G Model

ARTICLE IN PRESS

BIOMAC 5079 1–9

International Journal of Biological Macromolecules xxx (2015) xxx–xxx

Contents lists available at ScienceDirect

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

Nitric oxide-releasing chitosan film for enhanced antibacterial and in vivo wound-healing efficacy

1

2

Q1

3 4

Jong Oh Kim a,1 , Jin-Ki Noh b,1 , Raj Kumar Thapa a , Nurhasni Hasan b , Moonjeong Choi b , Jeong Hwan Kim a , Joon-Hee Lee b , Sae Kwang Ku c , Jin-Wook Yoo b,∗ a

College of Pharmacy, Yeungnam University, Gyeongsan 712-749, South Korea College of Pharmacy, Pusan National University, Busan 609-735, South Korea c College of Korean Medicine, Daegu Haany University, Gyeongsan 712-715, South Korea

5

b

6 7 8

9 21

a r t i c l e

i n f o

a b s t r a c t

10 11 12 13 14 15

Article history: Received 7 March 2015 Received in revised form 22 April 2015 Accepted 27 April 2015 Available online xxx

16

20

Keywords: Nitric oxide-releasing film Antibacterial activity Wound healing

22

1. Introduction

17 18 19

Nitric oxide (NO) is a promising therapeutic agent with antibacterial and wound-healing properties. However, the gaseous state and short half-life of NO necessitate a formulation that can control its storage and release. In this study, we developed NO-releasing films (CS/NO film) composed of chitosan (CS) and Snitrosoglutathione (GSNO) as a NO donor. Thermal analysis demonstrated molecular dispersion of GSNO in the films. In vitro release study revealed that NO release from CS/NO films followed Korsmeyer–Peppas model with Fickian diffusion kinetics. Moreover, the CS/NO film showed a stronger antibacterial activity against Pseudomonas aeruginosa (Gram-negative) and Staphylococcus aureus (Gram-positive) than the CS film. Further, the CS/NO film accelerated wound healing and epithelialization in a rat model of full-thickness wounds as compared to the CS film. Histopathological studies revealed that CS/NO films favorably enhanced the re-epithelialization and reconstruction of wounded skin. Therefore, our results suggest that CS/NO films could be a suitable formulation for treating full-thickness wounds. © 2015 Published by Elsevier B.V.

Q2 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38

Wound healing is a series of complex but well-coordinated processes including inflammation, cell proliferation, matrix deposition and tissue remodeling [1]. Progression from one phase of wound healing to the next is managed by various biological components such as cytokines, growth factors, and cellular elements, which are discharged coordinately into the wounds [2–5]. If any of the steps are not sufficiently completed, wound healing can be impaired, resulting in a chronic wound. Bacterial infection may also delay the process of wound healing. The two most common bacteria in wounds are Pseudomonas aeruginosa (Gram-negative) and Staphylococcus aureus (Gram-positive); these bacteria are easily sequestered at the surface of wounds and can access the underlying tissue [6]. Incomplete clearance of the bacteria from wounds can prolong the inflammatory phase, leading to chronic wounds. Bacteria are also known to form biofilms that make them more resistant to antibiotic treatment [7].

∗ Corresponding author. Tel.: +82 51 510 2807. E-mail address: [email protected] (J.-W. Yoo). 1 Both these authors contributed equally to this work.

Nitric oxide (NO), an endogenous bactericidal agent produced in large quantities by macrophages, exhibits antimicrobial properties against a broad spectrum of bacteria. NO has been reported to be capable of killing bacteria by direct or indirect oxidation via formation of peroxynitrite, the product of the reaction of NO with superoxide and by nitrosation of cysteine and tyrosine residues, which lead to dysfunction of bacterial proteins [8]. In addition to NO’s antimicrobial activity, there is an increasing evidence that NO plays a functional role in wound healing. Endogenous NO produced by inducible nitric oxide synthase (iNOS) in wounds is known to regulate collagen formation, cell proliferation and wound contraction [9]. Inhibition of iNOS by competitive inhibitors decreases collagen deposition, reduces the breaking strength of incisional wounds and impairs wound healing [10,11]. Studies have also demonstrated that NO-releasing formulations can improve incisional and excisional wound healing in rats [12–14]. These findings have facilitated the development of NO-releasing systems for wound healing. Several NO-releasing formulations have been developed to enhance wound healing. For example, NO-releasing poly(vinyl alcohol) hydrogel dressing improves dermal tissue regeneration in wound healing in diabetic mice [15]. Another NO-releasing hydrogel that combines the advantages of pluronic F-127 as a biologically inert polymer for controlling local NO release during prolonged time periods has also

http://dx.doi.org/10.1016/j.ijbiomac.2015.04.073 0141-8130/© 2015 Published by Elsevier B.V.

Please cite this article in press as: J.O. Kim, et al., Int. J. Biol. Macromol. (2015), http://dx.doi.org/10.1016/j.ijbiomac.2015.04.073

39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62

G Model BIOMAC 5079 1–9

J.O. Kim et al. / International Journal of Biological Macromolecules xxx (2015) xxx–xxx

2 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80

been reported [16]. Recently, an ointment containing NO-loaded zeolites was shown to exhibit faster wound healing in full-thickness cutaneous wounds in Zucker obese rats than that exhibited by a control ointment [17]. In the present study, we developed a NO-releasing film type dressing by using chitosan (CS) as a film-forming polymer and Snitrosoglutathione (GSNO) as a naturally occurring NO donor. CS is a well-known biopolymer with antimicrobial effects [18] and wound repair abilities [19]. GSNO has been identified as an endogenous NO donor and an enhancer of wound healing and antibacterial activity. GSNO decomposes by hydrolysis, producing NO and oxidized glutathione. A film-type dressing was chosen because it can provide protection of the wound, absorption of exudate and a moist environment as well as the stability of a NO donor during storage. We evaluated the physicochemical properties of NO-releasing film (CS/NO film) and determined its microbicidal activities against P. aeruginosa and S. aureus. The wound-healing efficacy of CS/NO film was also evaluated in a rat model of full-thickness wounds.

81

2. Materials and Methods

82

2.1. Materials

83 84 85 86 87 88 89 90 91 92

93

94 95 96 97 98 99 100 101 102 103

104

105 106 107 108 109 110 111 112 113 114 115 116

ARTICLE IN PRESS

Low-molecular-weight chitosan (viscosity, 20–200 cps; 85% acetylation), sodium nitrite, reduced l-glutathione and Griess reagent were purchased from Sigma-Aldrich (St. Louis, MO, USA). Lysogeny broth (LB) media were purchased from Daejung (Siheung, South Korea). Isopropyl ␤-d-1-thiogalactopyranoside (IPTG) was purchased from Duchefa-Biochemie (Haarlem, Netherlands). Zoletil 50® (tiletamine/zolazepam) was obtained from Virbac S.A. (Virbac, Carros, France) and Rompun® (xylazine hydrochloride) was obtained from Bayer (Bayer Korea, Korea). All other reagents and solvents were of analytical grade.

2.2. Synthesis of GSNO GSNO was synthesized as described previously [20]. Briefly, reduced l-glutathione was dissolved in 2 M HCl at 4 ◦ C to obtain a solution with a final concentration of 0.625 mM. Sodium nitrate was added and the mixture was placed in an ice bath under stirring for 30 min. The final solution was precipitated with cold acetone and stirred for another 10 min. The precipitate was collected by centrifugation and the pellet was washed once with 80% cold acetone, twice with 100% acetone, and thrice with cold diethyl ether. The pink solid GSNO was freeze-dried for 24 h and kept at −20 ◦ C until further use.

2.4. Characterization of the films 2.4.1. Film thickness The thickness of the films was measured using a digital outside micrometer (Bluebird Multinational Co.) at five different locations (center and four corners). The mean of the thickness measurements was considered as the film thickness. 2.4.2. Scanning electron microscopy (SEM) The surface morphology of the films was examined using a field emission scanning electron microscope (FE-SEM, S4800, Hitachi, Japan). Samples (1.5 × 1.5 cm2 ) were mounted on a double-sided carbon tape and coated with platinum for 2 min under vacuum. The samples were viewed under the FE-SEM at an acceleration voltage of 1–5 kV. 2.4.3. Loading efficiency and loading capacity After fabrication, the CS/NO films were cut, weighed and placed in 0.1 M cold HCl to prevent further decomposition of GSNO. The film samples were homogenized at 30.000 rpm for 1 min in an ice bath and kept at 4 ◦ C overnight to extract GSNO from the film. After centrifugation at 20.000 × g for 20 min, the clear supernatant was spectrophotometrically analyzed at a wavelength of 336 nm to determine the GSNO content in the film. Loading efficiency (LE) and loading capacity (LC) were calculated using the following formulas:

117

118 119 120 121 122

123 124 125 126 127 128 129

130 131 132 133 134 135 136 137 138 139

LE (%)

Amount of GSNO in the film × 100 Amount of initially loaded GSNO

140

LC (%)

Amount of GSNO in the film × 100 Total amount of chitosan and GSNO

141

2.4.4. Stability study Stability of GSNO in the CS/NO films was tested for 28 days at temperatures of −20, 4 and 25 ◦ C. Fifty milligram of CS/NO film (20 wt%) were placed in glass bottles in the presence of silica gel at different temperatures. All glass bottles were fully covered with aluminum foil to protect CS/NO films from light. At predetermined time intervals, the remaining amount of GSNO in the film was analyzed spectrophotometrically as described in the previous section. 2.4.5. Mechanical properties The CS and CS/NO films were cut into a specific dog bone shape (80 mm in length and 30 mm in width). Tensile strength (TS) and percentage elongation at break (E%) were measured using a tensile test machine (Instron 3345, Norwood, MA, USA). The tests were performed at a stretching rate of 10 mm/min and the thickness of the film was measured with a caliper just before the examinations. E% was calculated from the difference between the initial length of the sample (30 mm) and the extended length at the moment of breakage [21].

142 143 144 145 146 147 148 149

150 151 152 153 154 155 156 157 158 159

2.3. Preparation of NO-releasing chitosan (CS/NO) films Chitosan solution (1, w/v%, pH 4.4) was prepared by dissolving chitosan in 0.1 M acetate buffer (pH 4.4). Insoluble matters in the chitosan solution were removed by centrifugation at 20.000 × g for 30 min. Glycerol as a plasticizer was added to the chitosan solution at a final concentration of 1 wt%. Various amount of GSNO (2.5, 10 and 20 wt%) were added to 20 g of the chitosan solution. After removing air bubbles by sonication, the final solution was cast into a petri dish and dried at 37 ◦ C in an incubator (SI-300, Jeio Tech) in the dark for 2 days. A chitosan film (CS film) was prepared using the same procedure but without adding GSNO. The resulting films were stored at 4 ◦ C in a desiccator under dark conditions unless immediately used.

2.4.6. Thermal properties The thermal properties of GSNO, CS film and CS/NO film were examined using a differential scanning calorimeter (DSC, N-650, SCINCO, Korea). Each sample was heated in a completely sealed aluminum pan at a rate of 5 ◦ C/min from 30 to 200 ◦ C in a dynamic nitrogen atmosphere. 2.5. NO release study CS/NO films were cut into a square equivalent to 50 mg and immersed in 10 mL of phosphate-buffered saline (PBS, pH 7.4) at 37 ◦ C. NO released from the film was analyzed using Griess reagent. At predetermined time intervals, 100 ␮L of PBS was removed

Please cite this article in press as: J.O. Kim, et al., Int. J. Biol. Macromol. (2015), http://dx.doi.org/10.1016/j.ijbiomac.2015.04.073

160 161 162 163 164 165

166

167 168 169 170

G Model

ARTICLE IN PRESS

BIOMAC 5079 1–9

J.O. Kim et al. / International Journal of Biological Macromolecules xxx (2015) xxx–xxx 171 172 173 174 175 176 177 178 179 180 181

182

and replaced with fresh PBS. After an appropriate dilution, Griess reagent was added at a ratio of 1:1. The obtained mixtures (100 ␮L) were added to the wells of 96-well plates and placed for 30 min at room temperature in the dark. Finally, the amount of nitrite was measured using a microplate reader (iMark, Bio-Rad, Hercules, CA, USA) at a wavelength of 540 nm. Mathematical modeling was used to describe the kinetics of NO release from CS/NO films. The results were fitted to different models such as zero order, first order, Higuchi, and Korsmeyer–Peppas for comparison. Kinet DS 3 software was used to determine the R2 value for each of the kinetic models.

3

where W0 is the wound area at initial time 0, and Wt is the wound area at time t. Epithelialization rate(%) =

Et × 100 Wt + Et

where Et is the epithelialized area at time t, and Wt is the wound area at time t. Multiple comparison tests were performed to statistically clarify the differences between the groups. The acquired data were analyzed by one-way analysis of variance (ANOVA) followed by the least significant difference (LSD) method. Handling of all animals and the experimental procedures were approved by the Yeungnam University Animal Care and Use Committee.

184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209

231

232 233 234 235 236 237 238 239

2.6. Anti-bacterial activity assay 2.8. Histopathological studies

183

229 230

The strains of bacteria used for this study were P. aeruginosa PAO1 (wild-type prototroph) [22] and S. aureus RN4220 [23]. Both bacteria were cultured in lysogeny broth (LB) at 37 ◦ C for 15 h with vigorous shaking and grown to the mid-exponential phase (107 colony forming units, CFU/mL). The resulting bacterial suspension was centrifuged for 15 min at 8000 × g. The pellet was re-suspended in sterile DPBS and adjusted to an appropriate concentration. A total of 200 ␮L of the bacterial suspension (final concentration of 106 CFU/mL) was incubated with 1.8 mL of LB media. Then, a piece of the film (1.3 × 1.3 cm2 ) was placed in 12-well plates. A well without film was used as a control. All the samples were incubated at 37 ◦ C for 24 h in a shaking incubator, then centrifuged at 8000 × g and washed twice with 0.85% NaCl. After serial dilution, 200 ␮L aliquot of each dilution was plated on LB agar media and incubated at 37 ◦ C overnight. The number of colonies was enumerated, factoring in the number of viable bacteria at the time of plating. For the fluorescent images, a cover slip was placed on each well of a 12-well plate and 200 ␮L of bacteria-containing LB was inoculated on the cover slip, followed by 1 hour incubation at room temperature. Then, a piece of the film (1.3 × 1.3 cm2 ) was placed on the glass cover slip with fresh LB media was added. For P. aeruginosa, IPTG at a final concentration of 1 mM was added as a plasmid inducer. The plates were incubated at 37 ◦ C for a given time. After incubation, the glass cover slips were gently washed with sterile water to remove the non-adherent bacteria. Bacteria on the glass cover slips were observed under a fluorescence microscopy (Zeiss, Axioskop, Germany)

2.8.1. Histological process Samples were collected from full-thickness wound areas of the skin containing the dermis and hypodermis; these samples were trimmed into one or two parts per wound sample based on the granulation tissues, using central regions if possible. Each trimmed skin sample was fixed in 10% neutral-buffered formalin. After the samples were embedded in paraffin, 3–4-␮m-thick sections of tissues were prepared. Representative sections were stained with hematoxylin and eosin (H&E) for examination by light microscopy or Masson’s trichrome stain for detection of collagen fibers [24,27]. Thereafter, individual skin samples were observed under a light microscope (E400, Nikon, Japan) for a histological analysis. 2.8.2. Histomorphometry To identify more detailed histopathological changes, desquamated epithelial regions (mm), number of microvessels in granulation tissues (vessels/mm2 of field), number of infiltrated inflammatory cells in granulation tissues (cells/mm2 of field), percentages of collagen-occupied region in granulation tissues (%/mm2 of field) and granulation tissue areas (mm2 /cross trimmed central regions of wounds) were measured on the prepared cross-trimmed individual histological skin samples by using a computer-based automated digital image analyzer (iSolution FL ver 9.1, IMT isolution Inc., Quebec, Canada), according to previously published methods [24–26]. Eight wounds in each group were considered for further analysis in this experiment. In addition, re-epithelialization rates were also calculated as follows:

240

241 242 243 244 245 246 247 248 249 250 251 252

253 254 255 256 257 258 259 260 261 262 263 264 265 266 267

210

211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227

228

2.7. In vivo wound-healing assay To evaluate the in vivo wound-healing efficacy of the CS/NO film, male Sprague–Dawley rats (Orient Bio, Seongnam, Korea) weighing 250–280 g were chosen as an animal model. Prior to the development of wounds on the dorsal area, the rats were anesthetized using Zoletil 50 (tiletamine/zolazepam) and Rompun (xylazine hydrochloride) at a ratio of 5:2 [24]. Dorsal hair was removed with an electric razor. Subsequently, back skin was excised to create full-thickness wounds (1.5 cm × 1.5 cm). Each wound was covered with sterile gauze (control), CS film, or CS/NO film. All materials were covered and fixed with an elastic adhesive tape (Micropore, 3 M Consumer Health Care, St Paul, MN, USA). Every dressing on wound lesions was replaced with a new dressing at proper times. All rodents were cared for in separate cages, and digital images of the lesions were collected every 3 days by using a digital camera to detect macroscopic changes in the wounds. Using Adobe Acrobat 9 Professional [24–26], wound size reduction and epithelialization rates were determined and calculated as follows. Wound size reduction(%) =

W0 − Wt × 100 W0

Re-epithelization (%) Total length of wound (mm) − desquamated epithelial region (mm) = Total length of wound (mm)

268

269 270

2.9. Statistical analysis Multiple comparison tests for different dose groups were conducted. Variance homogeneity was examined using the Levene test [28]. If the Levene test indicated no significant deviations from variance homogeneity, the obtained data were analyzed by one-way ANOVA followed by LSD multiple comparison tests to determine which pairs of group comparisons were significantly different. In cases of significant deviations from variance homogeneity in the Levene test, nonparametric Kruskal–Wallis H-tests were conducted. When a significant difference was observed in the Kruskal–Wallis H-test, Mann–Whitney U (MW)-tests were conducted to determine the significance of differences between specific pairs of groups. Statistical analyses were conducted using SPSS for Windows (Release 14.0, SPSS Inc., USA) [29]. In addition, the changes between the gauze control group and groups treated

Please cite this article in press as: J.O. Kim, et al., Int. J. Biol. Macromol. (2015), http://dx.doi.org/10.1016/j.ijbiomac.2015.04.073

271

272 273 274 275 276 277 278 279 280 281 282 283 284 285

G Model

ARTICLE IN PRESS

BIOMAC 5079 1–9

J.O. Kim et al. / International Journal of Biological Macromolecules xxx (2015) xxx–xxx

4

Fig. 1. Surface morphology of the (a) CS film and (b) CS/NO film (20 wt%) determined by SEM analysis.

287

with test materials were assessed to determine the efficacy of test materials as follows:

288

Percentage changes from gauze control (%)

286

289

Data of tested group − Data of gauze control = × 100 Data of gauze control

290

291

3. Results and Discussion

292

3.1. Preparation and characterization of CS/NO films

323

CS/NO films were prepared by the solvent evaporation method. Since GSNO undergoes thermal decomposition, the films were dried at a low temperature (30 ◦ C) in an incubator with a dehumidifier to accelerate solvent evaporation. With conventional methods, in which the films were dried in a dry oven without dehumidification, it took over 3 days for drying at 37 ◦ C, and the loading efficiency of GSNO was less than 20% in the final films; this could be attributed to GSNO’s thermal decomposition, reducing the NO concentration in the films [30]. With the aim of maximizing the loading efficiency of GSNO in the films, drying was performed at a low temperature (30 ◦ C) with a dehumidifier, which resulted in faster evaporation of the solvent, thus improving the preservation of GSNO in the film during the drying process. Table 1 shows the characterization of CS/NO films with different loading doses. CS/NO films with up to 20 wt% loading exhibited a reddish, transparent, homogenous surface. Films loaded with over 20 wt% of GSNO occasionally exhibited a cloudy surface and phase separation. The quick decomposition of GSNO with NO release in the form of bubbles may have led to this result [20]. As presented in Table 1, the decreased loading efficiency of GSNO, concurrent with the increase in total GSNO weight could be attributed to the concentration-dependent decomposition of GSNO owing to the cage effect (greater decomposition at higher concentrations) [20,31] explained that this phenomenon was a result of autocatalytic effects of the reaction between the GSNO molecule and RS· radicals, leading to concentration-dependent decomposition of GSNO [31]. Although there was a reduction in the loading efficiency of GSNO with the increase in GSNO concentration, the loading capacity was increased to around 8.6% for 20 wt% GSNO. These results suggested that the maximum loading capacity of GSNO in the CS films was achieved with 20 wt% GSNO loading.

324

3.2. Characterization of the films

293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322

325 326 327 328 329 330

3.2.1. Thickness and surface morphology The loading dose of GSNO directly or indirectly influenced the morphology and thickness of the CS/NO film surface. As presented in Table 1, the mean thickness of the CS film was directly affected by the amount of polymer in the film. When the loading dose of GSNO was lowered (yielding a higher amount of chitosan), the thickness

of the film increased; the opposite effect was observed when the loading dose of GSNO was increased, suggesting that the film thickness depended on the amount of chitosan rather than the amount of GSNO in the film. SEM was used to evaluate the potential effects of GSNO (20 wt% loading dose) incorporation on the surface morphology of the films. As shown in Fig. 1, CS and CS/NO films had a homogenous, smooth surface morphology, indicating that the incorporation of GSNO did not alter the surface morphology of the CS film.

3.2.2. Mechanical properties Films for wound treatment should not only be durable to endure physical stresses but also be elastic for ease of application [32–34]. To investigate the influence of GSNO on the mechanical properties of the films, the tensile strength, elongation at break, and Young’s modulus were determined. As shown in Table 2, the elongation at break was not significantly affected by the addition of GSNO. There was a slight reduction in the stretchability of the CS film when GSNO was added to the film. Moreover, addition of GSNO resulted in the reduction of tensile strength and Young’s modulus of CS films. Thus, our data verified that addition of GSNO may decrease the mechanical strength of the film, leading to formation of films that are less stiff and more flexible. This may be explained by the formation of pores inside the films through decomposition of GSNO during the drying procedure [35]. Although the incorporation of GSNO resulted in a reduction in the mechanical strength of the film, the CS/NO films retained good mechanical properties, supporting the use of these films in wound treatment models. Notably, the CS/NO films with 2.5 and 10 wt% GSNO showed mechanical properties similar to those of the CS/NO film with 20 wt% GSNO (data not shown).

3.2.3. Water absorption Water uptake of the film is an important property in skin tissue engineering applications and is essential for maintaining a moist environment and removing excess exudates from wounds [36]. Table 2 presents the swelling ability of the CS and CS/NO films. Higher swelling ability (120%) was observed for the CS film; this may be explained by the observation that the chitosan structure contains many -NH2 and -OH groups, which increase chitosan’s affinity for water [37]. With the addition of GSNO in the film, the swelling ability decreased to about 104%. The observed reduction in water uptake in the CS/NO films would be due to the reduced amount of chitosan in the film because of the addition of GSNO. In another reason, chitosan’s affinity for water may have been affected by the lipophilic nature of NO [38]. Although there was a reduction in the water uptake ability of the CS/NO film, the increase in water uptake capacity to over 100% indicated that the CS/NO films could be used for exudative wounds that develop following bacterial infection.

Please cite this article in press as: J.O. Kim, et al., Int. J. Biol. Macromol. (2015), http://dx.doi.org/10.1016/j.ijbiomac.2015.04.073

331 332 333 334 335 336 337 338 339

340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360

361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378

G Model

ARTICLE IN PRESS

BIOMAC 5079 1–9

J.O. Kim et al. / International Journal of Biological Macromolecules xxx (2015) xxx–xxx

5

Table 1 Characterization of CS/NO films prepared with different loading doses. Films CS film CS/NO film

Thickness (␮m) 0 wt% 2.5 wt% 10 wt% 20 wt%

74 72 68 64

± ± ± ±

5 4 4 3

Loading efficiency (%)

Loading capacity (%)

Physical characteristics

– 48.4 ± 2.9 48.2 ± 2.4 43.0 ± 3.3

– 1.2 ± 0.1 4.8 ± 0.1 8.6 ± 0.2

Transparent and homogenous surface Reddish, transparent and homogenous surface

Values are expressed as mean ± SD (n = 3). Table 2 Mechanical properties of the films.

CS film CS/NO film (20 wt%)

Tensile strength (MPa)

Elongation at break (%)

Young’s modulus (MPa)

Water uptake (%)

7.87 ± 1.02 6.53 ± 0.70

134.4 ± 11.5 129.9 ± 18.1

7.18 ± 0.53 5.48 ± 0.31#

120 ± 4.2 104 ± 5.7

Values are expressed as mean ± SD (n = 3). # p < 0.05 as compared with the CS film.

Fig. 2. Thermal properties of the GSNO powder, CS film and CS/NO film determined by DSC analysis.

379 380 381 382 383 384 385 386 387 388

389 390 391 392 393 394 395 396 397 398 399 400 401 402

3.2.4. Thermal properties Next, the thermal properties of the CS/NO films were evaluated by DSC using the melting points of GSNO. As shown in Fig. 2, GSNO exhibited a strong endothermic peak at 195 ◦ C and chitosan exhibited a weaker endothermic peak compared to that of GSNO. However, the peak for the melting point of GSNO disappeared in CS/NO film, indicating that GSNO fully dissolved and molecularly dispersed in the film. A broad endothermic peak was observed around 100 ◦ C for both films; this could be attributed to the evaporation of absorbed moisture from the films [20]. 3.2.5. Stability of GSNO in the films The stability of GSNO in the CS/NO film was evaluated under different storage temperatures (−20, 4 and 25 ◦ C) for 4 weeks (Fig. 3). The results revealed that GSNO in the films remained almost intact when stored at temperatures of −20 and 4 ◦ C for 4 weeks. On the other hand, a reduction in the level of GSNO, up to ∼25%, was observed when the films were stored at room temperature. At low temperatures and low kinetic energy (−20 and 4 ◦ C), the process of disulfide bond formation slows owing to the low flexibility of polypeptide chains; hence, significantly less decomposition of GSNO occurs at lower temperatures compared to that at room temperature [30]. Considering that most of NO donors including GSNO decompose by hydrolysis in the presence of water, these results suggest that a film type dressing has an advantage over other

Fig. 3. Percentage GSNO remaining in the CS/NO film (20 wt%) at different storage temperatures. Values are expressed as means ± SDs (n = 3).

water-containing formulations such as hydrogel in maintaining stability of NO donors for a long-term storage. 3.3. In vitro NO release study NO release profiles from the CS/NO films (with different loading doses of GSNO) are presented in Fig. 4, which shows patterns of spontaneous decomposition of GSNO into NO. In the initial hours, the release of NO from the GS/NO film was slower. The release rate then increased dramatically over time; this effect can be attributed to the time required for hydration of the film. Since the CS/NO films need to swell for the release of NO, the lag time during the initial hours of NO release was expected. Among the films loaded with different doses of GSNO, the CS/NO film containing 20 wt% GSNO released the highest amount of NO in a sustained manner for over 48 h, presuming that the CS/NO film is able to release a sufficient amount of NO during the duration of wound treatment. Therefore, CS/NO films containing 20 wt% GSNO was selected for further studies. The NO release profile showed a dose-dependent pattern of drug release. Next, mathematical modeling was used to determine the kinetics of NO release from CS/NO films. The results of mathematical modeling, fitted to different models (i.e., zero order, first order, Higuchi, and Korsmeyer–Peppas), for three CS/NO films are presented in Table 3. All the films were found to follow the Korsmeyer–Peppas model for NO release based on higher R2

Please cite this article in press as: J.O. Kim, et al., Int. J. Biol. Macromol. (2015), http://dx.doi.org/10.1016/j.ijbiomac.2015.04.073

403 404

405

406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426

G Model

ARTICLE IN PRESS

BIOMAC 5079 1–9

J.O. Kim et al. / International Journal of Biological Macromolecules xxx (2015) xxx–xxx

6

values. The n values in all the cases were close to 0.5, suggesting that drug release from the CS/NO films followed Fickian diffusion [27]. 3.4. Anti-bacterial efficacy

Fig. 4. NO release profiles of CS/NO films with different GSNO loading doses. Values are expressed as means ± SDs (n = 3).

Table 3 Release kinetics model for in vitro NO release. GSNO loading

2.5 wt% 10 wt% 20 wt%

Zero order

First order

Higuchi

Korsmeyer–Peppas

R2

R2

R2

R2

n

0.6990 0.7827 0.7120

0.5118 0.5291 0.4362

0.8516 0.9196 0.8445

0.9499 0.9376 0.8936

0.526 0.534 0.701

The antibacterial activity of the CS film and CS/NO film against P. aeruginosa and S. aureus was evaluated. Untreated bacteria were used as a control. As shown in Fig. 5, when treated with the CS film, there was a noticeable reduction in the levels of bacterial viability confirmed by fluorescence microscopy and macroscopic images of bacterial density in LB media for both strains. Importantly, significantly more prominent reduction in viability of both strains was observed with the CS/NO film than the CS film, clearly indicating that NO is a key component of bactericidal activity against P. aeruginosa and S. aureus. These results were confirmed by quantitatively assess the ability of CS/NO film to reduce bacterial viability by 2 logs, representing ∼99% killing (Fig. 5C and F). The proposed mechanisms for the antibacterial activity of chitosan in both Gramnegative and Gram-positive bacteria are as follows: (i) cell wall leakage caused by ionic surface interaction, (ii) mRNA and protein synthesis inhibition caused by penetration into bacterial nuclei and (iii) external barrier formation preventing the passage of metals and nutrients into bacterial cells [39]. The results for CS/NO films were consistent with those of the in vitro release study, which showed that the cumulative NO release increased with time, leading to an increase in antibacterial activity over time. NO and other reactive nitrogen intermediates have been found to exert antibacterial effects by modifying DNA, proteins, and lipids of bacteria. The possible mechanisms mediating these effects include deamination of

Fig. 5. Antibacterial activities of the CS film and CS/NO film (20 wt%) against P. aeruginosa (A–C) and S. aureus (D–E). Fluorescence microscopy images of bacteria grown on a cover slip (A, D), macroscopic images of bacterial density in LB media (B, E) and the bacterial viability (C, F). Values are expressed as means ± SDs (n = 3).

Please cite this article in press as: J.O. Kim, et al., Int. J. Biol. Macromol. (2015), http://dx.doi.org/10.1016/j.ijbiomac.2015.04.073

427 428 429

430

431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454

G Model

ARTICLE IN PRESS

BIOMAC 5079 1–9

J.O. Kim et al. / International Journal of Biological Macromolecules xxx (2015) xxx–xxx

7

Fig. 6. Representative photographs of wounds treated with the gauze control, CS film and CS/NO film for 15 days.

472

bacterial DNA, oxidation of proteins, and inhibition of metabolic enzymes in bacteria [40,41]. These results suggested that the CS/NO films exerted strong antibacterial activity against a broad spectrum of bacteria (including both Gram-positive and Gram-negative bacteria). The different processes involved in wound healing, from inflammation to remodeling, may be affected by the bacteria that are present at the surface of the wound and within the deep tissues of wounds. While bacteria may be responsible for acceleration of wound healing by increasing granulation tissue formation, angiogenesis, and tensile strength of the wound, uncontrolled colonization may inhibit wound healing from the angiogenesis process to re-epithelialization [42]. Therefore, minimization of colonized bacteria may play a vital role in efficient wound healing. Hence, the combined use of CS (as a film-forming agent) and NO (as an active pharmaceutical agent) may have synergistic effects on the total antibacterial activity. Therefore, CS/NO films may be used to control bacterial growth in the wound area, leading to enhanced wound healing.

473

3.5. In vivo wound healing

455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471

474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497

Finally, the wound-healing abilities of CS and CS/NO films were evaluated in a rat model with full-thickness wounds. Estimation of the wound healing effects of the films, which were expected to accelerate wound repair, was performed following application of the CS and CS/NO films to the wounded area of the rat dorsum. The macroscopic appearances of the wounds treated with sterile gauze, CS film, and CS/NO film at different days after the operation are presented in Fig. 6. Each wound was observed for a period of 3, 6, 9, 12 and 15 days after the operation. All of the rats used in the study survived throughout the experimental period until sacrifice and there was no evidence of necrosis. Severe inflammation in the wound area was observed in each group of rats on postoperative day (POD) 3. High levels of hemorrhage, along with arid surfaces and scabs, were observed in wounds treated with sterile gauze only. In contrast, wounds treated with the films exhibited moist surfaces, supporting the ability of the CS film and CS/NO film to retain water. Compared to wounds treated with the CS film or gauze only, those treated with CS/NO films showed faster closure. Thus, these data supported that NO plays an important role in the wound healing process, as has been observed in previous studies showing that NO accelerates the growth and differentiation of epidermal keratinocytes [43]. On POD 6, increased bleeding was observed in the gauze control group during replacement of the materials on the wounds because of the dryness of

Fig. 7. (A) Size reduction (%) profiles and (B) epithelialization rate (%) profiles of fullthickness wounds treated with the gauze control (䊉), CS film (), and CS/NO film (). Values are expressed as means ± SDs (n = 8). a p < 0.01 and b p < 0.05 compared with the gauze control group. c p < 0.01 and d p < 0.05 compared with the CS film-treated group.

Please cite this article in press as: J.O. Kim, et al., Int. J. Biol. Macromol. (2015), http://dx.doi.org/10.1016/j.ijbiomac.2015.04.073

G Model BIOMAC 5079 1–9

ARTICLE IN PRESS J.O. Kim et al. / International Journal of Biological Macromolecules xxx (2015) xxx–xxx

8 Table 4 Histomorphometrical changes in this study. Histomorphometry Desquamated epithelium regions (mm) Re-epithelization (%) In granulation tissues Microvessels numbers Infiltrated inflammatory cell numbers Collagen occupied regions (%) Granulation tissue areas (mm2 )

Gauze control 2.93 ± 0.68 70.66 ± 6.79 154.24 441.13 34.32 6.39

± ± ± ±

28.36 122.71 8.88 1.58

CS film

CS/NO film

1.44 ± 0.22c 85.58 ± 2.19c 66.38 139.00 49.55 4.25

± ± ± ±

16.15a 21.53c 7.19a 0.55c

0.61 ± 0.10ce 93.91 ± 0.98ce 30.25 79.75 62.34 2.46

± ± ± ±

11.72ab 14.27ce 6.41ab 0.46ce

Values are expressed as mean ± SD of eight rat wounds. a p < 0.01 as compared with gauze control by the LSD test. b p < 0.01 as compared with CS film by the LSD test. c p < 0.01 and d p < 0.05 as compared with gauze control by the MW test. e p < 0.01 as compared with the CS film by the MW test.

498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519

the wound surface when treated with gauze only. From POD 9, the majority of the wounds began to heal. After POD 12, considerable closure of wounds was observed in the groups treated with the CS and CS/NO films compared to that observed in the gauze control group. Fig. 7A shows the percent size reduction of wounds in different groups treated with gauze, CS films, and CS/NO films. From POD 6, wounds in groups treated with CS and CS/NO films showed significantly reduced wound sizes compared to those in the gauze control group. From POD 9, the wound size in the rats treated with the CS/NO film significantly decreased compared to that in the rats treated with the CS film or gauze. At POD 12, the wounds in the rats treated with the CS/NO were almost closed (with significant differences in wound sizes compared to those in the control [p < 0.01] and the CS film group [p < 0.05]), thus indicating the beneficial effects of NO in wound-healing process [43]. There was a significant reduction in wound size in the rats treated with CS films compared to those in the rats treated with gauze only on POD 12. This can be attributed to acceleration of wound-healing activity by chitosan, as reported [41]. Wounds in the rats treated with the CS/NO film were almost completely healed at the end of POD 15, supporting the possible applicability of CS/NO films for the treatment of full-thickness

wounds. Fig. 7B shows the epithelialization rates in all three groups. On POD 3, film-treated wounds exhibited higher rates of epithelialization than gauze-covered wounds. On POD 6–12, wounds treated with the CS/NO film displayed significantly higher epithelialization rates (p < 0.01 or