Characterization and biocompatibility of chitosan nanocomposites

Colloids and Surfaces B: Biointerfaces 85 (2011) 198–206

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Characterization and biocompatibility of chitosan nanocomposites Shan-hui Hsu a,b,c,∗ , Yu-Bin Chang d , Ching-Lin Tsai e , Keng-Yen Fu a , Shu-Hua Wang f , Hsiang-Jung Tseng d a

Institute of Polymer Science and Engineering, National Taiwan University, Taipei, Taiwan Rehabilitation Engineering Research Center, National Taiwan University, Taipei, Taiwan c Institute of Biomedical Engineering, National Chung Hsing University, Taichung, Taiwan d Department of Chemical Engineering, National Chung Hsing University, Taichung, Taiwan e Department of Orthopaedics, National Taiwan University Hospital, Taipei, Taiwan f College of Life Science, National Chung Hsing University, Taichung, Taiwan b

a r t i c l e

i n f o

Article history: Received 9 October 2010 Received in revised form 20 February 2011 Accepted 22 February 2011 Available online 2 March 2011 Keywords: Chitosan Gold nanoparticles Nanocomposites Silver nanoparticles

a b s t r a c t Chitosan nanocomposites were prepared from chitosan and gold nanoparticles (AuNPs) or silver nanoparticles (AgNPs) of ∼5 nm size. Transmission electron microscopy (TEM) showed the NPs in chitosan did not aggregate until higher concentrations (120–240 ppm). Atomic force microscopy (AFM) demonstrated that the nanocrystalline domains on chitosan surface were more evident upon addition of AuNPs (60 ppm) or AgNPs (120 ppm). Both nanocomposites showed greater elastic modulus, higher glass transition temperature (Tg ) and better cell proliferation than the pristine chitosan. Additionally, chitosan-Ag nanocomposites had antibacterial ability against Staphylococcus aureus. The potential of chitosan-Au nanocomposites as hemostatic wound dressings was evaluated in animal (rat) studies. Chitosan-Au was found to promote the repair of skin wound and hemostasis of severed hepatic portal vein. This study indicated that a small amount of NPs could induce significant changes in the physicochemical properties of chitosan, which may increase its biocompatibility and potential in wound management. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Chitosan is a crystalline polysaccharide containing glucosamine and N-glucosamine. It is the deacetylation form of chitin, the second most abundant natural polymer next to cellulose, which can be obtained from crustaceans or fungal cell wall. The amino group in chitosan allows the polymer to be dissolved in acids, so chitosan films can be easily prepared from solution. The degree of deacetylation, which determines the content of free amino groups in the polysaccharide, can influence the performance of chitosan in many aspects [1]. Chitosan can be applied in vastly diverse fields, ranging from waste management to food processing and medicine because of its biocompatibility, biological activity and biodegradability [2,3]. Chitosan carries positive charge that can interact with the microbial surface, and therefore presents good antibacterial activities [4]. Chitosan also interacts with red blood cells and possesses a hemostatic effect [2]. The degradation rate of chitosan in vivo was slow, and with the higher degree of deacetylation, the slower degradation was observed [5]. The biomedical applications

∗ Corresponding author at: Institute of Polymer Science and Engineering, National Taiwan University, Taipei 10617, Taiwan. Tel.: +886 2 3366 5313; fax: +886 2 3366 5237. E-mail address: [email protected] (S.-h. Hsu). 0927-7765/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2011.02.029

of chitosan include novel drug delivery such as peptide or gene delivery [6–8] as well as in wound dressings and tissue engineering [9,10].Gold (Au) was a precious metal due to its inertness. The surface area of Au nanoparticles (AuNPs) increases in proportion tremendously, which makes them different in physical, chemical and biological properties from Au of the macroscopic scale. In a previous study, AuNPs were prepared in the presence of chitosan via reduction of HAuCl4 with sodium borohydride [11]. The activity of prepared Au/chitosan NPs was 80 times higher than that of ascorbic acid, which is well known as an antioxidant. In another study, Audendrimer NPs were prepared in the presence of poly(amidoamine) (PAMAM) dendrimer via reduction of HAuCl4 with sodium borohydride [12]. The highest activity for the Au-PAMAM dendrimer NPs was 85 times that of ascorbic acid. When AuNPs were added in polyurethane, the thermostability and mechanical properties of the polymer increased with Au contents up to 43.5 ppm, which was believed to be a result of induced crystallization in the presence of AuNPs [13]. Improved biocompatibility, biostability and greater free radical scavenging ability were also observed for the polymer nanocomposites [14,15]. As chitosan has many amino groups (polycations), it can serve as a mediator agent for synthesis of AuNPs or silver nanoparticles (AgNPs) and stabilize these NPs [16].AgNPs as well as the released Ag+ ion have a significant bactericidal effect. Due to the large surface area and fraction of surface atoms, AgNPs have exhibited higher antibacterial activities than bulk silver [17].

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AgNPs (∼10 nm in size) at the concentrations of 50–100 ppm inhibited 99.99% bacterial growth including Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) [18]. AgNPs were found to contact the bacterial wall of E. coli, form many small holes and cause the cell death [19]. AgNPs (∼5–50 nm) in polymethylmethacrylate could inhibit the growth such as S. epidermis, methicillin-resistant S. epidermidis, and methicillin-resistant S. aureus [20]. Polyurethane with 30 ppm AgNPs had better biostability and biocompatibility over the original polymer [21]. AgNPs were used in drinking water purification and antimicrobial dressing materials [22]. A few commercial products containing AgNPs have been approved by the US Food and Drug Administration (FDA) or the European Food Safety Authority (EFSA) for wound dressings and antibacterial applications [23].The crystalline structure of chitosan could be modified by metallic NPs formed in situ in the chitosan matrix [24]. The hemostatic properties of chitosan appeared to be associated with the crystalline structure of chitosan [25]. It was thus reasonable to assume that the addition of NPs could change the biocompatibility of chitosan. In this study, AuNPs or AgNPs were directly mixed in chitosan. The dispersion of these NPs in chitosan matrix was examined. The effects on physiochemical properties of chitosan and on human fibroblast proliferation were determined. The potential for the nanocomposites to serve as hemostatic wound dressings was evaluated preliminarily in vivo. 2. Materials and methods 2.1. Preparation of chitosan nanocomposites One percent of chitosan (average Mw 400 kDa, the degree of deacetylation 77.7%, Fluka) solution was dissolved in 0.5 mol/l acetic acid. AuNPs and AgNPs in aqueous solution were supplied by Gold NanoTech, Taipei, Taiwan. These NPs were produced by physical manufacturing and did not contain any surface modifiers or stabilizers [26]. Briefly, Au or Ag bulk material was cut or ground into the target material. Then the Au or Ag target was vaporized to the atomic level by an electrically gasified method under vacuum. The vapor was condensed in the presence of inert gas and then piled up to form AuNPs or AgNPs. The NP sizes can be effectively managed depending on the evaporation time and electric current used. The NPs were collected in a cold trap and centrifuged to obtain the final product. The average sizes of AuNPs and AgNPs were 5.52 ± 0.95 nm and 5.75 ± 1.12 nm, as observed by the transmission electron microscopy (TEM). After filtering the chitosan solution, a certain amount of AuNPs or AgNPs was added and the final concentration in the polymer was 30, 60, 120 and 240 ppm. To make the substrates for biological tests, 100 ␮l of the chitosan-Au (or chitosan-Ag) suspension was coated onto coverslip glass (15mm diameter; Matsunami, Osaka, Japan) by a spin coater (PM-490, Synrex, Taiwan) and dried at 37 ◦ C for 48 h. 2.2. Characterization of the chitosan-Au and chitosan-Ag nanocomposites The microstructure was examined by means of TEM. Thin sections (∼80 nm) of chitosan or chitosan nanocomposites were microtomed by an untramicrotome apparatus (Reichert-Jung Ultracut E, Leica Microsystems, Germany) equipped with a diamond knife, and were subsequently deposited on copper grids. The samples were detected using a JEOL JEM-1200 microscope operated at 110 kV. Topography and phase images were recorded simultaneously from an atomic force microscope (AFM) (CP-II, Veeco, USA) operated in tapping mode with a cantilever (spring constant 20–75 N/m) coated with Al on the reflective side and the diameter of the nee-

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dle tip was 10 nm (NSC 15/AIBS/50, MikroMasch, USA). The average roughness and average diameter of crystalline (brighter) domains were calculated using the packaged computer software (Image processing ver.2.0, Veeco, USA). The attenuated total reflection infrared spectroscopy (ATR-IR) was applied to determine the change in surface chemistry of nanocomposites. Samples were scanned by a Fourier transformed infrared spectrometer (Spectrum one FT-IR, Perkin-Elmer, USA) in the spectral region of 4000–650 cm−1 to obtain the IR adsorption spectra. The resolution was 2 cm−1 . The water contact angle was measured at 25 ◦ C and 70% relative humidity using a contact angle meter (First Ten Angstrom, FTA-125, USA). The sessile drop method (5 ␮l deionized water) was used. At least four readings were made for each sample and the values were calculated as the average from three independent detections. The dynamic mechanical analysis (DMA) was tested by a dynamic mechanical analyzer (DMA 2980, TA Instruments, USA). The specimen was cut as a dimension of 20 mm in length, 5 mm in width and 0.05 mm in thickness. The dynamic elastic modulus and glass transition temperature (Tg ) were determined in tensile mode. The specimen was cooled to 30 ◦ C and then heated to 225 ◦ C at a rate of 5 ◦ C/min. The dynamic frequency was 1 Hz and the amplitude was 20 ␮m. The dynamic elastic modulus and loss tangent (tan ı) at each temperature were recorded. The Tg of the specimen was obtained as the temperature at the highest point of tan ı curve. 2.3. Isolation of human gingival fibroblasts and cell proliferation tests Fresh human gingival tissue was obtained after the surgery for the periodontal flap operation in a nearby dental clinic by following the ethical guidelines. The age of the donors was 20–40 years old. The tissue was minced into 0.5 mm3 pieces and digested in the low glucose Dulbecco’s modified Eagle’s medium (DMEM, Gibco, USA) containing 1 mg/ml type I collagenase (Sigma, USA) in an incubator (37 ◦ C, 5% CO2 ) for 5 h. The suspension of gingival fibroblasts was filtered through a 200 ␮m mesh (BD Falcon, USA) and resuspended in DMEM containing 10% fetal bovine serum and antibiotics (penicillin G 100 U/ml, streptomycin 100 g/ml and gentamicin 50 g/ml) in a T25 flask and placed in the incubator. After 3–4 days, the primarily cultured cells were nearly confluent. They were trypsinized using 0.05% trypsin-EDTA solution and transferred to T75 flasks. Fibroblasts of the 5th passage were used in the experiment. The cast films on coverslip glass were sterilized by 70% ethanol and placed into the bottom of a 24-well tissue culture plate. Six wells were performed for each sample. One milliliter of cell suspension with a density of 5 × 104 cells/ml was added into each well of the culture plate. After 1, 3, 5 and 7 days of incubation, the number of viable cells was measured by the MTT assay. 0.5 mg/ml 3-(4,5dimethylthiazol-2-yl)-3,5-diphenyltetrazolium bromide (MTT) in phosphate buffered saline was added into each well and incubated for 4 h at 37 ◦ C. The supernatant was removed. An aliquot of dimethylsulfoxide (Tedia, Fairfield, OH) was added and reacted for 10 min to dissolve any resulting formazan crystals. The absorbance at 550 nm was determined by an ELISA reader (F-2500, Hitachi, Japan). The standard curve was made from different numbers of cells that the MTT assay was performed on. The values of absorbance were then converted into cell numbers from the standard curve in all cell experiments. 2.4. Antibacterial activities The antimicrobial activities were carried out followed the AATCC test method 90-1982. AATCC broth was prepared by pep-

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tone (Himedia, India), beef extract (Himedia) and NaCl (Sigma) that were dissolved in distilled water. The broth was adjusted to pH 6.8 by NaOH, and sterilized by an autoclave at 103 kPa for 20 min. The AATCC agar was prepared by adding the agar (BD, USA) to 1.5% final concentration on AATCC broth, adjusted pH to 7.0–7.2, and autoclaved for 20 min. When the agar temperature was cooled down to 45 ◦ C, 15 ml was poured into a 100 mm diameter petri dish and cooled to coagulate at room temperature. The test bacteria, Staphylococcus aureus, was cultured in AATCC broth for 24 h. Samples were cut into circular sharp with 28.6 mm in diameter. 100 ␮l of bacterial solution was spread on the surface of the agar plates. Specimens were gently pressed and intimately contacted with the agar. The plates were incubated at 37 ◦ C for 24 h. The width of the inhibition (W) zone was determined by the following equation: W = (T − D)/2, where T was the total diameter of the specimen and the clear zone (in mm), and D was the diameter (28.6 mm) of the specimen. The average was obtained on six specimens (n = 6). 2.5. Animal studies Only the chitosan-Au nanocomposite at the best AuNPs concentration and the plain chitosan were subjected to the preliminary animal evaluation. The samples were freeze-dried. Male Sprague–Dawley rats weighing 350–400 g were used for the animal studies. Animals were deeply anesthetized with isoflurane (Halo-carbon, River Edge, NJ) throughout the surgical procedures. Surgery was conducted after the hair shaving and disinfection by the treatment of iodine (povidone-iodine, China Chemical & Pharmaceutical) and 70% alcohol in the dorsal back. For wound repair experiments, ten rats were used for each sample. The total number of rats used in the study was 30. The rat dorsal skin was cut with sterile surgical procedure to create five wounds with the area of 1 cm2 and 2–3 mm depth. The samples of chitosan or chitosan-Au were cut as 1 cm × 1 cm in size and 3–5 mm in height and sterilized by ␥-ray (10 kGy). Each sample was rinsed in saline and then covered on the wound. The elastic bandage was used to fix the dressing. Tegaderm (3 M, the same size) was used as the control. The dressings were replaced and wound healing was recorded by photography every 2 days. The wound healing rate was evaluated as follows: wound healing ratio (%) = [(A0 − AH)/A0] × 100%, where A0 was the original wound area after cutting and AH was the area of the wound that was not repaired at the time. After 7 or 14 days, the rats (five rats for each time point for each sample) were euthanized by CO2 overdose treatment. The tissue of the wound including the dressing was obtained and fixed in formalin for histological evaluation. The samples were dehydrated in a graded series of ethanol solutions, and finally embedded. These embedded samples were cut into 3 mm thick sections, and H&E staining was performed. For hemostatic evaluation, three rats were used for each sample. The total number of rats used in the study was six. The abdominal cavity was exposed with sterile surgical procedure. A 23G needle was inserted into the hepatic portal vein. The vein was allowed to bleed for 10 s. The dressing was then held on the wound for 90 s. The dressing was removed and the situation of hemorrhage was recorded. Three rats for each sample were used in this study. 2.6. Statistical analysis Multiple samples were collected in each measurement (n = 3–6) and the data were expressed as mean ± standard deviations (SD). Single factor analysis of variance (ANOVA) method was used to assess the statistical significance of results. Statistical significance was indicated by p < 0.05.

Fig. 1. TEM images for different nanocomposites: (A) chitosan-Au 30 ppm, (B) chitosan-Au 60 ppm, (C) chitosan-Au 120 ppm, (D) chitosan-Au 240 ppm, (E) chitosan-Ag 30 ppm, (F) chitosan-Ag 60 ppm, (G) chitosan-Ag 120 ppm, and (H) chitosan-Ag 240 ppm. Scale bar = 20 nm.

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Fig. 2. AFM phase diagrams for (A) pure chitosan, (B) chitosan-Au 30 ppm, (C) chitosan-Au 60 ppm, (D) chitosan-Au 120 ppm, (E) chitosan-Ag 30 ppm, (F) chitosan-Ag 120 ppm, (G) chitosan-Ag 240 ppm. A is the topography diagram for chitosan, which did not vary significantly upon addition of NPs. Scale bar = 100 nm. The nanometric bright spots (nanocrystalline hard domains) were indicated by the black arrows.

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Table 1 The average roughness, average diameter of crystalline domains on the surface, contact angle, dynamic elastic modulus and glass transition temperature of different nanocomposites. Materials

Average diameter of hard spots (nm)

Pure chitosan Chitosan-Au 30 ppm Chitosan-Au 60 ppm Chitosan-Au 120 ppm Chitosan-Au 240 ppm Chitosan-Ag 30 ppm Chitosan-Ag 60 ppm Chitosan-Ag 120 ppm Chitosan-Ag 240 ppm

13.38 14.56 26.09 17.25 16.14 21.08 34.05 41.85 21.12

*

± ± ± ± ± ± ± ± ±

0.75 0.83 3.35* 0.49* 0.41* 1.64 3.21* 2.56* 1.42*

Average roughness (Ra) (nm)

Contact angle (◦ )

0.43 0.70 0.40 0.46 0.52 0.43 0.38 0.44 0.47

84.50 79.08 78.22 78.26 78.09 77.90 77.50 76.29 77.42

± ± ± ± ± ± ± ± ±

1.34 1.52* 2.11* 2.23* 2.35* 2.44* 2.68* 2.76* 1.84*

Elastic modulus (MPa) at 37 ◦ C and 1 Hz

Glass transition temperature (◦ C)

2321 2727 2987 2794 2433 3052 3359 3865 2779

178.25 184.20 185.41 184.15 183.57 181.43 182.86 183.36 180.73

p < 0.05 relative to pure chitosan.

3. Results Fig. 1 shows TEM images of the nanocomposites. The sizes of AuNPs and AgNPs in these images varied, depending on the status of aggregation. It was observed that the aggregation of AuNPs occurred in the nanocomposites when the concentration of NPs was beyond 120 ppm, while the aggregation of AgNPs occurred when it was beyond 240 ppm. Judging from these images, the dispersion of AgNPs in chitosan was better than that of AuNPs. Fig. 2 shows the AFM surface phase diagrams of chitosan and chitosan nanocomposites. In the diagrams, the brighter spots (harder domains) were distributed homogeneously in the matrix. The sizes of these brighter spots increased with an increase in the concentration of NPs. The maximal average diameter in each type of nanocomposites was about 26 nm at 60 ppm of AuNPs and about

42 nm at 120 ppm of AgNPs respectively, as listed in Table 1. These harder spots were suspected to be the nanocrystalline domains on the chitosan surface. A typical topography is shown in Fig. 2A . The average surface roughness calculated from the topography diagrams was similar (in the range of 0.3–0.6 nm) among chitosan and different nanocomposites. Contact angle analysis provides the surface hydrophobicity/hydrophilicity and molecular mobility at the air–solid–water interface for biomaterials. As shown in Table 1, the contact angle of nanocomposites fell into the range of 76–79◦ and was lower than that of chitosan (∼84.5◦ ). This indicated that each nanocomposite was more hydrophilic than the original chitosan. The values of dynamic elastic modulus and glass transition temperature for chitosan and chitosan nanocomposites are listed in Table 1. Judging from the data of elastic modulus, the highest mechanical proper-

Fig. 3. ATR-FTIR spectra for (A) chitosan-Au and (B) chitosan-Ag in the range 4000–650 cm−1 (left) and 3600–3000 cm−1 (right), respectively.

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Fig. 4. The proliferation of human gingival fibroblasts up to 7 days on (A) chitosanAu and (B) chitosan-Ag nanocomposites. *p < 0.05 relative to pure chitosan.

ties of chitosan occurred at 60 ppm of AuNPs or 120 ppm of AgNPs, but declined by further ascending the concentration of NPs. The elastic modulus of chitosan at 120 ppm of AgNPs (3865 MPa) was about 1.3-fold higher than that at 60 ppm of AuNPs (2987 MPa). The glass transition temperature, associated with their thermal stability, was a few degrees higher in the nanocomposites than that in pure chitosan. The ATR-IR absorption spectra for the nanocomposites are shown in Fig. 3. The pristine chitosan had an absorption peak of –NH band at 3278 cm−1 . This peak location shifted in nanocomposites. The maximal shift (to ∼3262–3263 cm−1 ) in chitosan-Au was at 30–60 ppm of AuNPs, whereas that (to ∼3254–3256 cm−1 ) in chitosan-Ag was at 120–240 ppm of AgNPs. The proliferation of fibroblasts on chitosan and chitosan nanocomposites is shown in Fig. 4. At 24 h, only chitosan-Au 60 ppm and chitosan-Ag 120 ppm showed significantly more cells than pure chitosan. From 3 to 7 days, the chitosan-Au 120 ppm as well as chitosan-Ag at all AgNP concentrations (30–240 ppm) also demonstrated significantly more cells. Among all nanocomposites, chitosan-Au 60 ppm and chitosan-Ag 120 ppm still had the greatest number of cells. The width of antibacterial zone is shown in Fig. 5. No statistically significant difference was demonstrated between chitosan and chitosan-Au nanocomposites in all concentrations. On the other hand, a wider antibacterial zone was observed for all chitosan-Ag nanocomposites. The addition of AgNPs at 120 ppm had the greatest inhibition effect on bacterial growth, followed by that at 240 ppm. The effect of chitosan-Au on skin wound healing is shown in Fig. 6. Chitosan-Au promoted the rate of wound healing closure more than control (Tegaderm) and the original chitosan after surgery at 7 and 11 days, but not at 14 days (Fig. 6A). The histology at 7 days (Fig. 6C and D) showed the debris of chitosan (indicated by circles). Meanwhile, the epithelium started to grow over the wound (Fig. 6D and E). At 14 days post surgery, the inflammatory cells decreased and capillary formation increased in the control (Fig. 6F). The phenomena of epithelial migration and collagen secretion by

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Fig. 5. Antibacterial activities as evaluated by the width of antibacterial zone at 24 h after exposure to different nanocomposites. *p < 0.05 relative to pure chitosan.

fibroblasts were present in the chitosan group (Fig. 6G). The epithelium that covered the wound was obvious at 7 and 14 days in the group of chitosan-Au (Fig. 6D and H; indicated by boxes). At 14 days, the hyperemia status of microvessels disappeared and connective tissue formed loosely, suggesting the primary repair has been achieved in chitosan-Au (Fig. 6H). The wound epithelialization process in the group of chitosan-Au was more complete than that in the chitosan. (Fig. 6H vs. Fig. 6G). In the hemostatic experiment, none of the original chitosan could stop the bleeding from hepatic portal vein when held on the wound. On the other hand, the dressings made from chitosanAu stopped the bleeding effectively (n = 3). The typical images are displayed in Fig. 7. 4. Discussion The characteristics of chitosan were significantly modified by the existence of a small amount of Au or Ag NPs in the chitosan matrix. Based on the TEM images, AuNPs at 60 ppm and AgNPs at 120 ppm both showed good dispersion in chitosan. When the concentration of NPs was higher, aggregation could occur. AFM phase diagrams also suggested that the addition of an appropriately small amount of AuNPs or AgNPs improved the homogeneity and increased the size of nanocrystalline domains on chitosan probably through nucleation. It has been reported that metallic NPs induced the formation of crystalline structure in chitosan, which may be due to the nucleation effect of these NPs [24]. In a previous study [14], the thermal and mechanical properties of polyurethane were significantly improved by adding 43.5 ppm of AuNPs into the polymer matrix, which was suspected to be an effect partially from crystallization. In the current study, the mechanical strength (elastic modulus) of chitosan containing 120 ppm of AgNPs or 60 ppm of AuNPs was the highest. This may be associated with the greater sizes (∼42 and ∼26 nm) of nanocrystalline domains in each nanocomposite upon addition of AuNPs and AgNPs, with AgNPs being more effective. The increased glass transition temperature of the nanocomposites also reflected such a tendency, but

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Fig. 6. (A) The wound repair for the rat dorsal skin (for n = 5) evaluated as the wound healing ratio from different dressings. *p < 0.05 between the two groups. (B–E) The histology of the wound obtained at 7 days post surgery for: (B) control group (Tegaderm) (100×), (C) chitosan group (100×), (D) chitosan-Au group (100×), and (E) chitosan-Au group at higher magnification (200×). (F–H) The histology of the wound at 14 days post surgery for: (F) control group (Tegaderm) (100×), (G) chitosan group (100×), and (H) chitosan-Au group (100×). Symbols v: vascularization, circle: chitosan debris, box: epithelialization.

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Fig. 7. Hemostatic evaluation for chitosan: (A) bleeding (marked by the white arrow) from the rat hepatic portal vein and (A ) the appearance of dressing after removal; and that for chitosan-Au nanocomposites: (B) hemostasis in the rat portal vein (blot clot marked by the black arrow) and (B ) the appearance of dressing after removal.

with AuNPs being more effective. The sizes of NPs could be crucial because they may influence the nanocrystalline structure of chitosan. In a preliminary study, NPs of greater sizes (∼25 nm) were tested but showed less an effect. Pure chitosan has an IR absorption peak at around 3278 cm−1 , which is assigned to –NH2 band. The shift in the peak location of this band from 3278 cm−1 to 3263 cm−1 or to 3254 cm−1 was observed upon addition of AuNPs 60 ppm or AgNPs 120 ppm as shown in Fig. 3, suggesting that hydrogen bonding may form in the presence of AuNPs or AgNPs. As in the previous polyurethane system [27], the addition of AuNPs may play as a nucleating agent of crystallization, enhancing the effect of hydrogen bonding and stabilizing the phase separation in the polyurethane matrix. Therefore, the largest shift (∼24 cm−1 ) in the peak location of the –NH band in the presence of 120 ppm AgNPs suggested the formation of additional bonds, which also gave the nanocomposite (PU-Ag 120 ppm) the largest mechanical strength (3865 MPa vs. 2321 MPa for the original chitosan). The shift of –NH peak in IR spectra may be due to the interaction between the lone pair electrons of nitrogen and the metal NPs, which has been observed for polyurethane coated with AgNPs [28]. The nanocomposites with AuNPs or AgNPs enhanced the proliferation of human fibroblasts on chitosan. Among all the nanocomposites investigated, chitosan-Au 60 ppm and chitosanAg 120 ppm had the greatest cell number. This significant enhancement of biocompatibility may be due to the altered surface morphology [15]. The size of the surface nanometric domains could have an impact on the cellular response. Especially, enhanced fibroblast response to surface nanoislands (featured 27 nm) was reported [29]. There existed a possibility of modified cellular or tissue responses by the nanocomposite surfaces. The greater average size of hard domains in chitosan-Au 60 ppm and chitosan-Ag

120 ppm showed the most remarkable biocompatibility in this study. In the measurements of inhibition circles, the chitosan-Au nanocomposites failed to detect any antibacterial activities toward S. aureus. On the other hand, the chitosan-Ag nanocomposites gained significant antibacterial activities at the concentration of 60 or 120 ppm, but decreased in higher concentration. The antibacterial mechanism of AgNPs was attributed that the positively charged AgNPs could bind with the negatively charged bacterial wall and interfere with the catabolism and growth of bacteria. Another mechanism could be through the interaction between Ag+ and bacterial protein thiol groups [30]. The greatest antimicrobial efficacy was found in chitosan with 120 ppm of AgNPs but not at the higher concentration, suggesting the interaction may be related to the exposure of individual AgNPs that were not aggregated in the matrix. In another experiment (data not shown), the presence of AuNPs or AgNPs enhanced the free radical scavenging ratio over the original chitosan, especially for chitosan containing 120 ppm AgNPs. Because AgNPs could scavenge free radicals [31], this result also suggested the exposure of individual AgNPs to the surface of chitosan. The release of free silver from chitosan-Ag nanocomposite containing 120 ppm AgNPs was measured by ICP-MS for samples soaked and shaken at 100 rpm at 37 ◦ C for 2 days. Only ∼0.1% of the total AgNPs in the specimen was released after 48 h. Therefore, the antibacterial and antioxidant effects in short term were not likely to arise from the free Ag+ or AgNPs released to the environment. Animal studies showed that chitosan-Au nanocomposites had better performance than pure chitosan in wound repair and in hemostasis. The better wound healing and hemostaticity of the nanocomposite may be attributed to the surface crystalline structure [25] or simply due to the extra hydrogen bonds and water retention. The chitosan-Ag nanocomposite was not tested because

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of the recent concerns regarding the cytotoxicity and genotoxicity of AgNPs [32], which could be released from the degradable chitosan matrix. Although our data showed that the amount of free silver released to the environment was insignificant in short term, the release over an extensive period of time in vivo could be significant, which may lead to toxicity. On the other hand, if these concerns could be resolved, the use of chitosan-Ag nanocomposites to treat infectious skin wound should worth further investigations. AuNPs and AgNPs have demonstrated effectively anti-inflammatory efficacy [33]. Therefore, appropriate dressings developed from chitosan containing AuNPs or AgNPs possess the potential for treatment of wounds. References [1] A. Baxter, M. Dillon, K.D.A. Taylor, G.A.F. Roberts, Int. J. Biol. Macromol. 14 (1992) 166. [2] Y.L. Kuen, S.H. Wan, H.P. Won, Biomaterials 16 (1995) 1211. [3] A. Bernkop-Schnurch, C.E. Kast, Adv. Drug Deliv. Rev. 52 (2001) 127. [4] J. Rhoades, S. Roller, Appl. Environ. Microbiol. 66 (2000) 80. [5] C. Claire, D. Odile, D. Alain, Biomaterials 22 (2001) 261. [6] K. Aiedeh, E. Gianasi, I. Orienti, V. Zecchi, J. Microencapsul. 14 (1997) 567. [7] P. Giunchedi, I. Genta, B. Conti, R. Muzzareli, Biomaterials 19 (1998) 157. [8] S. Miyazaki, K. Ishii, T. Nadai, Chem. Pharm. Bull. 29 (1981) 3067. [9] F.L. Mi, S.S. Shyu, Y.B. Wu, S.T. Lee, J.Y. Shyong, R.N. Huang, Biomaterials 22 (2001) 165. [10] S.W. Whu, C.L. Tsai, S.H. Hsu, J. Med. Biol. Eng. 29 (2009) 52.

[11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] [33]

K. Esumi, N. Takei, T. Yoshimura, Colloids Surf. B Biointerfaces 32 (2003) 117. K. Esumi, H. Houdatsu, T. Yoshimura, Langmuir 20 (2004) 2536. S.H. Hsu, C.W. Chou, S.M. Tseng, Macromol. Mater. Eng. 289 (2004) 1096. S.H. Hsu, C.W. Chou, Polym. Degrad. Stabil. 85 (2004) 675. S.H. Hsu, C.M. Tang, H.J. Tseng, J. Biomed. Mater. Res. A 79 (2006) 759. D. Wei, W. Qian, Colloids Surf. B Biointerfaces 62 (2008) 136. S.K. Bajpai, Y.M. Mohan, M. Bajpai, R. Tankhiwale, V. Thomas, J. Nanosci. Nanotechnol. 7 (2007) 2994. K.H. Cho, J.E. Park, T. Osaka, S.G. Park, Electrochim. Acta 51 (2005) 956. I. Sondi, B. Salopek-Sondi, J. Colloid Interface Sci. 275 (2004) 177. V. Alt, T. Bechert, P. Steinruke, M. Wagener, P. Seidel, E. Dingeldein, E. Domann, R. Schnettler, Biomaterials 25 (2004) 4383. C.W. Chou, S.H. Hsu, H. Chang, S.M. Tseng, H.R. Lin, Polym. Degrad. Stabil. 91 (2006) 1017. A. Petica, S. Gavriliu, M. Lungua, N. Burunteaa, C. Panzarub, Mater. Sci. Eng. B 152 (2008) 22. E. Ulkur, O. Oncul, H. Karagoz, E. Yeniz, B. Celikoz, Burns 31 (2005) 874. H. Huang, Q. Yuan, X. Yang, Colloids Surf. B Biointerfaces 39 (2004) 31. A. Hoekstra, H. Struszczyk, O. Kivekäs, Biomaterials 19 (1998) 1467. R. Y. Wu, B.S. Chen, S.W. Tang, W.Y. Kao, P.N. Sui, C.C. Hsu, J.H. Liu, H.Y. Chang, Taiwanese pending patent. Appl. no. 093141926. S.H. Hsu, C.M. Tang, H.J. Tseng, Acta Biomater. 4 (2008) 1797. P. Jain, T. Pradeep, Biotechnol. Bioeng. 90 (2005) 59. M. Dalby, D. Giannaras, M. Riehle, N. Gadegaard, S. Affrossman, A. Curtis, Biomaterials 25 (2004) 77. J.R. Morones, J.L. Elechiguerra, A. Camacho, K. Holt, J.B. Kouri, J.T. Ramírez, M.J. Yacaman, Nanotechnology 16 (2005) 2346. S.H. Hsu, H.J. Tseng, Y.C. Lin, Biomaterials 31 (2010) 6796. R. Foldbjerg, D.A. Dang, H. Autrup, Arch. Toxicol. (2010) (Epub ahead of print). M.M. Kemp, A. Kumar, S. Mousa, T.J. Park, P. Ajayan, N. Kubotera, S.A. Mousa, R.J. Linhardt, Biomacromolecules 10 (2009) 589.