G Model
ARTICLE IN PRESS
BIOMAC 4350 1–5
International Journal of Biological Macromolecules xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Synthesis and characterization of antimicrobial crosslinked carboxymethyl chitosan nanoparticles loaded with silver
1
2
3 4
Q1
Riham R. Mohamed ∗ , Magdy W. Sabaa Department of Chemistry, Faculty of Science, Cairo University, Giza 12613, Egypt
5
6 19
a r t i c l e
i n f o
a b s t r a c t
7 8 9 10 11 12
Article history: Received 7 March 2014 Received in revised form 30 April 2014 Accepted 7 May 2014 Available online xxx
13
18
Keywords: Hydrogels Silver Carboxymethyl chitosan Antimicrobial activity
20
1. Introduction
14 15 16 17
21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42
Carboxymethyl chitosan (CMCh)–silver nanoparticle (Ag) hydrogels with high antibacterial activity against three Gram +ve bacteria (Staphylococcus aureus, Bacillus subtilis and Streptococcus faecalis), three Gram −ve bacteria (Escherichia coli, Pseudomonas aeruginosa and Neisseria gonorrhoeae) and a Candida albicans fungus were prepared. The in situ preparation reaction involved crosslinking of CMCh with epichlorohydrin in alkaline medium containing silver nitrate to yield silver nanoparticles loaded CMCh hydrogel giving pale brown or darker hydrogels when the silver content increases. FTIR spectroscopy, SEM and TEM were done for the prepared hydrogels. Silver nanoparticles hydrogels exhibited higher antimicrobial activity than virgin CMCh. TEM analysis showed the small size of the prepared hydrogels to be in the range of 9–16 nm in size. © 2014 Published by Elsevier B.V.
Hydrogels are an important class of polymeric materials that have been utilized in a wide variety of biomedical and pharmaceutical applications. Hydrogels are three dimensional, hydrophilic, polymeric networks that can absorb up to thousands of times their dry weight in water or biological fluids [1,2]. They consist of polymeric chains with either physical or chemical cross-links that prevent dissolution of the hydrogel structure and instead result in swelling of the material upon interaction with aqueous solutions. Hydrogel nanocomposites involve the incorporation of various nanoparticulate materials within a versatile hydrogel matrix which can provide easy, straight forward methods for enhancing the properties of hydrogels. The use of silver and other metal ions for their sustained antifungal, antibacterial and antiviral effects have been practiced for a long time. Silver ion has been known to be effective against a broad range of microorganisms. Their synthesis and highly effective observed antibacterial activity make them a very attractive form of silver administration. Nanocomposite hydrogels have been shown to modify and improve a variety of material properties of the polymer through the addition of conducting particles such as carbon nanotubes (CNTs), clay, ceramics, magnetic nanoparticles, hydroxyapatite (HA), and semiconducting nanoparticles [3].
∗ Corresponding author. E-mail address: [email protected] (R.R. Mohamed).
Metallic nanoparticles are toxic to microorganisms. Silver nanoparticles exhibit antimicrobial effects by binding to microbial DNA, preventing bacterial replication, and also causing inactivation of bacteria function [4,5]. Silver nanoparticles with higher surface to volume ratio compared to common metallic silver have shown better antimicrobial activity. Due to unique biological properties of silver nanoparticles such as biocompatibility and antibacterial affinity they have been applied for various medical purposes such as implants, catheters, and healing of wounds. Different methods of incorporating silver nanoparticles in hydrogels have been reported in the literature. Nanocomposites can be synthesized by incorporation of particles prior to polymerization, during polymerization, or after polymerization of hydrogels [6]. Chitosan, an amino polysaccharide, is composed of ,1-4 glucosidic bonds. Due to its unique polycationic nature, chitosan and its derivatives have been proposed for various applications in biomedical, food, agricultural, biotechnological and pharmaceutical fields [7]. The antibacterial activity of chitosan and its antibacterial mechanism have been researched extensively. Due to the low solubility of chitosan in water, its hydrogels do not show high binding water capacities. Carboxymethyl chitosan, an amphoteric material with high hydrophilic characteristics. It is a very important chitosan derivative showing very good water solubility. Recently, hydrogels with high water binding capacity have been successfully produced from carboxymethyl chitosan by its crosslinking in aqueous media, followed by drying of the resultant gel [8].
http://dx.doi.org/10.1016/j.ijbiomac.2014.05.025 0141-8130/© 2014 Published by Elsevier B.V.
Please cite this article in press as: http://dx.doi.org/10.1016/j.ijbiomac.2014.05.025
R.R.
Mohamed,
M.W.
Sabaa,
Int.
J.
Biol.
Macromol.
(2014),
43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70
G Model
ARTICLE IN PRESS
BIOMAC 4350 1–5
R.R. Mohamed, M.W. Sabaa / International Journal of Biological Macromolecules xxx (2014) xxx–xxx
2 71
2. Materials and methods
72
2.1. Materials
76
Chitosan (code KB-002) was purchased from Funakoshi Co. LTD, Japan. Deacetylation content = 88.2%, epichlorohydrin (ECH) (98%), sodium hydroxide and silver nitrate of laboratory grade chemicals were used.
77
2.2. Methods
73 74 75
78 79 80 81 82 83 84 85 86 87 88 89 90
91 92 93 94 95 96 97 98
2.2.1. Preparation of carboxymethyl chitosan (CMCh) Carboxymethyl chitosan was prepared following the method reported previously [9] where chitosan (10 g), sodium hydroxide (13.5 g) and solvent isopropanol (100 ml) were suspended in a flask to swell and alkalize at room temperature for 1 h. The monochloroacetic acid (15 g) was dissolved in isopropanol, and added to the reaction mixture dropwisely within 30 min and reacted for 4 h at 55 ◦ C. Then the reaction was stopped and isopropanol was discarded. Ethyl alcohol (80%) was added and the solid product was filtered and rinsed with 80–90% ethyl alcohol to desalt and dewater, and vacuum dried at 50 ◦ C. The degree of substitution of carboxymethyl chitosan was determined by pH-titration [10] and found to be 0.75. 2.2.2. Preparation of crosslinked CMCh hydrogels Definite amount of CMCh was dissolved in 1% NaOH solution with continuous mechanical stirring until a homogeneous viscous mixture was obtained [11]. A definite concentration of epichlorohydrin (10% based on weight of CMCh) was added dropwisely with continuous stirring. The formed paste, was transferred to Petri dish, dried in an oven at 80 ◦ C for 5 min then cured for 3–7 min at different temperature (120–140 ◦ C).
2.2.3. Loading of CMCh hydrogels with silver nanoparticles (in situ process) 100 Definite amount of CMCh was dissolved is 1% NaOH Solu101 tion with continuous mechanical stirring until a homogeneous 102 viscous mixture was obtained, then epichlorohydrin was added 103 dropwisely. Silver nitrate solution was then added in different con104 centration (1–3%). 105 The prepared hydrogels were pale brown in color; darker hydro106 gels were obtained when larger amounts of Ag were added. The 107 paste was dried in an oven at 80 ◦ C for 15 min, and then cured at 108 Q2 130 ◦ C [11] (Scheme 1). 109 99
110
3. Characterization and analysis
111
3.1. FTIR spectroscopy
114
FTIR spectra were recorded using KBr discs on Testcan Shimadzu IR-Spectrometer (FTIR model 8000) at room temperature within the wave number range of 4000–400 cm−1 .
115
3.2. Scanning electronic microscopy
112 113
aqueous particles dispersed in 5 mL acetone to become slightly turbid solution onto the copper grid and allowing it to dry well. The images of representative areas were captured at suitable magnifications which clarify the morphology and the size of the nanoparticles.
3.4. Antibacterial activity
119
The dry sample, spread on a double sided conducting adhesive tape, pasted on a metallic stub, was coated (100 ) with gold in an ion sputter coating unit (JEOL S150A) for 2 min and observed in a JEOL-JXA-840A Electron probe microanalyzer at 20 kV.
120
3.3. Transmission electron microscopy (TEM)
117 118
121 122 123
Micrographs of the colloidal nanogel particles were taken using a JEM-100S Transmission Electron Microscope (TEM, Japan). The TEM sample was prepared by mixing one dilute drop of prepared Please cite this article in press as: http://dx.doi.org/10.1016/j.ijbiomac.2014.05.025
R.R.
Antibacterial activity of the investigated hydrogels was evaluated using a modified Kirby–Bauer disc diffusion method [12]. Briefly, 100 L of the test bacteria were grown in 10 mL of fresh media until they reached a count of approximately 108 cells/mL for bacteria [13]. 100 L of microbial suspension was spread onto agar plates corresponding to the broth in which they were maintained. Isolated colonies of each organism that might be playing a pathogenic role should be selected from primary agar plates and tested for susceptibility by disc diffusion method [14,15]. Of the many media available, National Committee of Clinical Laboratory Standards (NCCLS) recommends Mueller–Hinton agar due to its results in good batch-to-batch reproducibility. Antibacterial activity of the prepared hydrogels was carried out against three Gram +ve bacteria (S. aureus, B. subtilis and S. faecalis), three Gram −ve bacteria (E. coli, P. aeruginosa and N. gonorrhoeae) and a Candida albicans fungus. Plates are inoculated with bacteria at 35–37 ◦ C for 24–48 h then the diameters of the inhibition zones were measured in millimeters [12]. Standard discs of Tetracycline and Ampicillin (Antibacterial agents) served as positive controls for antibacterial activity but filter discs impregnated with 10 L of solvent (distilled water, chloroform, DMSO) were used as a negative control. The agar used is Meuller–Hinton agar that is rigorously tested for composition and pH. Further the depth of the agar in the plate is a factor to be considered in the disc diffusion method. This method is well documented and standard zones of inhibition have been determined for susceptible and resistant values. Blank paper discs (Schleicher and Schuell, Spain) with a diameter of 8.0 mm were impregnated with 10 of tested concentration of the stock solutions. When a filter paper disc – impregnated with a tested chemical – is placed on Agar, the chemical will diffuse from the disc into the agar. This diffusion will place the chemical on the agar only around the disc. The solubility of the chemical and its molecular size will determine the size of the area of chemical in filtration around the disc. If an organism is placed on the agar; it will not grow around the disc if it is susceptible to the chemical. This area of no growth around the disc is known as a “zone of inhibition” or “clear zone”. For the disc diffusion, the zone diameters were measured with slipping calipers of the NCCLS [14]. Agar-based methods such as Etest and disc diffusion can be good alternatives because they are simpler and faster than broth-based methods [16,17].
M.W.
Sabaa,
126 127 128
130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171
172
The sterilized filter paper discs impregnated with a solution of the test compound in DMSO (1 mg/ml) were placed on nutrient agar plate seeded with the appropriate test organism in triplicates. Standard conditions of 104 CFU/mL (colony forming U/mL)were used for antifungal assay, respectively. Petri dishes (9 cm in diameter) were used and the discs of filter paper were inoculated in each plate. The utilized test organisms were C. albicans as fungi. Amphotericin B were used as reference drug against fungi. DMSO alone was used as control at the same abovementioned concentration and during this, there was no visible change in bacterial growth. The plates were incubated at 37 ◦ C for 48 h for fungi.
Mohamed,
125
129
3.5. Antifungal activity
116
124
Int.
J.
Biol.
Macromol.
(2014),
173 174 175 176 177 178 179 180 181 182 183
G Model
ARTICLE IN PRESS
BIOMAC 4350 1–5
R.R. Mohamed, M.W. Sabaa / International Journal of Biological Macromolecules xxx (2014) xxx–xxx
3
Scheme 1. Crosslinking reaction of CMCh with epichlorohydrin in alkaline medium.
4.2. FTIR spectroscopy
197
Fig. 1 represents FTIR spectra of (a) the CMCh and (b) the crosslinked CMCh hydrogel with epichlorohydrin. The bands at 1422, 1608, 2931, and 3420 cm−1 are assigned to the stretching vibration of COO (symmetric), COO (asymmetric),CH (aliphatic), and O H, respectively. The bands observed at 1608 and 1422 cm−1 in the spectrum of crosslinked CMCh hydrogels with epichlorohydrin indicate that the carboxyl groups of CMCh exist in the hydrogels after crosslinking. It can also be seen from the spectrum of crosslinked hydrogel some typical peaks appear at 1327, 1262, and 1064 cm−1 . The peaks at 1437 and 1155 cm−1 belong to stretching vibration of C O C and C C stretching vibration respectively, of the reacted epichlorohydrin with CMCh, whereas the band appeared at 1065 cm−1 is characteristic for bending vibration of OH group.
4.3. Scanning electron microscopy (SEM)
Fig. 1. FTIR spectra of (a) CMCh (b) the crosslinked CMCh hydrogel with epichlorohydrin.
4. Results and discussion
185
4.1. In situ formation of silver nanoparticles
186 187 188 189 190 191 192 193 194 195 196
Nanoparticles can be incorporated into the hydrogel matrix by mixing the silver with the preformed hydrogel, by adding the AgNO3 salt during the formation of the hydrogel or during the swelling of the hydrogel [18]. In this research, the formation of silver nanoparticles inside the chains of CMCh was performed during the hydrogel formation. Our approach depends on the dual function of CMCh as both a reducing and a stabilizing agent for silver nanoparticles during the formation of CMCh hydrogel containing nanoparticles. The negatively charged carboxyl groups in CMCh attract the positively charged silver cations.
Please cite this article in press as: http://dx.doi.org/10.1016/j.ijbiomac.2014.05.025
R.R.
Scanning electron microscopy images of CMCh and crosslinked CMCh hydrogel containing silver nanoparticles prepared by in situ technique are shown in Fig. 2. CMCh has a surface full of lumps as bulky COOH groups are formed on the surface. On the other hand, in the hydrogel CMCh–silver nanoparticles, the silver nanoparticles are clearly visible as spherical particles onto the surface of CMCh in Fig. 2. The prepared hydrogels were pale brown in color, darker hydrogels were obtained when larger amounts of Ag nanoparticles were reacted with the CMCh.
M.W.
Sabaa,
Int.
J.
Biol.
200 201 202 203 204 205 206 207 208 209 210 211
Macromol.
213 214 215 216 217 218 219 220 221 222
223
TEM images, done for CMCh–Ag nanoparticles prepared by in situ technique, were shown in Fig. 3 to demonstrate the shape of silver nanoparticles within the hydrogel network. It reveals Ag nanoparticles in the composites are dispersed in the CMCh matrix. The nanoparticles size range from 9–16 nm via the in situ preparation technique. It is also obvious that, the nanoparticles do not form aggregates showing that the particle size of the Ag nanoparticles in the CMCh–Ag composites was controlled. This may be due to the excellent stabilization of silver nanoparticles by carboxylate anions present in the CMCh chains. The increased antimicrobial activity of the Ag nanoparticles could be attributed to their smaller size due to the fact that smaller particles have an easier ability getting through the cell membrane and cell wall relative to larger particles.
Mohamed,
199
212
4.4. Transmission electron microscopy (TEM) 184
198
(2014),
224 225 226 227 228 229 230 231 232 233 234 235 236 237
G Model
ARTICLE IN PRESS
BIOMAC 4350 1–5
R.R. Mohamed, M.W. Sabaa / International Journal of Biological Macromolecules xxx (2014) xxx–xxx
4
Fig. 2. SEM images of CMCh and crosslinked CMCh hydrogel containing different ratios of silver nanoparticles.
Table 1 Antimicrobial activity of CMCh–Ag hydrogels. Sample
Non-crosslinked CMCh CMCh crosslinked CMCh/Ag (1%) CMCh/Ag (2%) CMCh/Ag (3%) Tetracycline (standard antibacterial agent) Ampicillin (standard antibacterial agent) Amphotricin B (antifungal agent)
238
239 240 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257
Induction zone diameter (mm/mg sample) Bacillus subtilis (G+ )
Escherichia coli (G− )
Pseudomonas aeruginosa (G− )
Staphylococcus aureus (G+ )
Neisseria gonorrhoeae (G− )
Streptococcus faecalis (G+ )
Canida albicans (Fungus)
11.3 30 30 34 36 30 20 –
NA 36 32 34 32 31 22 –
NA 34 32 32 32 30 17 –
10.1 38 32 32 36 28 18 –
28 27 26 30 26 30 21 –
31 31 33 27 25 31 23 –
15 18 15 16 19 – – 19
4.5. Antimicrobial activity The antibacterial activity of hydrogels was carried out against three Gram −ve bacteria (E. coli, P. aeruginosa and N. Gonorrhoeae) and against three Gram +ve bacteria (S. aureus, B. subtilis and S. Faecalis) using disc plate technique. The results obtained are shown in Table 1. It is seen that, compared to CMCh, crosslinked CMCh has higher antibacterial effect (almost 3 to 4 times higher) toward Gram +ve and Gram −ve bacteria. Several mechanisms elucidating the antimicrobial activity of chitosan have been postulated. The most acceptable mechanism is mediated by the electrostatic forces between the protonated NH3 + groups of chitosan and the electronegative charges on the microbial cell surface [19]. As such mechanism is based on electrostatic interaction, it suggests that the greater the number of cationized amines, the higher will be the antimicrobial activity. Carboxymethylation of chitosan allowed the synthesis of CMCh with more hydrophilicity, with better solubility in aqueous media and with greater positive charge density; as in CMCh the COOH groups may react with the NH2 groups and converted these NH2 groups into NH groups leading to increased polycationic character (non-pH dependent positive charges on
Please cite this article in press as: http://dx.doi.org/10.1016/j.ijbiomac.2014.05.025
R.R.
CMCs). Another proposed mechanism is the binding of chitosan with microbial DNA, which leads to the inhibition of the mRNA and protein synthesis via penetration of chitosan into the nuclei of the microorganisms [20]. The prepared hydrogels containing silver nanoparticles has high antibacterial properties than virgin CMCh as proved by their higher inhibition zone, regardless to the kind of bacteria used. Also the antibacterial activity of the hydrogels improved with the increase in Ag % in the hydrogels. Although the antimicrobial properties of silver have been known for centuries, the mechanisms by which silver inhibits bacterial growth were just recently discussed. Most of the proposed mechanisms involve silver entering the cell in order to cause damage. It is thought that silver atoms bind to thiol groups ( SH) in enzymes and subsequently cause the deactivation of enzymes. Silver forms stable S Ag bonds with thiol-containing compounds in the cell membrane that are involved in transmembrane energy generation and ion transport [21]. It is also believed that silver can take part in catalytic oxidation reactions that result in the formation of disulfide bonds (R S S R). Silver does this by catalyzing the reaction between oxygen molecules in the cell and hydrogen atoms of thiol groups: water is released as a product
Mohamed,
M.W.
Sabaa,
Int.
J.
Biol.
Macromol.
(2014),
258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278
G Model BIOMAC 4350 1–5
ARTICLE IN PRESS R.R. Mohamed, M.W. Sabaa / International Journal of Biological Macromolecules xxx (2014) xxx–xxx
5
proposed [21]. It was proposed that Ag+ enters the cell and intercalates between the purine and pyrimidine base pairs disrupting the hydrogen bonding between the two anti-parallel strands and denaturing the DNA molecule. Also those CMCh–Ag nanoparticles have higher antifungal activity compared to virgin CMCh and all of them have good antifungal activity compared to Amphotericin B “the antifungal drug reference”. 5. Conclusions
References
279 280 281 282 283 284
and two thiol groups become covalently bonded to one another through a disulfide bond [22]. The silver-catalyzed formation of disulfide bonds could possibly change the shape of cellular enzymes and subsequently affect their function, leading to changes in protein structure and the inactivation of key enzymes, such as those needed for cellular respiration which causes cell death. Another suggested mechanism of the antimicrobial activity of silver was
Please cite this article in press as: http://dx.doi.org/10.1016/j.ijbiomac.2014.05.025
R.R.
M.W.
287 288 289 290 291 292
294 295 296 297 298 299 300 301 302 303
304
[1] A.S. Hoffman, Adv. Drug Deliv. Rev. 43 (2002) 3–12. [2] N.A. Peppas, P. Bures, W. Leobandung, H. Ichikawa, Eur. J. Pharm. Biopharm. 50 (2000) 27–46. [3] B.C. Kim, G. Spinks, C.O. Too, G.G. Wallace, Y.H. Bae, React. Funct. Polym. 44 (2000) 31–40. [4] V. Thomas, M.M. Yallapu, B. Sreedhar, S.K. Bajpai, J. Colloid Interface Sci. 315 (1) (2007) 389–395. [5] I. Sondi, B. Salopek-Sondi, J. Colloid Interface Sci. 275 (1) (2004) 177–182. [6] S.K. Bajpai, Y.M. Mohan, M. Bajpai, R. Tankhiwale, V. Thomas, J. Nanosci. Nanotechnol. 7 (2007) 2994–3010. [7] I.S. Arvanitoyannis, A. Nakayama, S.-I. Aiba, Carbohydr. Polym. 37 (4) (1998) 371–382. [8] H. Bidgoli, A. Zamani, M.J. Taherzadeh, Carbohydr. Res. 345 (2010) 2683–2689. [9] X.G. Chen, H.J. Park, Carbohydr. Polym. 53 (2003) 355–359. [10] R.W. Eyler, E.D. Kluge, F. Diephuis, Anal. Chem. 79 (1947) 24–27. [11] A. Hebeish, M. Hashem, M.M. Abd El-Hady, S. Sharaf, Carbohydr. Polym. 92 (2013) 407–413. [12] A.W. Bauer, W.M. Kirby, C. Sherris, M. Turck, Am. J. Clin. Pathol. 45 (1996) 493–496. [13] M.A. Pfaller, L. Burmeister, M.A. Bartlett, M.G. Rinaldi, J. Clin. Microbiol. 26 (1988) 1437–1441. [14] National Committee for Clinical Laboratory Standards, Approved standard M7A3, NCCLS, Villanova, PA, 1993. [15] National Committee for Clinical Laboratory Standards, Performance 41 (1997). Q3 [16] L.D. Liebowitz, H.R. Ashbee, E.G.V. Evans, Y. Chong, N. Mallatova, M. Zaidi, D. Gibbs, Global Antifungal Surveillance Group, Diagn. Microbiol. Infect. Dis. 4 (2001) 27–33. [17] M.L. Matar, L. Ostrosky-Zeichner, V.L. Paetznick, J.R. Rodriguez, E. Chen, J.H. Rex, Antimicrob. Agents Chemother. 47 (2003) 1647–1651. [18] L.M. Hamming, R. Qiao, P.B. Messersmith, L.C. Brinson, Comput. Sci. Technol. 69 (2009) 1880–1886. [19] Q.L. Feng, J. Wu, G.Q. Chen, F.Z. Cui, T.N. Kim, J.O. Kim, J. Biomed. Mater. Res. 52 (2000) 662–668. [20] L.A. Hadwiger, D.F. Kendra, B.W. Fristensky, W. Wagoner, R.A.A. Muzzarelli, C. Jeuniaux, C.W. Gooday (Eds.), Chitin in Nature and Technology, Plenum Press, New York, 1986, pp. 209–214. [21] U. Klueh, V. Wagner, S. Kelly, A. Johnson, J.D. Bryers, J. Biomed. Mater. Res. B: Appl. Biomater. 53 (2000) 621–631. [22] R.L. Davies, S.F. Etris, Catal. Today 36 (1997) 107–114.
Mohamed,
286
293
CMCh–Ag hydrogels with high antibacterial activity against four different types of bacteria were synthesized. The hydrogels were obtained as pale brown in color; darker hydrogels were obtained when larger amounts of Ag were added. The particle size of the nanoparticles was determined using TEM to be as small as 9–16 nm in size. SEM showed the silver nanoparticles as spherical shapes on the surface of the hydrogels. The antimicrobial activity of the CMCh–Ag hydrogels was estimated to be higher than CMCh itself, the antimicrobial activity of the hydrogels increased as the amount of Ag increased.
Fig. 3. TEM images of CMCh–Ag nanoparticles prepared by in situ technique.
285
Sabaa,
Int.
J.
Biol.
Macromol.
(2014),
305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343
ARTICLE IN PRESS
BIOMAC 4350 1–5
International Journal of Biological Macromolecules xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Synthesis and characterization of antimicrobial crosslinked carboxymethyl chitosan nanoparticles loaded with silver
1
2
3 4
Q1
Riham R. Mohamed ∗ , Magdy W. Sabaa Department of Chemistry, Faculty of Science, Cairo University, Giza 12613, Egypt
5
6 19
a r t i c l e
i n f o
a b s t r a c t
7 8 9 10 11 12
Article history: Received 7 March 2014 Received in revised form 30 April 2014 Accepted 7 May 2014 Available online xxx
13
18
Keywords: Hydrogels Silver Carboxymethyl chitosan Antimicrobial activity
20
1. Introduction
14 15 16 17
21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42
Carboxymethyl chitosan (CMCh)–silver nanoparticle (Ag) hydrogels with high antibacterial activity against three Gram +ve bacteria (Staphylococcus aureus, Bacillus subtilis and Streptococcus faecalis), three Gram −ve bacteria (Escherichia coli, Pseudomonas aeruginosa and Neisseria gonorrhoeae) and a Candida albicans fungus were prepared. The in situ preparation reaction involved crosslinking of CMCh with epichlorohydrin in alkaline medium containing silver nitrate to yield silver nanoparticles loaded CMCh hydrogel giving pale brown or darker hydrogels when the silver content increases. FTIR spectroscopy, SEM and TEM were done for the prepared hydrogels. Silver nanoparticles hydrogels exhibited higher antimicrobial activity than virgin CMCh. TEM analysis showed the small size of the prepared hydrogels to be in the range of 9–16 nm in size. © 2014 Published by Elsevier B.V.
Hydrogels are an important class of polymeric materials that have been utilized in a wide variety of biomedical and pharmaceutical applications. Hydrogels are three dimensional, hydrophilic, polymeric networks that can absorb up to thousands of times their dry weight in water or biological fluids [1,2]. They consist of polymeric chains with either physical or chemical cross-links that prevent dissolution of the hydrogel structure and instead result in swelling of the material upon interaction with aqueous solutions. Hydrogel nanocomposites involve the incorporation of various nanoparticulate materials within a versatile hydrogel matrix which can provide easy, straight forward methods for enhancing the properties of hydrogels. The use of silver and other metal ions for their sustained antifungal, antibacterial and antiviral effects have been practiced for a long time. Silver ion has been known to be effective against a broad range of microorganisms. Their synthesis and highly effective observed antibacterial activity make them a very attractive form of silver administration. Nanocomposite hydrogels have been shown to modify and improve a variety of material properties of the polymer through the addition of conducting particles such as carbon nanotubes (CNTs), clay, ceramics, magnetic nanoparticles, hydroxyapatite (HA), and semiconducting nanoparticles [3].
∗ Corresponding author. E-mail address: [email protected] (R.R. Mohamed).
Metallic nanoparticles are toxic to microorganisms. Silver nanoparticles exhibit antimicrobial effects by binding to microbial DNA, preventing bacterial replication, and also causing inactivation of bacteria function [4,5]. Silver nanoparticles with higher surface to volume ratio compared to common metallic silver have shown better antimicrobial activity. Due to unique biological properties of silver nanoparticles such as biocompatibility and antibacterial affinity they have been applied for various medical purposes such as implants, catheters, and healing of wounds. Different methods of incorporating silver nanoparticles in hydrogels have been reported in the literature. Nanocomposites can be synthesized by incorporation of particles prior to polymerization, during polymerization, or after polymerization of hydrogels [6]. Chitosan, an amino polysaccharide, is composed of ,1-4 glucosidic bonds. Due to its unique polycationic nature, chitosan and its derivatives have been proposed for various applications in biomedical, food, agricultural, biotechnological and pharmaceutical fields [7]. The antibacterial activity of chitosan and its antibacterial mechanism have been researched extensively. Due to the low solubility of chitosan in water, its hydrogels do not show high binding water capacities. Carboxymethyl chitosan, an amphoteric material with high hydrophilic characteristics. It is a very important chitosan derivative showing very good water solubility. Recently, hydrogels with high water binding capacity have been successfully produced from carboxymethyl chitosan by its crosslinking in aqueous media, followed by drying of the resultant gel [8].
http://dx.doi.org/10.1016/j.ijbiomac.2014.05.025 0141-8130/© 2014 Published by Elsevier B.V.
Please cite this article in press as: http://dx.doi.org/10.1016/j.ijbiomac.2014.05.025
R.R.
Mohamed,
M.W.
Sabaa,
Int.
J.
Biol.
Macromol.
(2014),
43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70
G Model
ARTICLE IN PRESS
BIOMAC 4350 1–5
R.R. Mohamed, M.W. Sabaa / International Journal of Biological Macromolecules xxx (2014) xxx–xxx
2 71
2. Materials and methods
72
2.1. Materials
76
Chitosan (code KB-002) was purchased from Funakoshi Co. LTD, Japan. Deacetylation content = 88.2%, epichlorohydrin (ECH) (98%), sodium hydroxide and silver nitrate of laboratory grade chemicals were used.
77
2.2. Methods
73 74 75
78 79 80 81 82 83 84 85 86 87 88 89 90
91 92 93 94 95 96 97 98
2.2.1. Preparation of carboxymethyl chitosan (CMCh) Carboxymethyl chitosan was prepared following the method reported previously [9] where chitosan (10 g), sodium hydroxide (13.5 g) and solvent isopropanol (100 ml) were suspended in a flask to swell and alkalize at room temperature for 1 h. The monochloroacetic acid (15 g) was dissolved in isopropanol, and added to the reaction mixture dropwisely within 30 min and reacted for 4 h at 55 ◦ C. Then the reaction was stopped and isopropanol was discarded. Ethyl alcohol (80%) was added and the solid product was filtered and rinsed with 80–90% ethyl alcohol to desalt and dewater, and vacuum dried at 50 ◦ C. The degree of substitution of carboxymethyl chitosan was determined by pH-titration [10] and found to be 0.75. 2.2.2. Preparation of crosslinked CMCh hydrogels Definite amount of CMCh was dissolved in 1% NaOH solution with continuous mechanical stirring until a homogeneous viscous mixture was obtained [11]. A definite concentration of epichlorohydrin (10% based on weight of CMCh) was added dropwisely with continuous stirring. The formed paste, was transferred to Petri dish, dried in an oven at 80 ◦ C for 5 min then cured for 3–7 min at different temperature (120–140 ◦ C).
2.2.3. Loading of CMCh hydrogels with silver nanoparticles (in situ process) 100 Definite amount of CMCh was dissolved is 1% NaOH Solu101 tion with continuous mechanical stirring until a homogeneous 102 viscous mixture was obtained, then epichlorohydrin was added 103 dropwisely. Silver nitrate solution was then added in different con104 centration (1–3%). 105 The prepared hydrogels were pale brown in color; darker hydro106 gels were obtained when larger amounts of Ag were added. The 107 paste was dried in an oven at 80 ◦ C for 15 min, and then cured at 108 Q2 130 ◦ C [11] (Scheme 1). 109 99
110
3. Characterization and analysis
111
3.1. FTIR spectroscopy
114
FTIR spectra were recorded using KBr discs on Testcan Shimadzu IR-Spectrometer (FTIR model 8000) at room temperature within the wave number range of 4000–400 cm−1 .
115
3.2. Scanning electronic microscopy
112 113
aqueous particles dispersed in 5 mL acetone to become slightly turbid solution onto the copper grid and allowing it to dry well. The images of representative areas were captured at suitable magnifications which clarify the morphology and the size of the nanoparticles.
3.4. Antibacterial activity
119
The dry sample, spread on a double sided conducting adhesive tape, pasted on a metallic stub, was coated (100 ) with gold in an ion sputter coating unit (JEOL S150A) for 2 min and observed in a JEOL-JXA-840A Electron probe microanalyzer at 20 kV.
120
3.3. Transmission electron microscopy (TEM)
117 118
121 122 123
Micrographs of the colloidal nanogel particles were taken using a JEM-100S Transmission Electron Microscope (TEM, Japan). The TEM sample was prepared by mixing one dilute drop of prepared Please cite this article in press as: http://dx.doi.org/10.1016/j.ijbiomac.2014.05.025
R.R.
Antibacterial activity of the investigated hydrogels was evaluated using a modified Kirby–Bauer disc diffusion method [12]. Briefly, 100 L of the test bacteria were grown in 10 mL of fresh media until they reached a count of approximately 108 cells/mL for bacteria [13]. 100 L of microbial suspension was spread onto agar plates corresponding to the broth in which they were maintained. Isolated colonies of each organism that might be playing a pathogenic role should be selected from primary agar plates and tested for susceptibility by disc diffusion method [14,15]. Of the many media available, National Committee of Clinical Laboratory Standards (NCCLS) recommends Mueller–Hinton agar due to its results in good batch-to-batch reproducibility. Antibacterial activity of the prepared hydrogels was carried out against three Gram +ve bacteria (S. aureus, B. subtilis and S. faecalis), three Gram −ve bacteria (E. coli, P. aeruginosa and N. gonorrhoeae) and a Candida albicans fungus. Plates are inoculated with bacteria at 35–37 ◦ C for 24–48 h then the diameters of the inhibition zones were measured in millimeters [12]. Standard discs of Tetracycline and Ampicillin (Antibacterial agents) served as positive controls for antibacterial activity but filter discs impregnated with 10 L of solvent (distilled water, chloroform, DMSO) were used as a negative control. The agar used is Meuller–Hinton agar that is rigorously tested for composition and pH. Further the depth of the agar in the plate is a factor to be considered in the disc diffusion method. This method is well documented and standard zones of inhibition have been determined for susceptible and resistant values. Blank paper discs (Schleicher and Schuell, Spain) with a diameter of 8.0 mm were impregnated with 10 of tested concentration of the stock solutions. When a filter paper disc – impregnated with a tested chemical – is placed on Agar, the chemical will diffuse from the disc into the agar. This diffusion will place the chemical on the agar only around the disc. The solubility of the chemical and its molecular size will determine the size of the area of chemical in filtration around the disc. If an organism is placed on the agar; it will not grow around the disc if it is susceptible to the chemical. This area of no growth around the disc is known as a “zone of inhibition” or “clear zone”. For the disc diffusion, the zone diameters were measured with slipping calipers of the NCCLS [14]. Agar-based methods such as Etest and disc diffusion can be good alternatives because they are simpler and faster than broth-based methods [16,17].
M.W.
Sabaa,
126 127 128
130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171
172
The sterilized filter paper discs impregnated with a solution of the test compound in DMSO (1 mg/ml) were placed on nutrient agar plate seeded with the appropriate test organism in triplicates. Standard conditions of 104 CFU/mL (colony forming U/mL)were used for antifungal assay, respectively. Petri dishes (9 cm in diameter) were used and the discs of filter paper were inoculated in each plate. The utilized test organisms were C. albicans as fungi. Amphotericin B were used as reference drug against fungi. DMSO alone was used as control at the same abovementioned concentration and during this, there was no visible change in bacterial growth. The plates were incubated at 37 ◦ C for 48 h for fungi.
Mohamed,
125
129
3.5. Antifungal activity
116
124
Int.
J.
Biol.
Macromol.
(2014),
173 174 175 176 177 178 179 180 181 182 183
G Model
ARTICLE IN PRESS
BIOMAC 4350 1–5
R.R. Mohamed, M.W. Sabaa / International Journal of Biological Macromolecules xxx (2014) xxx–xxx
3
Scheme 1. Crosslinking reaction of CMCh with epichlorohydrin in alkaline medium.
4.2. FTIR spectroscopy
197
Fig. 1 represents FTIR spectra of (a) the CMCh and (b) the crosslinked CMCh hydrogel with epichlorohydrin. The bands at 1422, 1608, 2931, and 3420 cm−1 are assigned to the stretching vibration of COO (symmetric), COO (asymmetric),CH (aliphatic), and O H, respectively. The bands observed at 1608 and 1422 cm−1 in the spectrum of crosslinked CMCh hydrogels with epichlorohydrin indicate that the carboxyl groups of CMCh exist in the hydrogels after crosslinking. It can also be seen from the spectrum of crosslinked hydrogel some typical peaks appear at 1327, 1262, and 1064 cm−1 . The peaks at 1437 and 1155 cm−1 belong to stretching vibration of C O C and C C stretching vibration respectively, of the reacted epichlorohydrin with CMCh, whereas the band appeared at 1065 cm−1 is characteristic for bending vibration of OH group.
4.3. Scanning electron microscopy (SEM)
Fig. 1. FTIR spectra of (a) CMCh (b) the crosslinked CMCh hydrogel with epichlorohydrin.
4. Results and discussion
185
4.1. In situ formation of silver nanoparticles
186 187 188 189 190 191 192 193 194 195 196
Nanoparticles can be incorporated into the hydrogel matrix by mixing the silver with the preformed hydrogel, by adding the AgNO3 salt during the formation of the hydrogel or during the swelling of the hydrogel [18]. In this research, the formation of silver nanoparticles inside the chains of CMCh was performed during the hydrogel formation. Our approach depends on the dual function of CMCh as both a reducing and a stabilizing agent for silver nanoparticles during the formation of CMCh hydrogel containing nanoparticles. The negatively charged carboxyl groups in CMCh attract the positively charged silver cations.
Please cite this article in press as: http://dx.doi.org/10.1016/j.ijbiomac.2014.05.025
R.R.
Scanning electron microscopy images of CMCh and crosslinked CMCh hydrogel containing silver nanoparticles prepared by in situ technique are shown in Fig. 2. CMCh has a surface full of lumps as bulky COOH groups are formed on the surface. On the other hand, in the hydrogel CMCh–silver nanoparticles, the silver nanoparticles are clearly visible as spherical particles onto the surface of CMCh in Fig. 2. The prepared hydrogels were pale brown in color, darker hydrogels were obtained when larger amounts of Ag nanoparticles were reacted with the CMCh.
M.W.
Sabaa,
Int.
J.
Biol.
200 201 202 203 204 205 206 207 208 209 210 211
Macromol.
213 214 215 216 217 218 219 220 221 222
223
TEM images, done for CMCh–Ag nanoparticles prepared by in situ technique, were shown in Fig. 3 to demonstrate the shape of silver nanoparticles within the hydrogel network. It reveals Ag nanoparticles in the composites are dispersed in the CMCh matrix. The nanoparticles size range from 9–16 nm via the in situ preparation technique. It is also obvious that, the nanoparticles do not form aggregates showing that the particle size of the Ag nanoparticles in the CMCh–Ag composites was controlled. This may be due to the excellent stabilization of silver nanoparticles by carboxylate anions present in the CMCh chains. The increased antimicrobial activity of the Ag nanoparticles could be attributed to their smaller size due to the fact that smaller particles have an easier ability getting through the cell membrane and cell wall relative to larger particles.
Mohamed,
199
212
4.4. Transmission electron microscopy (TEM) 184
198
(2014),
224 225 226 227 228 229 230 231 232 233 234 235 236 237
G Model
ARTICLE IN PRESS
BIOMAC 4350 1–5
R.R. Mohamed, M.W. Sabaa / International Journal of Biological Macromolecules xxx (2014) xxx–xxx
4
Fig. 2. SEM images of CMCh and crosslinked CMCh hydrogel containing different ratios of silver nanoparticles.
Table 1 Antimicrobial activity of CMCh–Ag hydrogels. Sample
Non-crosslinked CMCh CMCh crosslinked CMCh/Ag (1%) CMCh/Ag (2%) CMCh/Ag (3%) Tetracycline (standard antibacterial agent) Ampicillin (standard antibacterial agent) Amphotricin B (antifungal agent)
238
239 240 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257
Induction zone diameter (mm/mg sample) Bacillus subtilis (G+ )
Escherichia coli (G− )
Pseudomonas aeruginosa (G− )
Staphylococcus aureus (G+ )
Neisseria gonorrhoeae (G− )
Streptococcus faecalis (G+ )
Canida albicans (Fungus)
11.3 30 30 34 36 30 20 –
NA 36 32 34 32 31 22 –
NA 34 32 32 32 30 17 –
10.1 38 32 32 36 28 18 –
28 27 26 30 26 30 21 –
31 31 33 27 25 31 23 –
15 18 15 16 19 – – 19
4.5. Antimicrobial activity The antibacterial activity of hydrogels was carried out against three Gram −ve bacteria (E. coli, P. aeruginosa and N. Gonorrhoeae) and against three Gram +ve bacteria (S. aureus, B. subtilis and S. Faecalis) using disc plate technique. The results obtained are shown in Table 1. It is seen that, compared to CMCh, crosslinked CMCh has higher antibacterial effect (almost 3 to 4 times higher) toward Gram +ve and Gram −ve bacteria. Several mechanisms elucidating the antimicrobial activity of chitosan have been postulated. The most acceptable mechanism is mediated by the electrostatic forces between the protonated NH3 + groups of chitosan and the electronegative charges on the microbial cell surface [19]. As such mechanism is based on electrostatic interaction, it suggests that the greater the number of cationized amines, the higher will be the antimicrobial activity. Carboxymethylation of chitosan allowed the synthesis of CMCh with more hydrophilicity, with better solubility in aqueous media and with greater positive charge density; as in CMCh the COOH groups may react with the NH2 groups and converted these NH2 groups into NH groups leading to increased polycationic character (non-pH dependent positive charges on
Please cite this article in press as: http://dx.doi.org/10.1016/j.ijbiomac.2014.05.025
R.R.
CMCs). Another proposed mechanism is the binding of chitosan with microbial DNA, which leads to the inhibition of the mRNA and protein synthesis via penetration of chitosan into the nuclei of the microorganisms [20]. The prepared hydrogels containing silver nanoparticles has high antibacterial properties than virgin CMCh as proved by their higher inhibition zone, regardless to the kind of bacteria used. Also the antibacterial activity of the hydrogels improved with the increase in Ag % in the hydrogels. Although the antimicrobial properties of silver have been known for centuries, the mechanisms by which silver inhibits bacterial growth were just recently discussed. Most of the proposed mechanisms involve silver entering the cell in order to cause damage. It is thought that silver atoms bind to thiol groups ( SH) in enzymes and subsequently cause the deactivation of enzymes. Silver forms stable S Ag bonds with thiol-containing compounds in the cell membrane that are involved in transmembrane energy generation and ion transport [21]. It is also believed that silver can take part in catalytic oxidation reactions that result in the formation of disulfide bonds (R S S R). Silver does this by catalyzing the reaction between oxygen molecules in the cell and hydrogen atoms of thiol groups: water is released as a product
Mohamed,
M.W.
Sabaa,
Int.
J.
Biol.
Macromol.
(2014),
258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278
G Model BIOMAC 4350 1–5
ARTICLE IN PRESS R.R. Mohamed, M.W. Sabaa / International Journal of Biological Macromolecules xxx (2014) xxx–xxx
5
proposed [21]. It was proposed that Ag+ enters the cell and intercalates between the purine and pyrimidine base pairs disrupting the hydrogen bonding between the two anti-parallel strands and denaturing the DNA molecule. Also those CMCh–Ag nanoparticles have higher antifungal activity compared to virgin CMCh and all of them have good antifungal activity compared to Amphotericin B “the antifungal drug reference”. 5. Conclusions
References
279 280 281 282 283 284
and two thiol groups become covalently bonded to one another through a disulfide bond [22]. The silver-catalyzed formation of disulfide bonds could possibly change the shape of cellular enzymes and subsequently affect their function, leading to changes in protein structure and the inactivation of key enzymes, such as those needed for cellular respiration which causes cell death. Another suggested mechanism of the antimicrobial activity of silver was
Please cite this article in press as: http://dx.doi.org/10.1016/j.ijbiomac.2014.05.025
R.R.
M.W.
287 288 289 290 291 292
294 295 296 297 298 299 300 301 302 303
304
[1] A.S. Hoffman, Adv. Drug Deliv. Rev. 43 (2002) 3–12. [2] N.A. Peppas, P. Bures, W. Leobandung, H. Ichikawa, Eur. J. Pharm. Biopharm. 50 (2000) 27–46. [3] B.C. Kim, G. Spinks, C.O. Too, G.G. Wallace, Y.H. Bae, React. Funct. Polym. 44 (2000) 31–40. [4] V. Thomas, M.M. Yallapu, B. Sreedhar, S.K. Bajpai, J. Colloid Interface Sci. 315 (1) (2007) 389–395. [5] I. Sondi, B. Salopek-Sondi, J. Colloid Interface Sci. 275 (1) (2004) 177–182. [6] S.K. Bajpai, Y.M. Mohan, M. Bajpai, R. Tankhiwale, V. Thomas, J. Nanosci. Nanotechnol. 7 (2007) 2994–3010. [7] I.S. Arvanitoyannis, A. Nakayama, S.-I. Aiba, Carbohydr. Polym. 37 (4) (1998) 371–382. [8] H. Bidgoli, A. Zamani, M.J. Taherzadeh, Carbohydr. Res. 345 (2010) 2683–2689. [9] X.G. Chen, H.J. Park, Carbohydr. Polym. 53 (2003) 355–359. [10] R.W. Eyler, E.D. Kluge, F. Diephuis, Anal. Chem. 79 (1947) 24–27. [11] A. Hebeish, M. Hashem, M.M. Abd El-Hady, S. Sharaf, Carbohydr. Polym. 92 (2013) 407–413. [12] A.W. Bauer, W.M. Kirby, C. Sherris, M. Turck, Am. J. Clin. Pathol. 45 (1996) 493–496. [13] M.A. Pfaller, L. Burmeister, M.A. Bartlett, M.G. Rinaldi, J. Clin. Microbiol. 26 (1988) 1437–1441. [14] National Committee for Clinical Laboratory Standards, Approved standard M7A3, NCCLS, Villanova, PA, 1993. [15] National Committee for Clinical Laboratory Standards, Performance 41 (1997). Q3 [16] L.D. Liebowitz, H.R. Ashbee, E.G.V. Evans, Y. Chong, N. Mallatova, M. Zaidi, D. Gibbs, Global Antifungal Surveillance Group, Diagn. Microbiol. Infect. Dis. 4 (2001) 27–33. [17] M.L. Matar, L. Ostrosky-Zeichner, V.L. Paetznick, J.R. Rodriguez, E. Chen, J.H. Rex, Antimicrob. Agents Chemother. 47 (2003) 1647–1651. [18] L.M. Hamming, R. Qiao, P.B. Messersmith, L.C. Brinson, Comput. Sci. Technol. 69 (2009) 1880–1886. [19] Q.L. Feng, J. Wu, G.Q. Chen, F.Z. Cui, T.N. Kim, J.O. Kim, J. Biomed. Mater. Res. 52 (2000) 662–668. [20] L.A. Hadwiger, D.F. Kendra, B.W. Fristensky, W. Wagoner, R.A.A. Muzzarelli, C. Jeuniaux, C.W. Gooday (Eds.), Chitin in Nature and Technology, Plenum Press, New York, 1986, pp. 209–214. [21] U. Klueh, V. Wagner, S. Kelly, A. Johnson, J.D. Bryers, J. Biomed. Mater. Res. B: Appl. Biomater. 53 (2000) 621–631. [22] R.L. Davies, S.F. Etris, Catal. Today 36 (1997) 107–114.
Mohamed,
286
293
CMCh–Ag hydrogels with high antibacterial activity against four different types of bacteria were synthesized. The hydrogels were obtained as pale brown in color; darker hydrogels were obtained when larger amounts of Ag were added. The particle size of the nanoparticles was determined using TEM to be as small as 9–16 nm in size. SEM showed the silver nanoparticles as spherical shapes on the surface of the hydrogels. The antimicrobial activity of the CMCh–Ag hydrogels was estimated to be higher than CMCh itself, the antimicrobial activity of the hydrogels increased as the amount of Ag increased.
Fig. 3. TEM images of CMCh–Ag nanoparticles prepared by in situ technique.
285
Sabaa,
Int.
J.
Biol.
Macromol.
(2014),
305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343
