for another 24 h to promote dehydration of the copolymer salts and the polymerization of glycolic acid. At last, the samples were extracted with methanol to remove unreacted glycolic acid and oligo(glycolic acid). 2.3. Preparation of chitosan film Chitosan was dissolved in acetic acid solutions with different concentrations and the mixtures were then standing overnight at room temperature. The solution was poured into a frame mold and dried at 45 °C to a constant weight. The samples were then extracted with methanol to remove acetic acid, and then the films were immersed in 2 M sodium hydroxide solution for 4 h. All prepared films were sterilized with 70% alcohol and washed with sterilized phosphate buffered saline (PBS, 0.1 M, pH 7.4) prior to use. 2.4. In vitro degradation of poly(glycolic acid) grafted chitosan films and chitosan film The enzymatic degradation of poly(glycolic acid) grafted chitosan films in vitro was investigated in the lysozyme solution. The poly(glycolic acid) grafted chitosan films (0.1 g) were immersed in 10 mL of lysozyme solution (2 mg/mL) in phosphate buffered saline (PBS) (pH 7.4) at 37 °C. For the sake of comparison, chitosan film was treated in the same manner. All the solutions were changed weekly, and the films were taken out at the predetermined time points. The degraded samples were washed three times with distilled water, dried under vacuum at 40 °C and weighted. The measurements were performed in triplicate and the results were the average of the three times. The degradation ratio was determined according to the following equation:
Degradation ratio ¼
2.6. In vitro cytotoxicity According to ISO-10993, the MTT [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide] assay was performed to determine the in vitro cytotoxicity of the extracts of films. L929 cells were seeded into a 96-well plate at a density of 5000 cells per well and incubated with 100 μL culture medium containing either in the DMEM medium or the film extract fluid at 37 °C for various time periods. After the incubation, the culture medium in each well was removed and the cells were washed three times with PBS. 20 μL of MTT solution (5 mg/mL) was added to each well and cells were cultured for another 4 h. The supernatant was discarded and then 100 μL of DMSO was added to each well. The OD values of the plate were measured on an EIX-800 Microelisa reader at 570 nm (Bio-Tek Inc., USA). 2.7. Bright-field microscopy measurement of cells The cells were seeded onto the films, which had been placed into a 6-well plate at a density of 2.5 × 10 5 cells per well, and incubated for 12 h. Thereafter, the cells were observed by optical microscopy. 3. Results and discussion The poly(glycolic acid) grafting chitosan were obtained after the reaction between chitosan and glycolic acid without using a catalyst (Scheme 1). When chitosan was dissolved in a glycolic acid aqueous solution, the amino groups of chitosan were protonated and the chitosan amino glycolic salt was formed [19]. The dehydration of the salt was done to form amide groups between chitosan and glycolic acid by heating the solution, and the polycondensation of glycolic acid was carried out simultaneously. The different poly(glycolic acid) grafted chitosan films were prepared via the variation of the feeding molar ratios of glucosamine unit to glycolic acid (the molar ratios of glucosamine unit to glycolic acid was changed from 1/1 to 1/10). Fig. 1 shows the IR spectra of chitosan and the poly(glycolic acid) grafted chitosan copolymer films extracted with methanol. For the chitosan film (Fig. 1a), the absorption peaks at 1595 and 1658 cm−1 are attributed to the N\H bending vibrations of non-acylated NH2 group and the carbonyl stretching of secondary amide (amide I band), respectively. Compared with chitosan film, the film of poly(glycolic acid) grafted chitosan (CS:GA = 1:1) (Fig. 1b) has new peaks appearing at 1639 and 1087 cm−1 corresponding to the carbonyl stretching of secondary amide (the NH2 groups of chitosan unit are acylated by glycolic acid) and C\O stretching of glycolic acid (CH2–OH), respectively. With the molar ratios of glucosamine unit to glycolic acid increased to 1/5 and 1/10 (Fig. 1c and d), the peaks located at 1749 and 1184 cm−1 can be assigned to the ester carbonyl stretching and C\O stretching of oligo(glycolic acid), indicating the formation of oligo(glycolic acid) attached to the chitosan. Additionally, no peak corresponding to ether groups from the reaction between the hydroxyl groups was found in the spectrum of the poly(glycolic acid) grafted
Initial dry weightdry weight after degradation : Initial dry weight
2.5. Cell culture The mouse fibroblast cells (L-929) were grown at 37 °C in a humidified atmosphere of 5% CO2 in Dulbecco's modified Eagle's medium (DMEM). The culture medium was supplemented with 10% fetal calf serum, 100 μg/mL streptomycin, 100 U/mL penicillin and 4 × 10 −3 M L-glutamine.
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Scheme 1. Grafting copolymerization of chitosan and glycolic acid.
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a
Transmission
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d 1640 1749
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Fig. 1. FT-IR spectra of chitosan (a) and the poly(glycolic acid) grafted chitosan (b, CS: GA = 1:1; c, CS:GA = 1:5; d, CS:GA = 1:10; the feeding molar ratio of glucosamine unit (CS) to glycolic acid(GA)).
chitosan, suggesting the successful synthesis of poly(glycolic acid) grafted chitosan. In order to confirm the chemical composition, XPS was used to obtain the information about different elements at the surface. Fig. 2 shows that the XPS N 1s region corresponding to the atomic orbital 1s of nitrogen occurred in the spectra of chitosan and poly(glycolic acid) grafted chitosan films. For the chitosan films after the NaOH treatment,
5600 5400
a
Pos. 399
the N 1s narrow scan (Fig. 2a) displayed only one peak at 399 eV approximately, corresponding to the amine and amide chemical binding [20]. In contrast to the chitosan film, the N 1 s narrow scan of the poly(glycolic acid) grafted chitosan (CS:GA= 1:1, Fig. 2b) displayed two peaks located at 399 eV and 401 eV approximately, corresponding to the amide and protonated amine [20]. The atomic ratios of these peaks were about 54% and 46%. With the molar ratios of glucosamine unit to glycolic acid increased to 1/5 and 1/10, the atomic ratios of the protonated amine decreased to 35% and 29% [Fig. 2c and d], respectively, indicating that the formation of amide groups between the chitosan and glycolic acid. The XPS C 1s spectrum of the chitosan film and chitosan graft copolymer films were also shown in Fig. 3. The C 1s narrow scan (Fig. 3a) at 284 eV approximately, was mainly assigned to the carbon surface contaminant \CH2- for the chitosan films [21]. The C 1s peak at 286 eV approximately was assigned to the C\N\C_O in the spectrum of the poly(glycolic acid) grafted chitosan (CS:GA = 1:1, Fig. 3b), suggesting that the glycolic acid has grafted the chitosan [22]. When the molar ratio of chitosan and glycolic acid in the poly(glycolic acid) grafted chitosan came to 1/5, a new peak located at 288 eV approximately emerged (Fig. 3c), demonstrating that more glycolic acid was grafted onto chitosan [20]. With the molar ratio decreasing to 1/10, the atomic ratio of the peak at 284 eV increased and can be reasoned that more glycolic acid were grafted onto the chitosan (Fig. 3d). The controllable biodegradability and biocompatibility are the important requirements for the development of any medical implant materials [23]. For example, the biomaterial degradation rate is regarded as an important design consideration for artificial nerve scaffolds, since non-degradable tubes could compress regenerated nerves in the long term. Proper degradation rate is an important factor for a tissue-engineered scaffold to resist rearing and stretching
10000
%Area 100
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%Area 54.37 45.63
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Fig. 2. XPS N 1s narrow scans with the curve fit of chitosan (a) and poly(glycolic acid) grafted chitosan (b, CS:GA = 1:1; c, CS:GA = 1:5; d, CS:GA = 1:10; the feeding molar ratio of glucosamine unit (CS) to glycolic acid(GA)).
L. Zhang et al. / Materials Science and Engineering C 33 (2013) 2626–2631
80000 70000
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%Area 100
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b
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%Area 68.98 31.02
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Fig. 3. XPS C 1s narrow scans with the curve fit of chitosan (a) and poly(glycolic acid) grafted chitosan (b, CS:GA = 1:1; c, CS:GA = 1:5; d, CS:GA = 1:10; the feeding molar ratio of glucosamine unit (CS) to glycolic acid(GA)).
forces during nerve regeneration process [24]. To evaluate the performance of the poly(glycolic acid) grafted chitosan in biomedical application, the degradation behaviors of the chitosan or poly(glycolic acid) grafted chitosan films in vitro were investigated in the presence of lysozyme at 37 °C. Since hen egg white (HEW) lysozyme as well as human lysozyme could cleavage the β(1-4)-linked unit of chitosan
[25], the in vitro degradation behavior of chitosan-g-poly(glycolic acid) has been investigated by using HEW lysozyme in this study. Fig. 4 shows the degradation behavior of the poly(glycolic acid) grafted chitosan samples, including chitosan film, poly(glycolic acid) grafted chitosan films (the molar ratios of glycolic acid to glucosamine unit was changed from 1/1, 3/1, 5/1, 10/1). It can be observed that only 0.70% weight was lost in the group of chitosan film within
100
chitosan CS:GA=1:1 CS:GA=1:3 CS:GA=1:5 CS:GA=1:10
Weight loss (%)
80
60
40
20
0 0
7
14
21
28
Degradation Time (days) Fig. 4. Dry weight loss after in vitro degradation of poly(glycolic acid) grafted chitosan films and chitosan film in 4 mg/mL lysozyme solution in PBS (pH 7.4) incubated at 37 °C for various time periods (the feeding molar ratio of glucosamine unit to glycolic acid, CS:GA).
Fig. 5. SEM images of the chitosan film and the poly(glycolic acid) grafted chitosan film before (a, chitosan; c, poly(glycolic acid) grafted chitosan and after degradation (b, chitosan; d, poly(glycolic acid) grafted chitosan) for 4 weeks. The chitosan-g-poly(glycolic acid) molar ratio of glucosamine unit to glycolic acid is 1:10.
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1.5
OD value
1.2
CS:GA=1:1 CS:GA=1:5 CS:GA=1:10 chitosan DMEM
0.9
0.6
0.3
0.0 1
3
5
7
Time (Days) Fig. 6. The changes in cell viability of L929 cells after they were cultured in the DMEM medium or different poly(glycolic acid) grafted chitosan extraction fluids for 1 d, 3 d, 5 d, 7 d, respectively (CS:GA, the molar ratio of glucosamine unit to glycolic acid).
28 days [1,26]. Thus, the chitosan film has a very slow degradation rate. Compared to the chitosan film, the weight loss percentage of poly(glycolic acid) grafted chitosan significantly increased. The mean percentage of weight loss at the termination of the experiment for the groups of poly(glycolic acid) grafted chitosan (1/1) and poly(glycolic acid) grafted chitosan (10/1) are 4.67% and 85.60%. The percentage of weight loss gradually increased with the increasing of the molar ratio of glycolic acid to chitosan. This phenomenon can be attributed to the fact that the poly(glycolic acid) is easy to be degraded [27] and the amide of chitosan will make the degradation faster [26]. Additionally, the pH values did not change significantly during the degradation in the PBS. The various degradation rates would help to design poly(glycolic acid) grafted chitosan based biomedical materials with the predetermined degradation time. Therefore, the degradation rate of chitosan based materials could be regulated by varying the molar ratio of glycolic acid to chitosan. The SEM analysis further showed the morphological change of poly(glycolic acid) grafted chitosan after degradation over 4 weeks (Fig. 5). It is found that the surface of chitosan and poly(glycolic
acid) grafted chitosan films were smooth before degradation. The sample of chitosan film degraded in PBS containing lysozyme shows slightly rough surface, while the sample of poly(glycolic acid) grafted chitosan film shows obviously rough surface under the same condition. These results demonstrated that the poly(glycolic acid) grafted chitosan films have the faster degradation rate than chitosan film, which is consistent with the results of the mean weight loss of poly(glycolic acid) grafted chitosan after 4 weeks. To examine the compatibility of the poly(glycolic acid) grafted chitosan, the in vitro cytotoxicity tests of the film extracts against the mouse fibroblast cells (L929) were conducted according to ISO-10993. Since L929 cells are easy to prepare and culture, it is routinely used for cytotoxicity studies [28]. The L929 cells were cultivated in the DMEM culture medium and the poly(glycolic acid) grafted chitosan film extract fluid, for 1, 3, 5, and 7 days (Fig. 6), respectively. It is found that the viability of L929 cells cultured in the film extract fluid was not significantly different from that in DMEM supplemented with 10% fetal calf serum (blank control) after 1, 3, 5, and 7 days, indicating that the films of poly(glycolic acid) grafted chitosan have no obvious cytotoxic effect at all used conditions. Furthermore, the cells were observed by optical microscopy after being cultured for 12 h on the chitosan and different poly(glycolic acid) grafted chitosan films (Fig. 7). Bright-field images show that all cells proliferate very well and maintain their normal configuration on all the used films (The normal L929 cells should be mainly adhered to the surface of the culture plate, plump, spindle-shaped and glossy, usually accompanied by some round dividing cells.). Therefore, the poly(glycolic acid) grafted chitosan films showed no obvious cytotoxicity and presented a good substrate for the growth of L929 cells. 4. Conclusion The poly(glycolic acid) grafted chitosan films were prepared without using a catalyst via dehydration reaction, and characterized by FT-IR and XPS analysis. The poly(glycolic acid) grafted chitosan films degraded much faster than chitosan film, and the degradation rate of poly(glycolic acid) grafted chitosan films gradually increased with the increasing of the molar ratio of glycolic acid to chitosan. In vitro cytotoxicity tests exhibited that the poly(glycolic acid) grafted chitosan films have good biocompatibility. These results suggested that poly(glycolic acid) grafted chitosan could be a good biocompatible and biodegradable materials. The poly(glycolic acid) grafted
Fig. 7. Bright-field microscopic images of L929 cells after 12 h cultured in the DMEM medium (a), chitosan extraction film (b) and different extraction films of poly(glycolic acid) grafted chitosan (c, CS:GA = 1:1; d, CS:GA = 1:5; e, CS:GA = 1:10) (CS:GA, the molar ratio of glucosamine unit to glycolic acid). The scale bar represents 50 μm.
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chitosan would be an effective material with controllable degradation rate to meet the diverse needs in biomedical fields. Acknowledgments
[8] [9] [10] [11] [12]
This study was supported by the Hi-Tech Research and Development Program of China (863 Program, No. 2012AA020502), Natural Science Foundation of China (No. 81171457, 21242005, 30970996 and 30970713), Natural Science Foundation of Nantong City (No. BK2012089, AS2011016), the Priority of Academic Program Development of Jiangsu Higher Education Institutions (PAPD) and Natural Science Foundation of Nantong University (No. 03080470, 10Z014).
[13] [14]
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