Antibacterial activity and biocompatibility of a chitosan–γ-poly(glutamic acid) polyelectrolyte complex hydrogel

Carbohydrate Research 345 (2010) 1774–1780

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Antibacterial activity and biocompatibility of a chitosan–c-poly(glutamic acid) polyelectrolyte complex hydrogel Ching Ting Tsao a, , Chih Hao Chang b,c, , Yu Yung Lin a, Ming Fung Wu d, Jaw-Lin Wang a, Jin Lin Han e Kuo Huang Hsieh a,* a

Institute of Polymer Science and Engineering, College of Engineering, National Taiwan University, No. 1, Sec. 4, Roosevelt Road, Taipei City 10617, Taiwan Institute of Biomedical Engineering, College of Engineering, National Taiwan University, No. 1, Sec. 4, Roosevelt Road, Taipei City 10617, Taiwan c Department of Orthopedics, National Taiwan University Hospital and National Taiwan University College of Medicine, No. 1, Jen-Ai Road, Taipei City 10018, Taiwan d Animal Medicine Center, College of Medicine, National Taiwan University, No. 1, Jen-Ai Road, Taipei City 10018, Taiwan e Department of Chemical and Materials Engineering, College of Engineering, National Ilan University, No.1, Sec. 1, Shennong Road, Ilan City, Ilan County 26047, Taiwan b

a r t i c l e

i n f o

Article history: Received 21 April 2010 Received in revised form 31 May 2010 Accepted 6 June 2010 Available online 16 June 2010 Keywords: Chitosan c-PGA Polyelectrolyte complex (PEC) Antibacterial ability Biocompatibility

a b s t r a c t In this study, we prepared a polyelectrolyte complex (PEC) hydrogel comprising chitosan as the cationic polyelectrolyte and c-poly(glutamic acid) (c-PGA) as the anionic polyelectrolyte. Fourier transform infrared spectroscopy revealed that ionic complex interactions existed in the chitosan–c-PGA PEC hydrogels. The compressive modulus increased upon increasing the degree of complex formation in the chitosan–cPGA PEC hydrogel; the water uptake decreased upon increasing the degree of complex formation. At the same degree of complex formation, the compressive modulus was larger for the chitosan-dominated PEC hydrogels; the water uptake was larger for the c-PGA-dominated ones. Scanning electron microscopy images revealed the existence of interconnected porous structures (pore size: 30–100 lm) in all of the chitosan–c-PGA PEC hydrogels. The chitosan–c-PGA PEC hydrogels also exhibited antibacterial activity against Escherichia coli and Staphylococcus aureus. In addition, in vitro cell culturing of 3T3 fibroblasts revealed that all the chitosan–c-PGA PEC hydrogels were effective in promoting cell proliferation, especially the positively charged ones (chitosan-dominated). Therefore, the chitosan–c-PGA polyelectrolyte hydrogel appears to have potential as a new material for biomedical applications. Ó 2010 Elsevier Ltd. All rights reserved.

1. Introduction A hydrogel is a material that exhibits the ability to swell in water and to retain a significant fraction of water within its structure.1 Because their hydrophilic surfaces have low interfacial free energy when contacting with body fluid, giving them good biocompatibility, hydrogels have attracted much interest recently for use as drug carriers and artificial tissue scaffolds.2 Although they possess hydrophilic polymeric backbones, hydrogels are kept from dissolution by the presence of radical, chemical, or physical crosslinks.3 Although radical crosslinks provide a high crosslink quality, there is always the possibility of radical residues remaining in the hydrogels.4 Chemical crosslinks involve covalent bond formation between different polymer chains; because it requires a toxic crosslinker, it is also unfavorable for biological applications.5 The use of non-covalent interactions (physical crosslinks) removes the need for radicals or toxic chemical crosslinkers.

* Corresponding author. Tel.: +886 2 3366 3043; fax: +886 2 3366 3044. E-mail address: [email protected] (K.H. Hsieh).   These authors contributed equally to this work. 0008-6215/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.carres.2010.06.002

Several non-covalent modalities have been exploited in the design of hydrogels, including coiled–coil interactions,6,7 hydrophobic interactions,8 antigen–antibody interactions,9 stereocomplex interactions,10 and ionic interactions.11 For ionic crosslinked hydrogels, swelling occurs as a result of ionic interactions between free ions and the charged polymer, which may feature carboxylic acid, sulfonic acid, or amino groups, thereby rendering the polymer hydrophilic and leading to its high water uptake. In this study, we exploited ionic interactions to form homogeneous polyelectrolyte complex (PEC) hydrogels from pairs of oppositely charged agents. Chitosan, derived from chitin through alkaline deacetylation, is a polysaccharide constituted by N-glucosamine and N-acetylglucosamine units, in which the number of N-glucosamine units exceeds 50%.12 Its slightly crystalline character makes chitosan insoluble when the pH is around 7 or greater. Because the free amine groups of chitosan become protonated in acidic environments, the positively charged polymer is soluble at low pH.13,14 The net positive charge of chitosan in acidic environments allows the formation of PEC with polyanionic species.15 In addition, some antibacterial activities have been described for chitosan and chitosan derivatives. The main factor affecting the antibacterial ability of chitosan is its molecular weight.16,17 Chitosan oligomers are reported to be more

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effective in inhibiting the growth of bacteria than polymeric chitosan. The antibacterial ability of chitosan is greatly dependent on its molecular weight, especially in the range between 5 and 10 kDa.18,19 c-Poly(glutamic acid) (c-PGA) is an unusual anionic, natural polyamide made of D- and L-glutamic acid units, connected through amide bonds between the a-amine and c-carboxylic acid groups.20 Because c-PGA is water-soluble, biodegradable, and edible, it has been applied as animal feed supplement, biopolymer flocculant, humectant, or moisturizer in cosmetics,21 and a natural bactericide or fungicide.22 Through culturing of bacteria via fermentation, c-PGA is already produced on an industrial scale in high yield.23,24 The anionic nature of c-PGA allows it to form PEC with chitosan at appropriate values of pH. In this study, we used a simple ionic interaction to prepare homogeneous PEC hydrogels of several compositions by varying molar ratios of amine groups of chitosan to carboxylic acid groups of c-PGA. We examined the physicochemical characteristics of these chitosan–c-PGA PEC hydrogels using X-ray diffraction (XRD), Fourier transform infrared (FTIR) spectroscopy, and scanning electron microscopy (SEM). In terms of physical behavior, we measured the compressive modulus and the water uptake of each chitosan–c-PGA PEC hydrogel. In terms of in vitro biological behavior, we investigated the antibacterial ability of chitosan– c-PGA PEC hydrogels against Escherichia coli (E. coli, Gram-negative bacteria) and Staphylococcus aureus (S. aureus, Gram-positive bacteria). The biocompatibility of the chitosan–c-PGA PEC hydrogels with 3T3 fibroblasts was also evaluated. 2. Experimental 2.1. Reagents Chitosan (average molecular weights: 3  104 and 3  105 Da; degree of deacetylation: 97%) was purchased from G-HT Co. (Hsinchu, Taiwan). c-PGA (average molecular weight: 1250 kDa) was purchased from VEDAN Co. (Taichung, Taiwan). Fetal bovine serum (FBS), Dulbecco’s modified Eagle’s medium (DMEM F-12 medium) and 3-[4,5-dimethylthiazolyl-2]-2,5-diphenyl tetrazolium bromide (MTT) were obtained from Gibco Invitrogen (Taipei, Taiwan). Ace-

Table 1 Compositions of chitosan–c-PGA PEC hydrogels Nomenclature

Neat chitosan C75P25 C50P50 C25P75 Neat c-PGA a

Composition Weight ratio chitosan/ c-PGA

Molar ratio [–NH2] of chitosana/ [–COOH] of c-PGA

4/0 3.06/0.94 2.09/1.91 1.07/2.93 0/4

100/0 75/25 50/50 25/75 0/100

Degree of complex formation (%)

Charge of PEC hydrogel

0 25 50 25 0

Positive Positive Neutral Negative Water-soluble

All the chitosan–c-PGA PEC hydrogels were fabricated with chitosan molecular weight of 3  105 Da, except for that a set of PEC hydrogels were fabricated with chitosan molecular weight of 3  104 Da for antibacterial activity test.

tic acid (AcOH), sodium hydroxide (NaOH), phosphate-buffered saline (PBS), and other reagents used in the study were purchased from Sigma–Aldrich Chemical Co. (St. Louis, MO, USA.) 2.2. Preparation of PEC hydrogels The chitosan–c-PGA PEC hydrogels containing varied molar ratios of amine groups of chitosan to carboxylic acid groups of c-PGA ([–NH2]:[–COOH] = 75:25, 50:50, 25:75) were prepared for this study. First, chitosan powder was well dispersed in a previously prepared c-PGA aqueous solution. The weight percentage of chitosan and c-PGA in the prepared solution was 4, and then 1% acetic acid was added. The chitosan powder dissolved immediately due to its protonated amine groups. The homogeneous PEC hydrogel is subsequently formed through a complex formation between –NH2 of chitosan and –COOH of c-PGA. These PECs were then immersed in a 1 N NaOH aqueous solution and washed with deionized water to a pH value around 7. The neutral PEC hydrogel was further freeze-dried so a porous structure was formed; the freeze-dried PEC hydrogel was found to shrink to 75–80% of its original size. The PEC hydrogels were categorized into five groups according to the molar ratios of amine groups (–NH2) of chitosan (named C) to carboxylic acid groups (–COOH) of c-PGA (named P), and thus, the degree of complex formation was defined as complex formed in an ionic solution.

Degree of complex formation ¼ ½—NH3 þ OOC—=ð½—NH2 C þ ½—COOHP Þ  100%

ð1Þ

The description of the chitosan–c-PGA PEC hydrogels is listed in Table 1; the schematic representation of the ionic interaction formation between chitosan and c-PGA is illustrated in Figure 1. As an example of the nomenclature used herein, C50P50 indicates that the molar ratio of amine groups (–NH2) of chitosan to carboxylic acid groups (–COOH) of c-PGA in this specimen was 50:50; the degree of complex formation = 50, which indicates that a complex formation between –NH2 of chitosan and –COOH of c-PGA was theoretically completed without free –NH2 or –COOH groups in the PEC hydrogels. The neat chitosan was prepared by the immersion–precipitation method.25 In brief, the neat chitosan was formed with solidification by immersing the chitosan solution (4 wt % in 1% acetic acid) into 1 N NaOH. The solidified neat chitosan was then neutralized with deionized water, and freeze-dried to remove any excess water. All the specimens tested were fabricated with chitosan molecular weight of 3  105 Da, except that a set of specimen were fabricated with chitosan molecular weight of 3  104 Da for antibacterial activity test. The neat c-PGA cannot be formed as hydrogel, as it is water soluble. 2.3. Characterization of PEC hydrogels 2.3.1. FTIR Spectroscopy FTIR spectroscopy (Fourier transform infrared spectroscopy, Perkin–Elmer Spectrum RX1 System) was used to examine the signal variations of the amine and carboxylic acid groups of the

Figure 1. Schematic representation of the ionic interaction formation between chitosan and c-PGA.

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prepared chitosan–c-PGA PEC hydrogels. The powdered PEC hydrogels were mixed with dry KBr (at a ratio of 1:10) and pressed into a transparent disc prior to FTIR spectroscopic analysis. Because of its simplicity, the most discussed technique is FTIR spectroscopy when quantifying the absorption of a specific peak, but it needs a calibration for the absolute value. The normalized absorption area of a specific peak (Aspecific/Astandard) reflects its quantity of existence. In our research, a functional group that does not change in the fabrication process is chosen as the standard absorption. The absorption area was calculated with the software of PeakFiT™ v4.12 (Systat Software, Inc., San Jose, CA, USA). 2.3.2. XRD The crystalline forms of neat chitosan, the chitosan–c-PGA PEC hydrogel (C50P50), and neat c-PGA were determined using an X-ray diffractometer (MXP18, MAC Science, Japan), operated at 40 kV and 35 mA. The diffraction patterns were determined over a diffraction angle (2h) range of 5–40°. 2.3.3. Compressive modulus A universal testing machine (5900 Systems, InstronÒ, Kingsport, TN, USA) was used to determine the compressive modulus of the chitosan–c-PGA PEC hydrogels by pressing the sample discs (diameter: 13 mm; thickness: 3 mm) at a constant rate of 5 mm/min.26 Six samples were measured for each type of the chitosan–c-PGA PEC hydrogel. The slopes of the compressive stress–strain curves from 5 to 35% deformation were used to calculate the compressive modulus. The results are presented as mean value with standard deviation (n = 6). 2.3.4. Morphology The morphologies of the chitosan–c-PGA PEC hydrogels were examined using a scanning electron microscope (JOEL JSM-6700f FE-SEM), operated at an accelerating voltage of 10 kV. Cross-sections of the PEC hydrogels were coated with an ultrathin layer of gold-palladium through ion sputtering prior to SEM examination. 2.3.5. Water uptake The water uptake was determined by weighing the initial and swollen chitosan–c-PGA PEC hydrogels at steady state. To calculate the water uptake, pre-weighed dry PEC hydrogels were immersed in 0.05 M PBS solution at room temperature. After reaching steady state, the excess surface water was removed using filter paper and the swollen samples were weighed. Six samples were measured for each type of the chitosan–c-PGA PEC hydrogel. The water uptake was calculated using the equation:

Water uptake ¼ ðW swollen  W dry Þ=W dry  100% where Wdry and Wswollen represent the weights of the PEC hydrogels before and after the immersion, respectively. The results are presented as mean values with standard deviation (n = 6).

containing 106 CFU/mL E. coli (or S. aureus) and then the medium was incubated in a humidified atmosphere (5% CO2, 100% humidity, 37 °C). Three samples were measured for each type of chitosan–c-PGA PEC hydrogel; the suspensions untreated with chitosan–c-PGA PEC hydrogel were set as control. During the incubation process, the O.D. value of the medium was measured at 650 nm after 24 h. The bacterial proliferation was illustrated in terms of the O.D. value; each operation was performed under aseptic conditions using aseptic techniques.27 The results are presented as mean values with standard deviation (n = 3). 2.5. Biocompatibility The biocompatibility of the chitosan–c-PGA PEC hydrogels was evaluated by assessing their abilities to support the proliferation of 3T3 fibroblasts (Bioresource Collection and Research Center, Hsinchu, Taiwan).28 Pieces (1 cm  1 cm) of a chitosan–c-PGA PEC hydrogel, sterilized in an autoclave in distilled water (2 mL) for 15 min at 121 °C, were placed in a 24-well polystyrene plate and treated with DMEM containing 10% (v/v) FBS and 1% (v/v) penicillin and 1% (v/v) streptomycin. The 3T3 fibroblasts (1  106 cells/mL) were seeded onto the PEC hydrogel, which was then maintained in a humidified atmosphere (5% CO2, 100% humidity, 37 °C). A tissue culture polystyrene dish (TCPS) was used as a control. After a culture period of 2 or 3 days, a solution of MTT (5 mg/mL in 0.05 M PBS solution, 50 lL) was added to each well and then the system was incubated at 37 °C for 4 h to form the MTT formazan complex. The medium was then replaced gently with dimethylsulfoxide (DMSO, 500 lL) to solubilize the formazan complex. The O.D. value of the solution in each well was then measured at a wavelength of 570 nm using a microplate reader. The results are presented as mean values with standard deviation (n = 3). The morphologies of the cells cultured on the PEC hydrogels were examined using SEM. The chitosan–c-PGA PEC hydrogels were fixed in formaldehyde (4%) for 4 h at room temperature and then dehydrated stepwise with 50%, 75%, 95%, and 100% ethanol, each for 2 h. The PEC hydrogels were coated with an ultrathin layer of gold-palladium through ion sputtering prior to SEM examination.

3. Statistical analysis All calculations have been done with SigmaStat statistical software (Jandel Science Corp., San Rafael, CA, USA). Statistical significance in the Student’s t-test corresponded to a confidence interval (CI) of 95%. The results were presented as the mean value with standard deviation (mean ± SD). Differences were considered statistically significant at p <0.05. 4. Results and discussion

2.4. Antibacterial ability 4.1. Characterization of the chitosan–c-PGA PEC hydrogels Antibacterial ability of the chitosan–c-PGA PEC hydrogels was evaluated using the optical density (O.D.) method, which was measured by shake flask testing (according to ASTM E2149-01 Standard Test Method for Determining the Antimicrobial Activity of Immobilized Antimicrobial Agents under Dynamic Contact Conditions). The bacteria tested were E. coli (E. coli, ATCC 25992, Bioresource Collection and Research Center, Hsinchu, Taiwan) and S. aureus (S. aureus, ATCC 6538P, Bioresource Collection and Research Center, Hsinchu, Taiwan). Briefly, a dry chitosan–c-PGA PEC hydrogel (0.2 g) was added into Nutrient Broth (Difico, Michigan, USA) medium (10 mL)

Figure 2 displays the X-ray diffractograms of neat chitosan, the chitosan–c-PGA PEC hydrogels (C50P50), and neat c-PGA. Two strong crystalline peaks appeared in the X-ray diffractogram of neat chitosan; they are associated with the most ordered region involving acetamide and amine groups at 10.4° and 21.8°, respectively.29 No significant crystalline peaks appeared in the X-ray diffractograms of neat c-PGA or of the chitosan–c-PGA PEC hydrogel. Hence, we deduced that the crystalline structure within chitosan was disrupted after the formation of the ionic complex interactions with c-PGA.

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a

A1400/A1080 A1400/A1650a

C25P75

C50P50

C75P25

1.34 0.419*

1.422 0.865

0.674* 0.814

a *

Figure 2. X-ray diffractograms of neat chitosan, the chitosan–c-PGA PEC hydrogel (C50P50), and neat c-PGA.

The formation of the ionic complex interactions between chitosan and c-PGA was further confirmed through FTIR spectroscopic analysis (Fig. 3). The characteristic peaks of chitosan appeared at 3450, 3300, 1560, 1080 cm1, corresponding to its hydroxyl (OH), amine (NH2), amide II (N–H bending vibrations coupled to C–N stretching vibrations) groups, and glycosidic linkage (ether bond), respectively.30,31 The characteristic peaks of c-PGA appeared at 3400 and 1700 cm1, corresponding to its N–H bending and carboxylic acid group (–COOH) vibrations, respectively.29 The FTIR spectra of the chitosan–c-PGA PEC hydrogels at various molar ratios featured similar characteristic peaks as those of their parent polymers; the carbonyl group (C@O) on c-PGA shifted to 1650 cm1. Furthermore, a peak, at 1400 cm1, arose in each of the chitosan–c-PGA PEC hydrogels but was not observed for neat chitosan or neat cPGA. We attribute this peak to the —NH3 þ groups of chitosan complex with the –COO groups of c-PGA.32 To quantify the degree of complex formation in chitosan–c-PGA PEC hydrogels, the absorption ratio at 1400 cm1 compared with that of 1080 and 1560 cm1 were calculated, respectively, and the results are shown in Table 2. The largest A1400/A1080 appeared in C50P50, reflecting the complete complex formation in this specimen. The A1400/A1080 of C25P75 is close to that of C50P50; there is no statistic difference between the two specimens, also reflecting

Figure 3. FTIR spectra of neat c-PGA, various chitosan–c-PGA PEC hydrogels (C25P75, C50P50, C75P25), and neat chitosan.

Absorption ratios calculated by infrared spectroscopy. Indicates a significant difference from the C50P50 in the same category (p <0.05).

the complete complex formation in C25P75. Furthermore, the A1400/A1080 of C75P25 is lower than those in C50P50 and C25P75, reflecting the quantity of glycosidic linkage on chitosan, in C75P25, outnumbered that of carbonyl groups on c-PGA. Besides, with the absorption of the carbonyl groups (C@O) on c-PGA set as internal standard bands, the degree of complex formation was also observed with A1400/A1560. The greatest A1400/A1560 appeared in C50P50, reflecting the complete complex formation in this specimen. The A1400/A1560 of C75P25 is close to that of C50P50; there is no statistic difference between the two specimens, also reflecting the complete complex formation in C75P25. The A1400/A1560 of C25P75 is lower than those in C50P50 and C75P25, reflecting the quantity of carbonyl groups on c-PGA, in C25P75, outnumbered that of glycosidic linkage on chitosan. As a result, from the theoretical calculation (Table 1 and Eq. 1)) and the structural investigation (Fig. 3 and Table 2), we deduced C50P50 had the largest degree of complex formation of 50%; C25P75 and C75P25 had the same degree of complex formation of 25%. These phenomena confirmed that ionic complex interactions, existing between chitosan and c-PGA, resulted in the formation of chitosan–c-PGA PEC hydrogels. We determined the mechanical properties of the chitosan– c-PGA PEC hydrogels and the neat chitosan from the compressive modulus (Fig. 4). The compressive modulus of C50P50 (largest degree of complex formation) was the largest among all the chitosan–c-PGA PEC hydrogels, whereas that of neat chitosan (no complex formation) was the smallest. These findings suggested that the ionic complex interactions between the two polyelectrolyte polymers were critical in affecting the mechanical properties of their PEC hydrogels. Notably, at the same degree of complex formation, the compressive modulus of C75P25 was larger than that of C25P75, presumably because of the ready packing ring structure in chitosan. Hydrophilicity is an important characteristic of PEC hydrogels. Excellent water uptake ability of a material would facilitate both cell attachment and penetration during in vitro cell culturing. To determine their hydrophilicities, we measured the water uptake

Figure 4. Compressive moduli of various chitosan–c-PGA PEC hydrogels (C25P75, C50P50, C75P25) and neat chitosan. Data are presented as mean ± SD (n = 6).

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of the chitosan–c-PGA PEC hydrogels. Table 3 displays the water uptake of the chitosan–c-PGA PEC hydrogels in 0.05 M PBS solution (pH 7.4) at room temperature. All the PEC hydrogels swelled rapidly, reaching equilibrium within 1 h. The water uptake percentages, with the value decreasing upon increasing the degree of complex formation, all fell within the range from 200% to 1000%. C50P50, with the highest degree of complex formation (no free water-bonding sites in c-PGA available), had the lowest water uptake (232 ± 44%). Although having the same degree of complex formation, the water uptake of C25P75 (959 ± 97%, c-PGA-dominated) was larger than that of C75P25 (675 ± 55%, chitosan-dominated), consistent with the hydrophilic character of c-PGA (greater number of water-bonding sites). In tissue engineering, biomaterials must have porous structures to ensure cell attachment, cell proliferation, tissue growth, and the passage of nutrients. The three dimensional porous structure of chitosan–c-PGA PEC hydrogels was achieved through the processes of complex formation, which squeezed out water and subsequently induced solidification; after the freeze-drying process, the pores were the result of water-vacancy. As revealed in SEM images (Fig. 5), the cross-sections of these PEC hydrogels were all highly porous, with pore sizes of around 30–100 lm, which are structurally favorable for cell attachment.33,34 Among all of our chitosan–cPGA PEC hydrogels (Fig. 5a–c), C50P50 had the smallest pore size because of its greatest degree of complex formation. Although they had the same degree of complex formation, C25P75 had larger pore sizes than C75P25 did. The greater water uptake in C25P75 resulted in larger pore sizes after the water had been sublimed. Notably, the pore sizes of neat chitosan (Fig. 5d) were smaller than Table 3 Water uptake of chitosan–c-PGA PEC hydrogels Sample

Water uptake (%)

Neat chitosan C75P25 C50P50 C25P75

350 ± 86 675 ± 55 232 ± 44 959 ± 97

those of the chitosan–c-PGA PEC hydrogels (Fig. 5a–c), presumably because the structure lacking ionic complex interactions was readily destroyed during the sublimation process. 4.2. Antibacterial ability Antibacterial ability is an important characteristic of a material intended for biomedical applications. O.D. values versus various chitosan–c-PGA PEC hydrogels with different chitosan molecular weight (MW) against E. coli and S. aureus are shown in Table 4. With Gram-negative bacteria, as the portion of c-PGA increased, the O.D. values decreased in the experiment groups compared with those of the control. However, there was no significant difference in antibacterial activity between the PEC hydrogels with low chitosan MW of 3  104 Da and PEC hydrogels of 3  105 Da. A similar trend was observed with Gram-positive bacteria: as the portion of c-PGA increased, the O.D. values decreased in the experiment groups compared with those of the control. In contrast to the response of E. coli, the growth of S. aureus was obviously suppressed by the chitosan–c-PGA PEC hydrogels with low chitosan MW of 3  104 Da as compared with PEC hydrogels of 3  105 Da. The result meant the antibacterial activity of the chitosan–c-PGA PEC hydrogels increased with the increase of c-PGA content, against both E. coli and S. aureus. The effect of chitosan MW was more profound when against S. aureus, which increased with decreased chitosan MW. Although chitosan is known to exhibit antibacterial ability, there are some limitations regarding its MW. Liu et al. reported that the antibacterial activity of low MW chitosan is stronger than that of the high MW chitosan against E. coli.36 No et al. reported that among one series of chitosan with MW ranging from 28 to 1671 kDa, chitosan of 470 kDa appeared most effective against S. aureus.37 Besides, c-PGA has also been reported to possess antibacterial ability. Shown in Table 4, all the chitosan–c-PGA PEC hydrogels exhibited significant antibacterial activity relative to the control. It is known that hydrophobic materials are more susceptible to microorganism adhesion.35 Hydrophobic interactions between a microorganism and a substrate would enhance microbial

Figure 5. SEM images of cross-sections of (a) C25P75, (b) C50P50, (c) C75P25, and (d) neat chitosan.

C. T. Tsao et al. / Carbohydrate Research 345 (2010) 1774–1780 Table 4 Antibacterial activity of chitosan–c-PGA PEC hydrogels against E. coli and S. aureus Sample

Chitosan molecular weight (Da)

O.D. value at 650 nm Gram-negative E. coli

Gram-positive S. aureus

Control Neat chitosan C75P25 C50P50 C25P75

— 3  l05 3  l05 3  105 3  l05

2.650 ± 0.2616 1.563 ± 0.3287* 1.640 ± 0.0566* 1.568 ± 0.0601* 1.485 ± 0.0495*

0.180 ± 0.0001 0.171 ± 0.0051 0.186 ± 0.0171 0.200 ± 0.0164 0.150 ± 0.0006

Control Neat chitosan C75P25 C50P50 C25P75

— 3  l04 3  l04 3  l04 3  l04

2.849 ± 0.0397 1.734 ± 0.0672* 1.914 ± 0.0168* 1.399 ± 0.0127* 1.023 ± 0.0354*

0.162 ± 0.0327 0.058 ± 0.0022* 0.109 ± 0.0015* 0.083 ± 0.0342* 0.055 ± 0.0103*

Data are presented as mean ± SD (n = 3). * Indicates a significant difference from the control at the same category (p <0.05).

adhesion, subsequently leading to microorganism proliferation. For large chitosan MW of 3  105 Da, C25P75 was the more hydrophilic (water uptake: 959 ± 97%) among all the chitosan–c-PGA PEC hydrogels. C75P25 was relatively hydrophobic (water uptake: 675 ± 55%). Thus, the antibacterial ability of PEC hydrogel toward E. coli and S. aureus may be attributed to its hydrophilicity, which prevented microbial adhesion. The same reason is accurate for chitosan–c-PGA PEC hydrogels with low MW of 3  104 Da, against E. coli and S. aureus. For Gram-negative bacteria, it was found that the chitosan–cPGA PEC hydrogels with high chitosan MW (3  105 Da) and low chitosan MW (3  104 Da) showed an equal antibacterial activity against E. coli. It meant that the effect of chitosan MW on the antibacterial activity of chitosan–c-PGA PEC hydrogels was not pronounced against E. coli. However, for Gram-positive bacteria, it was found that chitosan–c-PGA PEC hydrogels with high chitosan MW (3  105 Da) and low chitosan MW (3  104 Da) showed different antibacterial activity for S. aureus. The antibacterial activity of PEC hydrogels with chitosan of 3  104 Da was more active that of 3  105 Da. This result showed the combined effect of hydrophilic environment and cell membrane structure. Gram-negative bacteria contain an outer membrane wherein lipopolysaccharide and proteins are held together by electrostatic interactions with bivalent metal ions, one to two layers of peptidoglycans (cell wall), and a cell membrane (containing lipid bilayer, trans-membrane proteins, and inner/outer membrane proteins). The negatively charged O-specific antigenic oligosaccharide repeating units of the E. coli lipopolysaccharide form an ionic-type of binding with the positively charged amino groups of chitosan, thus blocking the nutrient flow with bacterial death due to depletion of the nutrients.16 That is, chitosan having high —NH3 þ groups cannot pass through the microbial membrane and hence stack to the cell surface, which blocks nutrient transport or permeabilizes the microbial cell membrane, resulting in cell lysis. In Gram-positive bacteria, on the other hand, the cell membrane is covered by a cell wall made up of 30–40 layers of peptidoglycans, which contain GlcNAc and N-acetylmuramic acid as well as D- and L-amino acids, including isoglutamate and teichoic acid, to which the positively charged amino groups of chitosan can bind, resulting in cell wall disruption, and exudation of the cytoplasmic contents.16 In this situation, low MW chitosan showed a strong effect. Owing to its small size and being water soluble, it can traverse the microbial membrane and may bind and regulate DNA transcription. It is true that the chitosan has high concentration of —NH3 þ groups, and the deposition of cationic chitosan onto the cell surface, which leads to cell leakage, is more prominent in the case of Gram-negative bacteria, owing to a stronger association of

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O-chains to the outer membrane structure.38 However, in the case of Gram-positive bacteria, based on the chitosan–c-PGA PEC hydrogel had already created a hydrophilic environment, the lower MW chitosan, small and soluble in the hydrophilic environment, can pass through the microbial membrane, binding and resulting in cell wall disruption, is much important. Moreover, surface morphology—or, more specifically, surface roughness—is also the physical effect of bacterial adhesion. The neat chitosan served to provide a smoother surface (Fig. 5d), which made bacterial adhesion more difficult. Thus, the inhibitory effects of the neat chitosan against bacterial adhesion may be attributed to the surface smoothness. In summary, for E. coli (Gram-positive bacteria), the antibacterial activity of the chitosan–c-PGA PEC hydrogels was dominated by hydrophilicity. The antibacterial activity was strong in the group of high c-PGA portion; the higher hydrophilicity leads to less microbial adhesion. On the other hand, for S. aureus (Gram-negative bacteria), the antibacterial activity of the chitosan–c-PGA PEC hydrogels was dominated by chitosan MW: the low MW chitosan, being small and soluble, was able to penetrate the outer membrane and then may bind and regulate DNA transcription. The surface morphology of PEC hydrogel is the third effect, the one with rougher surface provide the better inhibitory effect. 4.3. Biocompatibility Biocompatibility is another important characteristic of a material intended for biomedical applications. Although chitosan and c-PGA are both biocompatible materials for various types of cells,20,39 the relationship between the charge of the chitosan–c-PGA PEC hydrogels and their biocompatibility has not been previously discussed. The cell proliferation, illustrated as the O.D. value, versus various chitosan–c-PGA PEC hydrogels with different culture periods is shown in Figure 6. All the chitosan–c-PGA PEC hydrogels had better biocompatibility for 3T3 fibroblasts than the control. After a 2-day culture, C25P75 expressed the greatest cell proliferation among these PEC hydrogels. When the culture period was extended to 3 days, C75P25 overcame the others. We interpret this phenomenon as that the presence of c-PGA in C25P75 caused the PEC hydrogels surface to retain a significant amount of water, thereby attracting more cell attachment during the first stage (2-day culture). When the culture period was extended further (3-day culture), C75P25, with its positively charged surface, attracted more of the cells having a negatively charged membrane to proliferate.

Figure 6. Proliferation of 3T3 fibroblasts cultured on various chitosan–c-PGA PEC hydrogels (C25P75, C50P50, C75P25). Data are presented as mean ± SD (n = 3).

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Figure 7. SEM images of 3T3 fibroblasts after 3-day culturing on chitosan–c-PGA PEC hydrogels (a) C25P75, (b) C50P50, and (c) C75P25.

The charge effect of the chitosan–c-PGA PEC hydrogels on 3T3 fibroblasts was further confirmed through the analysis of SEM images. Figure 7a–c display images of 3T3 fibroblasts after the 3day culture on C25P75, C50P50, and C75P25, respectively. The majority of cells on C75P25 (positively charged, chitosan-dominated) gathered together and formed clusters, while those on C50P50 (neutral) and C25P75 (negatively charged, c-PGA-dominated) were randomly attached onto the surface. Moreover, the 3T3 cells cultured on C75P25 exhibited flattened morphologies. In contrast, those on C50P50 and C25P75 exhibited round and spherical morphologies. The flattened morphologies indicated that the cells had better affinity for C75P25, attracted by the positive charge of the PEC hydrogel surface. Hsieh et al. have also reported the improved biocompatibility of chitosan with the addition of c-PGA.12 It was proposed that the improvements in hydrophilicity, which also enhanced the adsorption of serum proteins including cell attachment factors, are both beneficial for cell attachment and proliferation. However, Kang et al. have attributed the improved biocompatibility to its microstructure.40 They thought that with the addition of c-PGA, the interconnected 3-D porous structure of chitosan–c-PGA PEC is much more homogeneous than that of chitosan alone. The high uniformity is a crucial factor for cell attachment and proliferation. In this present study, the presence of c-PGA retained water, thereby attracting cells during the first stage. In the second stage, however, the positive charge of the PEC hydrogel membrane had a greater influence on the biocompatibility of PEC hydrogel. 5. Conclusions In this study, chitosan and c-PGA formed structurally stable PEC hydrogel through simple ionic complex interaction, between the amine groups of chitosan and the carboxylic acid groups of c-PGA, as confirmed using FTIR spectroscopy. The compressive modulus increased upon increasing the degree of complex formation in the chitosan–c-PGA PEC hydrogels. The water uptake decreased upon increasing the degree of complex formation. At the same the degree of complex formation, the compressive modulus was larger for the chitosan-dominated PEC hydrogels. The water uptake was larger for the c-PGA-dominated ones. Furthermore, the chitosan–c-PGA PEC hydrogels exhibited satisfactory antibacterial ability against E. coli and S. aureus. Biocompatibility studies revealed that the chitosan–c-PGA PEC hydrogel had good affinity for 3T3 fibroblast cells, especially the positive-charged ones (chitosan-dominated). As a result, the chitosan–c-PGA PEC hydrogel is a very promising material for biomedical applications. Acknowledgment This study was supported by a grant from the National Science Council, Republic of China (Project No. NSC, 95R0201).

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