International Journal of Biological Macromolecules 121 (2019) 707–717
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International Journal of Biological Macromolecules journal homepage: http://www.elsevier.com/locate/ijbiomac
Synthesis of novel chitosan-PVC conjugates encompassing Ag nanoparticles as antibacterial polymers for biomedical applications Samir T. Gaballah a,⁎, Hossam A. El-Nazer a, Reham A. Abdel-Monem a, Mohamed Azab El-Liethy b, Bahaa A. Hemdan b, Samira T. Rabie a a b
Photochemistry Department, National Research Centre, El Buhouth St., Dokki 12622, Giza, Egypt Environmental Microbiology Lab., Water Pollution Research Department, National Research Centre, El Buhouth St., Dokki 12622, Giza, Egypt
a r t i c l e
i n f o
Article history: Received 6 July 2018 Received in revised form 28 September 2018 Accepted 14 October 2018 Available online 16 October 2018 Keywords: Chitosan PVC Silver nanoparticles TiO2 nanoparticles Antibacterial
a b s t r a c t We herein describe the synthesis of four Cs-PVC conjugates three of them were functionalized with benzothiazole (BTh) derivative as an antibacterial agent. Two of these BTh-functionalized conjugates, namely Cs2 and Cs3, comprise silver nanoparticles (AgNPs) and Ag/TiO2 NPs, respectively. The structures were characterized via FTIR spectroscopic analysis, morphological investigation such as scanning (SEM) and transmission (TEM) electron microscopy, and thermal gravimetric analysis (TGA). Spectral data confirmed the introduction of the BTh to the Cs backbone as well as the coupling between the two polymers. SEM data showed homogenous polymer surfaces with well-distributed Ag nanoparticles. The Ag contents in the prepared samples Cs2 and Cs3 were, respectively, 0.61 and 0.21%, however, TEM analysis showed that the sizes of AgNPs and Ag/TiO2 NPs were in the range of 3–7 nm and 15–22 nm for the prepared conjugates, respectively. The antibacterial activity of the synthesized conjugates was investigated against two Gram-negative (E. coli, and S. typhimurium) and two Grampositive (S. aureus, and L. monocytogenes) bacteria. The antibacterial assay showed that all three Cs-PVC (Cs1, Cs2, and Cs3) conjugates modified with BTh exhibited excellent bacterial inhibition after 30, 60, and 120 min. © 2018 Elsevier B.V. All rights reserved.
1. Introduction Microbial infections are major risk factors in several areas including medical devices, hospitals and healthcare facilities, dental surgery equipment, hygienic applications, water purification systems, and food packaging [1]. Devices and equipment used in medical applications are often manufactured from synthetic polymers which are commonly called medical polymers. They play an important role in spreading infections throughout medical facilities and health applications consequently they have a direct massive impact on the patient's health. When these polymers are embedded in humans, bacteria may stick to the polymer surface and finally, reproduction followed by infection takes place. In health and medical applications, commercial production of antibacterial polymers have become interesting and represented a great challenge. Polymers with anti-infective properties are usually obtained by impregnation or complexation with some antibacterial reagents in a process which requires large amounts of the antimicrobial reagents. Polymeric medical devices that are contacting internal parts of a person's body are thought to be the most significant types of these devices. Thus, the biomedical polymers should fulfill certain standards to accomplish more safety during their use in the human body. One of ⁎ Corresponding author. E-mail address:
[email protected] (S.T. Gaballah).
https://doi.org/10.1016/j.ijbiomac.2018.10.085 0141-8130/© 2018 Elsevier B.V. All rights reserved.
these standards is the biocompatibility, which can be identified by the ability to implement without damaging effects, toxicity, or causing any host response [2–4]. There are common suggested polymeric materials used for biomedical applications than others. Examples of these polymers are polyolefins, polyurethanes, fluoropolymers, acrylic, and vinyl polymers [4]. PVC is considered to be a good example of such polymers which possesses unique properties needed for various medical applications. Some of its most substantial properties are high chemical and mechanical resistance, inertness against biological fluids, and a wide range of processing possibilities that enhances its use in biomedical applications [5–7]. PVC is used for cannulas, catheters, lung bypass sets, dialysis tubing, and endotracheal feeding. The growing usage of different polymeric materials in the hospital care is accompanied by an increase in fighting the so-called biomaterial-related infections (BRI) which results from the adhesion of bacteria or other microorganisms to the polymer surface to form a pathogenic biofilm [8–11]. The formed biofilm can defend the bacteria from the influence of the added antibacterial agents leading to some difficulties in curing the infections and hence this requires either higher doses or more potent antibiotics [8,9,12–15]. To prevent the biofilm development powerfully, many trials have been accomplished in both fields of scientific research and industry. Recently, modifications of the biomaterial surface by different techniques as coating with Ag, azidation, and impregnation of the antibacterial agents into the polymer matrix are considered to be effective efforts [16–23].
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The well-known biopolymer chitosan (Cs) can be easily produced from the shells of the crustaceans via simple chemical treatments. Chemically, Cs is composed of randomly distributed β-(1 → 4) linked 2-acetamido-2-deoxy-D -glucopyranose and 2-amino-2-deoxy- D glucopyranose with a degree of deacetylation greater than 60%. Cs is characterized by the presence of nucleophilic moieties (NH2 and OH) which are greatly reactive towards several chemical and mechanical modifications to obtain a modified substrate with novel properties, functions, and applications [24]. It was stated previously that the free NH 2 groups at C-2 are more reactive than both OH groups at C-3 and C-6 in the 2-amino-2-deoxy- D -glucopyranose units of chitosan towards electrophiles attack. The chemical reaction of NH2 of chitosan with aldehydes has been carried out to obtain the corresponding biopolymeric material in the form of Schiff base which is reduced by sodium cyanoborohydride to give aromatic derivative. However, this type of compounds shows a higher antibacterial efficiency against a wide variety of both Gram-positive and Gram-negative bacteria [25]. Another type of modification is the introduction of heterocyclic rings in the chitosan scaffold which enhances its pharmacological activity. Reductive amination reaction can be carried out by selective introduction of either alkyl or aryl groups at the amino group of chitosan [26]. Formation of piperazine heterocyclic derivatives of chitosan as antibacterial agents is proposed according to Másson et al. [27]. The amino groups of chitosan were protected by phthaloylation (A) whereas the hydroxyl groups by the introduction of trityl group (B). Deprotection of amino groups was attained by treatment with hydrazine hydrate (C), and the Nchloroacylation was carried out by using chloroacetyl chloride (D) that results in the formation of the intermediate N-chloroacyl6-O-triphenylmethyl chitosan. The final piperazine derivative of chitosan (F) was then prepared by deprotection of the hydroxyl groups (E). Consequently, due to its ease of functionalization, insignificant toxicity, biocompatibility, and biodegradability, a considerable attention has been paid to exploiting Cs in many biomedical and pharmaceutical applications [28] such as biosensor fabrications [29,30], tissue engineering [30,31], drug delivery [32], and gene delivery [33]. Silver nanoparticles (AgNPs) have been recognized as antimicrobial agent which is attributed to their high surface area-to-volume ratio. An advantage of using AgNPs as an antibacterial agent is due to their low toxicity in the human body and negligible danger is predictable due to body contact [34,35]. Synthesis of AgNPs using biopolymers or plant extract has received a great attention because it is eco-friendly, userfriendly, and cost-effective technique [36–38]. Incorporation of AgNPs in Cs aiming to produce antibacterial nanocomposites has produced a substantial number of publications [38–41]. Hitherto, there are a few number of research studies on the synthesis of covalently bonded CsPVC conjugates [42,43]. Another effort in this area has been achieved by incorporating AgNPs into a Cs/PVC blended matrix to form a novel Cs-PVC/Ag as antimicrobial self-sterilizing nanocomposite biomaterial [44]. Cs/PVC blend was obtained by the simultaneous casting of their separate solutions using 2:1 proportions for Cs and PVC, respectively, in suitable solvents. Glutaraldehyde was used to ensure the occurrence of crosslinking between the two polymers. However, to ensure monophasic homogeneous distribution of the newly synthesized conjugate, Cs was covalently linked to PVC via substitution reaction. Thus, we present a facile methodology to synthesize novel BThfunctionalized Cs-PVC containing AgNPs or Ag/TiO2 NPs aiming to obtain new conjugates that have satisfactory antibacterial properties which can be used in medical applications. The synthesized Cs-PVC conjugates were achieved in a one-pot reaction using bromoacetyl bromide as a linking agent. The synthesized materials were characterized by FTIR spectroscopy (FTIR), scanning electron microscopy (SEM), transmission electron microscopy (TEM), and energy dispersive spectroscopy (EDS). The antibacterial activities of the new polymers were also investigated against Gram-negative and Grampositive strains.
2. Experimental 2.1. Materials All fine chemicals were of analytical grade and purchased from common commercial suppliers. Chitosan (MW 100–300 kDa, 82% degree of deacetylation) and bromoacetyl bromide was obtained from Acros Organics, Belgium. Suspension PVC, with a K value of 70, was obtained from A1-Ameria Company for Petrochemicals, Alexandria, Egypt. Amino-PVC was prepared by modification of a previous procedure [45]. 2-Amino-N-(thiazol-2-yl)benzo[d]thiazole-6-sulfonamide was prepared according to the previously published procedure [46,47]. Tetrahydrofuran (THF) was distilled over potassium under N2. All other solvents were distilled before use. Hydroxylamine hydrochloride and silver nitrate were of laboratory grade chemicals. Ag/TiO2 NPs was prepared by photo-deposition technique as described previously [48]. All reactions were performed in the air unless otherwise determined. 2.2. Preparation of 2-amino-N-(thiazol-2-yl)benzo[d]thiazole-6-sulfonamide (BTh) Sulfathiazole (5.6 g, 22.2 mmol) (STh) and potassium thiocyanate (8.9 g, 88.8 mmol) were dissolved in acetic acid (35 mL) and stirred together until complete dissolution (~15–20 min). Then, bromine (1.1 mL, 22.2 mmol) dissolved in acetic acid (15 mL) was slowly added over 30 m at such a rate to keep the temperature of the solution below 10 °C throughout the addition. The reaction was then stirred at room temperature for 5 h. The reaction progress was monitored by TLC using toluene: acetone (8:2) as an eluent system. The hydrobromide salt thus separated out was filtered, washed with acetic acid, and dried. The solid product was dissolved in hot water and basified to pH 9–11 with NH4OH and the resulting precipitate was filtered by vacuum filtration, washed with water, and dried. The yellow solids were then dissolved in hot ethyl acetate and filtered again to produce a yellow powder; Yield 5.9 g (86%). FTIR (KBr, cm−1) 3429 (NH, NH2), 1629 (C_N, NH out of plane bend), 1521 (NH), 1450 (C_C), 1318 (NH-SO2), 1277 (C\\N amine), 1137 (SO2), 1090 (C\\N), 973 (_C\\H), 642 (C\\S). 2.3. Preparation of bromoacetyl chitosan (CsAc-Br) Chitosan (1 g, 6.2 mmol) was soaked in dry dimethylformamide (DMF) (50 mL) overnight. To suspended chitosan, triethylamine (TEA, 5 mL) was added with stirring. Bromoacetyl bromide (3.1 mmol) was added dropwise at 0 °C with stirring for 2 h and stirred further for 4 h at room temperature. The reaction mixture was poured on cold ice/ water to give a pale brown precipitate which separated by filtration, washed with methanol, acetone, and diethyl ether. CsAc-Br was finally dried in the oven until constant weight; FTIR (KBr, cm−1): 3437 (OH, NH2 amide), 2962 (CH), 2913 (CH2), 1634 (C_O amide), 1456 (CH2), 1382 (CH2), 1317 (C\\O), 1154 (C\\N, C\\O), 1022 (C\\O), 874 (NH), 548 (C\\Br). 2.4. Preparation of ethylenediamine-modified PVC (amino-PVC) The modification of PVC with ethylenediamine was performed by dissolving PVC (2 g, 32 mmol) in THF (50 mL) with stirring for 3 h at room temperature then ethylenediamine (1.1 mL, 16 mmol) was added to the mixture. The reaction mixture was refluxed for 5–6 h. Amino-PVC was precipitated by pouring the reaction mixture upon cold methanol/H2O (2:1) and the whole container was kept in the freezer overnight. The white solid polymer was separated by filtration followed by drying in an oven at 60 °C until constant weight; Yield 1.7 g. FTIR (KBr, cm−1): 3444 (NH, NH2), 2979 (CH), 2906 (CH2), 1634 (NH), 1427 (C\\C), 1326 (CH2), 1254 (CH, CH2), 1095 (C\\N), 961 (CH2), 697 (C\\Cl), 620 (C\\Cl).
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2.5. Synthesis of chitosan-PVC conjugates (CsAcPVC) To chitosan (0.5 g, 3.1 mmol) suspended in DMF (10 mL) and TEA (0.9 mL, 0.6 g, 6.2 mmol), bromoacetyl bromide (0.1 mL, 1.5 mmol) was sequentially added dropwise over 1 h with stirring at 0 °C. The mixture was further stirred at room temperature for 2 h then cooled to 0 °C before adding amino-PVC (0.8 g) dissolved in DMF 15 mL. The mixture was heated at 60 °C for 4 h, cooled, precipitated in cold methanol/water, and filtered. The product was washed with methanol, acetone, and ether then dried in the oven at 55 °C until constant weight to give a buff solid product; FTIR (KBr, cm−1): 3437 (O\\H and N\\H), 2962 (C\\H), 2913 (CH2), 1635 (C_O amide), 1432 and 1384 (C\\H), 1327 (CH2), 1253 (C\\H, CH2), 1156 (C\\O), 1024 (C\\C), 694 (C\\Cl), 613 (C\\Cl). 2.6. One-pot synthesis of BTh-functionalized Cs-PVC conjugates (Cs1) Chitosan (1.07 g, 6.2 mmol) was soaked in dry DMF (50 mL) overnight. TEA (2.3 mL, 16.1 mmol) was added to suspended chitosan solution. To the reaction mixture, bromoacetyl bromide (0.4 mL, 5 mmol) was added dropwise at 0 °C for 2 h with vigorous stirring then the reaction mixture was stirred further at room temperature for 5 h. BTh (0.2 g, 0.6 mmol) was added and the reaction mixture was stirred at room temperature overnight. Amino-PVC (1 g) dissolved in dry DMF (15 mL) was added to the reaction mixture and stirred at 55 °C for 10 h. The reaction mixture was poured on ice/water and filtered, then washed with methanol, acetone, and diethyl ether. Finally, the product was dried in the oven at 60 °C until constant weight to afford 1.64 g of light brown solid. FTIR (KBr, cm−1): 3433 (O\\H, N\\H), 2917 (C\\H), 1634 (C_O), 1560–1535 (C_C), 1426 (C\\H), 1382 (C\\H), 1326 (C\\H), 1249 (C\\H), 1080 (C\\O), 608 (C\\Cl). 2.7. One-pot synthesis of BTh-functionalized Cs-PVC/AgNPs (Cs2) Chitosan (1.07 g, 6.2 mmol) was soaked in dry DMF (50 mL) overnight. TEA (2 mL, 15 mmol) was added to suspended chitosan solution. To the reaction mixture, bromoacetyl bromide (0.4 mL, 5 mmol) was added dropwise at 0 °C for 2 h with vigorous stirring then the reaction mixture was stirred further at room temperature for 5 h. BTh (0.2 g, 0.6 mmol) was added and the reaction mixture was stirred at room temperature overnight. Amino-PVC (1 g) dissolved in dry DMF (15 mL) was added to the reaction mixture and stirred at 55 °C for 10 h. Finally, silver nitrate (0.3 g, 1.89 mmol; 3% w/w, relative to chitosan) and hydroxylamine hydrochloride (0.13 g, 1.9 mmol) was added to the reaction mixture and stirred for 10 h. The reaction mixture was poured on ice/water and filtered then washed with methanol, acetone, and diethyl ether. Finally, the product was dried in the oven at 60 °C until constant weight to afford 1.64 g of light brown solid. FTIR (KBr, cm−1): 3433 (O\\H, N\\H), 2919 (C\\H), 1633 (C_O), 1560–1530 (C_C), 1428 (C\\H), 1383 (C\\H), 1325 (C\\H), 1252 (C\\H), 1079 (C\\O), 612 (C\\Cl). 2.8. One-pot synthesis of BTh-functionalized Cs-PVC/Ag/TiO2 (Cs3) Chitosan (1.07 g, 6.2 mmol) was soaked in dry DMF (50 mL) overnight. TEA (2 mL, 15 mmol) was added to suspended chitosan solution. To the reaction mixture, bromoacetyl bromide (0.4 mL, 5 mmol) was added dropwise at 0 °C for 2 h with vigorous stirring then the reaction mixture was stirred further at room temperature for 5 h. BTh (0.2 g, 0.6 mmol) was added and the reaction mixture was stirred at room temperature overnight. Amino-PVC (1 g) dissolved in dry DMF (15 mL) was added to the reaction mixture and stirred at 55 °C for 10 h. Finally, Ag/TiO2 in DMF (5 mL) was sonicated for 5 min then added to the reaction mixture with stirring at room temperature for 10 h. The reaction mixture was poured on ice/water and filtered, then washed with methanol, acetone, and diethyl ether. Finally, the product
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was dried in the oven at 60 °C until constant weight to afford 1.64 g of light brown solid. FTIR (KBr, cm−1): 3433 (O\\H, N\\H), 2916 (C\\H), 1640 (C_O), 1560–1535 (C_C), 1430 (C\\H), 1379 (C\\H), 1328 (C\\H), 1250 (C\\H), 1093 (C\\O), 610 (C\\Cl). 2.9. Characterization techniques and analysis FTIR spectra were recorded on a Shimadzu IR-Spectrometer (FTIR 8201) at room temperature within the wave number (cm−1) range of 4000 to 400 using KBr disc. The morphology of the tested samples was imaged and investigated. Thus, the scanning electron microscope (SEM) micrographs were acquired using QUANTA FEG 250 ESEM. Energy dispersive X-ray spectroscopy (EDAX AMETEK Inc.; Mahwah, NJ, USA) analysis at an acceleration voltage of 15 kV was carried out to determine elemental content. The dry sample was spread on a doublesided conducting adhesive tape, pasted on a metallic stub. Samples were mounted on circular aluminum stubs with double-sided sticky tape. The films were fixed on the surface of the tape. The transmission electron microscope (TEM) images of the prepared powders were examined using high-resolution transmission electron microscope (HRTEM), JEOL, JEM-2100 working voltage at 200 kV. The TEM samples were prepared by mixing one dilute drop of prepared samples dispersed in the appropriate solvent onto the copper grid and allowing it to dry well. Thermogravimetric analysis was accomplished on TGA-50H Shimadzu thermogravimetric analyzer. Samples were heated from 0 to 600 °C in a platinum pan with a heating rate of 10 °C/min in an N2 atmosphere and flow rate of 25 mL/min. 2.10. Antibacterial activity 2.10.1. Bacterial strains preparation Both Escherichia coli American Typing Culture Collection (ATCC) 25,922 and Salmonella enterica serovar Typhimurium ATCC 14028, as Gram-negative bacteria and both Staphylococcus aureus ATCC 43300 and Listeria monocytogenes ATCC 25152 as Gram-positive bacteria were used in this study. The stock bacterial strains with 10% glycerol at −20 °C were inoculated in 50 mL of Tryptic Soya Broth (TSB) (Merck, Germany). The tubes were incubated at 37 °C for 24 h. Then the strains were washed three times using sterile distilled water by centrifugation at 2500 rpm for 20 min to remove any debris matters. The strain counts approximately 106 CFU/mL were chosen by ten serial dilutions using pour plate method according to APHA (2012) [49]. The resulting colonies were counted and expressed as Colony Forming Unit (CFU/mL). 2.10.2. Antibacterial effect of the tested materials Each sample (500 mg) was transferred to a tube containing 10 mL of sterile distilled water. Based on the EDX analysis, the AgNPs contents in the samples of Cs-PVC/AgNPs (Cs2) and Cs-PVC/AgNPs-TiO2 (Cs3) conjugates were estimated to be 3 and 1 mg, respectively. The tube containing the sample was inoculated by 10 μL from the previously prepared strain. The tubes were incubated in a DAIHAN Scientific Shaker at 250 rpm. One mL was withdrawn after 0, 30, 60 and 120 min contact time interval. The sample was withdrawn at zero min contact time to determine the initial bacterial strain. One mL from each tube was transferred to a sterile Petri dish then the melted plate count agar was poured onto the dishes. The plates were incubated at 37 °C for 24 h. The resulting colonies were counted and expressed as Colony Forming Unit (CFU/mL). 2.10.3. Transmission electron microscopy (TEM) used for examination of E. coli interaction with Cs-PVC conjugates E. coli suspension was diluted to a final concentration of 106 CFU/mL. E. coli suspension (10 μL) was inoculated with 500 mg of the tested CsPVC conjugate into a test tube containing 10 mL of sterile distilled water. The tube was incubated at room temperature and at 250 rpm shaking
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Scheme 1. Preparation of 2-amino-N-(thiazol-2-yl)benzo[d]thiazole-6-sulfonamide (BTh) and amino-PVC.
speed for 30 min. E. coli suspension alone (without any added material) was used as a control. After that, a drop of the mixture was placed on a glow-discharged formvar-coated copper grid for 1 min. The excess liquid was drained off with a filter paper, and the preparation was air dried for 5 min. The specimen was examined using high-resolution transmission electron microscopy model JEM 2100-HRTEM (JEOL, USA, Inc.) operated at an accelerating voltage of 200 kV [50]. 3. Results and discussion 3.1. Chemistry In this study, we aim to synthesize Cs-PVC conjugates modified with organic and/or AgNPs antibacterial agents [51]. 2-Amino-N-(thiazol-2yl)benzo[d]thiazole-6-sulfonamide, the organic antibacterial agent, was synthesized as reported in the literature (Scheme 1) [47]. An approach to join PVC with Cs is to use a spacer between the two reacting polymers to avoid the steric hindrance. However, the introduction of this spacer to PVC was achieved by reaction with ethylenediamine in THF at boiling temperature to form amino-PVC (Scheme 1) [45]. The chemical modification of chitosan with antibacterial agents was achieved followed by the reaction with PVC as illustrated in Scheme 2. However, to couple the two polymers, another linker on the chitosan backbone is required to accomplish our goal. We thought that bromoacetyl bromide would be an appropriate candidate that easily joins the terminal amino group of the amino-PVC to the Cs amino group. Therefore, bromoacetylated chitosan was first prepared
according to a modified procedure [52–54]. Thus, chitosan was soaked in dry DMF overnight. The swelling process was followed by the addition of TEA and the bromoacetylating agent and the reaction mixture was allowed to react at room temperature. Functionalization of Cs with an organic antibacterial agent was achieved by adding BTh dissolved in DMF to the previously prepared bromoacetyl chitosan. The coupling reaction of functionalized chitosan and amino-PVC was finalized in one-pot reaction by adding amino-PVC dissolved in dry DMF to the reaction mixture. Functionalized Cs-PVC/AgNPs (Cs2) was synthesized using one-pot reaction approach. Thus, to the previously prepared Cs1 conjugate, silver nanoparticles (AgNPs) were generated in situ by adding AgNO3 (3%, w/w equiv. to Cs) and hydroxylamine hydrochloride successively and the reaction was stirred for 10 h at room temperature [55]. The reaction mixture was poured on ice/water and the precipitate was filtered, washed with acetone and methanol, and dried in the oven to produce Cs2. Similarly, functionalized Cs-PVC/Ag/TiO2 nanocomposite was synthesized by adding previously prepared Ag/TiO2 NPs to the reaction mixture of functionalized Cs-PVC to produce Cs3. 3.2. Characterization 3.2.1. FTIR spectroscopy The structures of the synthesized materials were studied by FTIR spectroscopy. The amino-PVC spectrum showed a strong band appeared at 3444 which was assigned to NH and NH2 stretch whereas the band at 1634 cm−1 was assigned to NH bend which was indicative
Scheme 2. Synthesis of BTh-functionalized Cs-PVC (Cs1), BTh-functionalized Cs-PVC-containing AgNPs (Cs2), and BTh-functionalized Cs-PVC containing Ag/TiO2-NPs (Cs3).
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(D) (C) (B)
2000
1500
609
697
1053 1025
1156
1250
1430 1380
(A)
1635
of the presence of NH and NH2 groups. The bands appeared at 2979 (C\\H), 2906 (CH2), 1427 (C\\C), 1326 (CH2), 1254 (C\\H), 1095 (C\\N), 961 (CH2), 697 (C\\Cl), and 620 (C\\Cl) cm−1 were attributed to the PVC backbone (Fig. 1; C). The FTIR spectrum of the Cs is shown in Fig. 1; A. The band appeared at 3434 cm−1 was assigned to O\\H and N\\H stretch whereas bands appeared at 2924 and 2850 cm−1 were attributed to C\\H and CH2, respectively. It also showed the amide carbonyl stretch at 1634 cm−1 in addition to bands at 1426 and 1382 cm−1 which were attributed to C\\H bend. A complex band appeared at 1158, 1083, and 1025 cm−1 were assigned to stretch vibrations due to C\\O and C\\C of the glucopyranose ring in chitosan. The structure of CsAc-Br was studied by FTIR which revealed the progression of new bands at 1458, 874, 694, and 548 cm−1, in addition to the chitosan characteristic IR bands (Fig. 1; B). The band at 1458 is characteristic for C\\CH2 while bands appeared at 694 and 548 cm−1 are possibly due to C\\Br stretch. The bands appeared 1634 and 1022 cm−1 were assigned to C_O amide and C\\O, respectively, in bromoacetyl chitosan. The FTIR spectrum of CsAcPVC showed vibrational bands at 3437, 2962, 2913, and 1633 cm−1 which were ascribed to O\\H and N\\H, C\\H, CH2, and the carbonyl amide stretching, respectively. The bands at 1430 and 1384 cm−1 were characteristic for CH\\OH and CH2\\OH (Cs) and C\\CH2 (Cs and PVC) bend in Cs, respectively (Fig. 2; B). The bands that shown at 1327 and 1253 cm−1 could be attributed, respectively, to CH2 and C\\H bend vibrations in PVC backbone. The complex band appeared at 1156 and 1082 cm−1 were assigned to stretch vibrations due to C\\O and C\\C of the glucopyranose ring in chitosan. The band appeared around 1024 cm−1 was assigned to C\\O and C\\C in both Cs and PVC. Bands appeared at 694 (C\\Cl) and 613 (C\\Cl) cm−1 were attributed to the PVC backbone. The FTIR spectrum of Cs-amino-PVC blend is shown in Fig. 2; C. It indicated that there are some remarkable variations regarding the band shapes and intensities between the spectrum of CsAcPVC conjugate and Cs-PVC blend which indicates that the two polymers reacted to form the conjugate.
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1000
500
-1
Wavenumber (cm ) Fig. 2. FTIR spectra (KBr disc): (A) Cs, (B) CsAcPVC, (C) Cs-PVC blend, and (D) amino-PVC.
The FTIR spectra of Cs1, Cs2, and Cs3 are illustrated in Fig. 3 below. The comparison of the IR spectra of Cs and the three Cs-PVC conjugates showed the coincidence of a characteristic absorption band at 3433 cm−1 assigned to OH and NH stretch in both Cs and amino-PVC. The common C_O amide group of Cs appeared around 1640 cm−1 for all conjugates. The spectra also showed the absorption bands around 2918 cm−1 assigned to C\\H stretch in Cs and PVC for all conjugates whereas absorption bands around 1430, 1380, 1326, and 1253 cm−1
(C) 1458
(B)
(A)
609
1430 1326 1250 1156 1053 1025
620
1083 1022
1380
1635
2906
2918
874
1253
1430
3433
(A)
1640
(C) (B)
3444
(D)
4000 3500 3000 2500 2000 1500 1000 -1
Wavenumber (cm ) Fig. 1. FTIR spectra (KBr disc): (A) Cs and (B) CsAc-Br, and (C) amino-PVC.
500
4000 3500 3000 2500 2000 1500 1000 -1 Wavenumber (cm ) Fig. 3. FTIR spectra (KBr disc): (A) chitosan, (B) Cs1, (C) Cs2, and (D) Cs3.
500
Fig. 4. (a) SEM micrographs for Cs2 and (b) its EDX spectrum, and (c) SEM micrographs for Cs3 and (d) its EDX spectrum.
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Fig. 5. TEM images for (a) Cs2 and (b) Cs3.
3.2.2. Scanning electron microscopy (SEM) of Cs2 and Cs3 The SEM images of the prepared samples Cs2 and Cs3 are shown in Fig. 4. From these images, it could be concluded that the polymer surfaces are homogenous and the AgNPs are well distributed. The metal nanoparticles appeared in the images in the form of light spots over the darker Cs-PVC polymer surface which proposing that the Ag NPs are well-fixed to the surface of the polymeric matrix. The EDX spectrum of Cs2 and Cs3 reveals the characteristic peaks of Ag around 3 keV due to surface plasmon resonance which confirms the formation of AgNPs with elemental composition of Ag equivalent to 0.61% and 0.21% in Cs2 and Cs3, respectively. In the EDX spectrum of Cs3 the characteristic peaks of Ti and Ag around 4.5 and 3 keV, respectively, support the presence of both Ti and Ag as Ag/TiO2 NPs. Additionally, the EDX profile also showed other peaks for C, O, N, S, and Cl for elemental constituents of chitosan and PVC.
3.2.4. Thermogravimetric analysis Thermal gravimetric analysis for Cs2 and Cs3 samples is presented in Fig. 6. It is noticed that the degradation of Cs3 starts at higher temperature compared to Cs2, which implies higher thermal stability value of Cs3 compared to Cs2 conjugate. The observed weight loss of Cs3 at 248 °C was 9% where the weight loss of the other conjugate (Cs2) reached 17% at 214 °C. The thermal degradation of both conjugates at the first stage of degradation may be due to the elimination of HCl from PVC subunit. At elevated temperature, both conjugates suffer from further thermal degradation. The weight loss reached 45% for Cs3 at 280 °C, where at the same temperature Cs2 lost about 65% of its mass. This substantial weight loss may be attributed to the progress of depolymerization process of the Cs subunit to form low molecular weight fragments [56]. The relative higher thermal stability of Cs3 at either first or later degradation stages compared to Cs2 can be rationalized to the inclusion of Ag/TiO2 NPs. 3.3. Biology 3.3.1. Antibacterial activities Bacterial deactivations were assessed for several samples reported in this work against E. coli and S. typhimurium as Gram-negative
120 100
Weight (%)
corresponding to C\\H (in CH2) bend in PVC residue in the three conjugates whereas the band around 609 cm−1 for the new conjugates could be attributed to C\\Cl stretch of the PVC residue. It is noteworthy that the common absorption bands appeared for Cs and the three conjugates in the region 1156–1025 cm−1 was attributed to C\\O stretch of Cs residue. The most interesting feature of the spectra was the relative intense absorption bands around 1430, 1053, and 609 cm−1 for the new conjugates which could be attributed to the PVC residue. In the spectra of Cs1, Cs2, and Cs3, a shoulder appeared around 1535 cm−1 which could be attributed to the C_C of BTh modifier. A relative decrease of absorption bands was found in the region 3600–3000 cm−1 which can be correlated to the diminished concentration of the amino groups of chitosan residue upon the reaction with amino-PVC via bromoacetyl bromide to produce Cs1, Cs2, and Cs3 conjugates. There was no evidence of carbonyl ester IR band which gave an evidence for the lack of attack on the hydroxyl group. The selective acylation of the amino groups is probably due to its superior nucleophilic character compared to the hydroxyl group of the glucopyranose ring.
Cs2 Cs3
80 60 40 20
3.2.3. Transmission electron microscopy (TEM) of Cs2 and Cs3 The TEM affords additional detailed exploration of the morphologies of the Cs2 and Cs3 samples. Fig. 5 presents the typical TEM images of the samples demonstrating that the TiO2 and Ag particles possess nanoscale structures. The diameters of the TiO2 nanospheres are 15–22 nm and for Ag from 3 to 7 nm. According to the above results, it is inferred that the structures of TiO2 and Ag are in nanoscale sizes.
0
80
160
240
320
400 o
Temperature ( C) Fig. 6. TGA analysis for Cs2 and Cs3.
480
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bacteria, and S. aureus and L. monocytogenes as Gram-positive bacteria and the results were listed in Table 1. BTh exhibited effects on all tested bacterial strains (Table 1). Two-log10 reduction was observed after 30 min contact time. In addition to this, Gram-negative bacteria were more susceptible to BTh compared to Gram-positive after 30 and 60 min contact time. The results also revealed that amino-PVC had a poor antibacterial effect on all selected bacterial strains. Only onelog10 reduction was observed after 120 min contact time. CsAc-Br had low antibacterial activity against all the testing bacterial strains after 30, 60 and 120 min contact time. Only one-log10 reduction was observed after 120 min contact time against E. coli, S. typhimurium, and L. monocytogenes. However, S. aureus showed the least susceptibility as indicated by obtaining the same log10 reduction after 30, 60 and 120 min contact time. CsAc-PVC conjugate showed improved antibacterial activities against the bacterial strains after 30, 60 and 120 min contact time compared to CsAc-Br. Thus, one-log10 reduction was observed after 30 min contact time against E. coli, S. typhimurium, and L. monocytogenes. Moreover, two-log10 reduction for the testing strains was exhibited by CsAc-PVC after 60 and 120 min contact times (Fig. 7a). The results revealed that Cs1 had almost equivalent antibacterial effects against all tested bacterial strains. Four-log10 reduction was observed after 30 min contact time. In addition, five-log10 reductions were observed after 60 and 120 min contact time (Fig. 7b). Cs2 had antibacterial effects on all of the testing bacterial strains. Five-log10 reduction was observed after 30 and 60 min contact time. In addition, sixlog10 reduction was observed after 120 min contact time (Fig. 8a). E. coli, S. aureus, and L. monocytogenes were relatively more susceptible for Cs2 than S. typhimurium after both 30 and 60 min contact time. Cs3 had antibacterial effects on all testing bacterial strains. Five-log10 reduction was observed after 30 and 60 min contact time. In addition, sixlog10 reduction was observed after 120 min contact time (Fig. 8b). Comparing the maximum antibacterial activities of CsAc-PVC, Cs1, Cs2, and Cs3 it was noted that the maximum inhibition that CsAc-PVC could achieve was two-log10 reduction after 120 min contact time. On the other hand, the three BTh- functionalized Cs-PVC conjugates exhibited six-log10 reduction during the same period. This pronounced difference in activities could be attributed to the presence of BTh in all
3.00E+05 2.50E+05 2.00E+05 1.50E+05 1.00E+05 5.00E+04 0.00E+00 E. coli
S. typhimurium
CFU/mL (30 min)
S. aureus
CFU/mL (60 min)
L.monocytogenes
CFU/mL (120 min)
(a) 140 120 100 80 60 40 20 0 E. coli
S. typhimurium
CFU/mL (30 min)
S. aureus
CFU/mL (60 min)
L. monocytogenes
CFU/mL (120 min)
(b) Fig. 7. Bacterial deactivations by (a) CsAc-PVC and (b) Cs1 at 30, 60, and 120 min contact time under shaking.
Table 1 Antibacterial activities expressed in CFU/mL of all tested samples against E. coli, S. typhimurium, S. aureus, and L. monocytogenes after 0 (control), 30, 60, and 120 min contact time.
BTh
Amino-PVC
CsAc-Br
CsAc-PVC
Cs1
Cs2
Cs3
Strain
Control
30 min
60 min
120 min
E. coli S. typhimurium S. aureus L. monocytogenes E. coli S. typhimurium S. aureus L. monocytogenes E. coli S. typhimurium S. aureus L. monocytogenes E. coli S. typhimurium S. aureus L. monocytogenes E. coli S. typhimurium S. aureus L. monocytogenes E. coli S. typhimurium S. aureus L. monocytogenes E. coli S. typhimurium S. aureus L. monocytogenes
2.90E + 06 3.00E + 06 2.80E + 06 2.80E + 06 6.10E + 06 4.90E + 06 8.20E + 06 5.90E + 06 2.9 E + 06 3.0 E + 06 2.8E + 06 2.8E + 06 2.90E + 06 3.00E + 06 2.80E + 06 2.80E + 06 2.90E + 06 3.00E + 06 2.80E + 06 2.80E + 06 2.90E + 06 3.00E + 06 2.80E + 06 2.80E + 06 2.90E + 06 3.00E + 06 2.80E + 06 2.80E + 06
2.10E + 04 1.30E + 04 5.30E + 04 4.90E + 04 2.90E + 06 3.00E + 06 2.80E + 06 2.80E + 06 2.1E + 06 1.9E + 06 1.0E + 06 2.2E + 06 1.20E + 05 1.00E + 05 2.80E + 05 2.30E + 05 130 110 120 118 15 62 17 32 5 40 17 32
9.00E + 03 8.90E + 03 2.10E + 04 1.60E + 04 9.00E + 05 9.90E + 05 1.00E + 06 1.10E + 06 9.0E + 05 9.8E + 05 1.0E + 06 9.96E + 05 9.60E + 04 7.20E + 04 8.90E + 04 9.90E + 04 90 80 99 90 10 50 11 20 3 23 11 20
3.60E + 03 2.90E + 03 5.10E + 03 4.00E + 03 1.20E + 05 1.30E + 05 3.40E + 05 2.30E + 05 5.2E + 05 6.0E + 05 1.2E + 06 5.2E + 05 3.60E + 04 2.20E + 04 2.10E + 04 1.30E + 04 10 10 23 13 2 8 5 8 1 8 5 8
S.T. Gaballah et al. / International Journal of Biological Macromolecules 121 (2019) 707–717 70 60 50 40 30 20 10 0 E. coli
S. typhimurium
CFU/mL (30 min)
S. aureus
CFU/mL (60 min)
L. monocytogenes
CFU/mL (120 min)
(a) 45 40 35 30 25 20 15 10 5 0 E. coli
S. typhimurium
CFU/mL (30 min)
S. aureus
CFU/mL (60 min)
L. monocytogenes
CFU/mL (120 min)
(b) Fig. 8. Bacterial deactivations by (a) Cs2 and (b) Cs3 at 30, 60, and 120 min contact time under shaking.
functionalized conjugates as well as the presence of AgNPs and Ag/TiO2 NPs in Cs2 and Cs3, respectively. 3.3.2. TEM investigation of the interaction between E. coli and Cs2 Although the mechanism of the antibacterial activity of Cs is not fully understood, there are many plausible mechanisms that describe the interaction between Cs as antibacterial agent and bacteria. One of these mechanisms is recognized due to the presence of charged groups in the polymeric backbone and their ionic interactions with bacteria cell wall constituents [57]. This interaction suggests the hydrolysis of the peptidoglycans in the bacterial cell wall, triggering the leakage of intracellular electrolytes, leading to the bacterial death. The charges present in Cs chains are generated by protonation of amino groups to form (NH3)+ when present in an acid medium or they may be introduced via structural modification. According to this mechanism, this kind of ionic interaction between the positively charged Cs backbone and the negatively charged cell surface is unlikely to occur in our case due to two main reasons. Firstly, the Cs backbone is modified with neutral moiety and secondly, the pH of the medium during the assay was neutral and hence protonation of the remaining amino groups to form positively-charged backbone on Cs is excluded.
Fig. 9. TEM micrograph of (a) untreated E. coli (control) as a Gram-negative bacterium, (b) E. coli treated with Cs2 for 30 min illustrating partial cell membrane damage and shrinkage, and (c) E. coli treated with Cs2 illustrating cell shrinkage due to the destructed cell membrane and cytoplasm expulsion.
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To obtain a comprehensive understanding of the antibacterial action of Cs further verification by TEM was performed for E. coli subjected to Cs2 conjugate (Fig. 9). TEM images of Cs2 conjugate which contains AgNPs showed that the E. coli cell wall was disrupted. Our hypothesis is that the cell wall fault may be directly related to the binding between Cs and the protein of bacterial outer membrane via hydrogen bonding and electrostatic interactions [58]. These interactions change the cell membrane permeability which causes leakage of intracellular substances, hereby alters the membrane functionality and finally leads to cell death. The presence of BTh modifier is thought to enhance hydrogen bonding between the Cs-PVC conjugates and the bacterial cell membrane due to the presence of both H-bond donor and H-bond acceptor on the BTh moiety. However, AgNPs showed a low synergistic effect on the antibacterial action of Cs2 and Cs3 compared to Cs1. 4. Conclusions We herein address the synthesis of four Cs-PVC conjugates which can be used for biomedical applications. These conjugates were synthesized by reacting amino-PVC with Cs or BTh-functionalized Cs using bromoacetyl bromide as a linker and gave CsAc-PVC and BThfunctionalized conjugates (Cs1, Cs2, and Cs3), respectively. AgNPs were incorporated in Cs2 via in situ reduction of Ag salt using hydroxylamine hydrochloride whereas incorporation of Ag/TiO2 NPs in Cs3 conjugate occurred simply by mixing. All the synthesized materials were characterized by FTIR spectroscopic analysis, morphological investigations such as SEM and TEM electron microscopies, and thermal gravimetric analysis (TGA). Spectral data confirmed the introduction of the BTh to the Cs backbone as well as the coupling between the two polymers. SEM data showed homogenous polymer surfaces with welldistributed Ag nanoparticles. The EDX spectra of Cs2 and Cs3 showed the presence of AgNPs and Ag/TiO2 NPs among the polymeric matrix. The characteristic peaks were attributed to the C, O, N, S, Ag and Cl Kα lines. The characteristic maximum peak for Cl appeared at about 2.7 keV. The presence of AgNPs or Ag/TiO2 NP was confirmed and elemental Ag or Ag/TiO2 signal have been clearly identified. The Ag contents in the prepared samples Cs2 and Cs3 were 0.61 and 0.21%, respectively. TEM analysis showed that the AgNPs and Ag/TiO2 were of spherical with regular shapes while the sizes were in the range of 3–7 nm and 15–22 nm for the prepared conjugates, respectively. The antibacterial activity of the synthesized conjugates was investigated against two Gram-negative (E. coli, and S. typhimurium) and two Gram-positive (S. aureus, and L. monocytogenes) bacteria. The antibacterial assay showed that all three Cs-PVC (Cs1, Cs2, and Cs3) conjugates functionalized with BTh exhibited excellent bacterial inhibition compared to CsAc-PVC conjugate. Investigation of the mode of interaction between the synthesized conjugates and bacteria was recorded by TEM scan which revealed bacterial cell wall deterioration by the antibacterial agent. These results suggested that the use of these conjugates may provide a revolutionary antibacterial material that can be useful in biomedical applications. Acknowledgment The authors acknowledge the National Research Centre (NRC) Egypt for funding this work; Grant number (11090111). References [1] E.-R. Kenawy, S.D. Worley, R. Broughton, The chemistry and applications of antimicrobial polymers: a state-of-the-art review, Biomacromolecules 8 (5) (2007) 1359–1384. [2] D.F. Williams, The Williams Dictionary of Biomaterials, Liverpool University Press, 1999. [3] W.A.N. Dorland, Dorland's Illustrated Medical Dictionary, Saunders, 2000. [4] N. Akmal, A.M. Usmani, Medical polymers and diagnostic reagents, in: H. S (Ed.), Medical Polymers and Diagnostic Reagents, Marcel Dekkel Inc., New York 2000, pp. 485–495.
[5] R.R. Xu, L.X. Song, Y. Teng, J. Xia, Ferrous chloride-induced modification on thermal properties of polyvinyl chloride, Thermochim. Acta 565 (2013) 205–210. [6] N.R. James, A. Jayakrishnan, Surface thiocyanation of plasticized poly(vinyl chloride) and its effect on bacterial adhesion, Biomaterials 24 (13) (2003) 2205–2212. [7] M. Polaskova, M. Sowe, I. Kuritka, T. Sedlacek, M. Machovsky, P. Sáha, Medical-grade polyvinyl chloride modified with crystal violet and montmorillonite, Int. J. Polym. Anal. Charact. 15 (1) (2010) 18–26. [8] S. Lakshmi, S.S. Kumar, A. Jayakrishnan, Bacterial adhesion onto azidated poly(vinyl chloride) surfaces, J. Biomed. Mater. Res. 61 (2002) 26–32. [9] P. Vergidis, R. Patel, Novel approaches to the diagnosis, prevention, and treatment of medical device-associated infections, Infect. Dis. Clin. N. Am. 26 (1) (2012) 173–186. [10] D.S. Jones, J.G. McGovern, A.D. Woolfson, S.P. Gorman, Role of physiological conditions in the oropharynx on the adherence of respiratory bacterial isolates to endotracheal tube poly(vinyl chloride), Biomaterials 18 (6) (1997) 503–510. [11] J. Baptista, M. Simões, A. Borges, Effect of plant-based catecholic molecules on the prevention and eradication of Escherichia coli biofilms: a structure activity relationship study, Int. Biodeterior. Biodegrad. (2018). https://doi.org/10.1016/j.ibiod.2018.02.004. [12] Y.H. An, R.J. Friedman, Prevention of sepsis in total joint arthroplasty, J. Hosp. Infect. 33 (2) (1996) 93–108. [13] Y.H. An, R.J. Friedman, Concise review of mechanisms of bacterial adhesion to biomaterial surfaces, J. Biomed. Mater. Res. 43 (3) (1998) 338–348. [14] A. Gristina, Biomaterial-centered infection: microbial adhesion versus tissue integration, Science 237 (4822) (1987) 1588–1595. [15] A.G. Gristina, C.D. Hobgood, L.X. Webb, Q.N. Myrvik, Adhesive colonization of biomaterials and antibiotic resistance, Biomaterials 8 (6) (1987) 423–426. [16] N.P. Desai, S.F.A. Hossainy, J.A. Hubbell, Surface-immobilized polyethylene oxide for bacterial repellence, Biomaterials 13 (7) (1992) 417–420. [17] K.D. Park, Y.S. Kim, D.K. Han, Y.H. Kim, E.H.B. Lee, H. Suh, K.S. Choi, Bacterial adhesion on PEG modified polyurethane surfaces, Biomaterials 19 (7) (1998) 851–859. [18] Z. Zdanowski, B. Koul, E. Hallberg, C. Schalén, Influence of heparin coating on in vitro bacterial adherence to poly(vinyl chloride) segments, J. Biomater. Sci. Polym. Ed. 8 (11) (1997) 825–832. [19] D.J. Balazs, K. Triandafillu, Y. Chevolot, B.O. Aronsson, H. Harms, P. Descouts, H.J. Mathieu, Surface modification of PVC endotracheal tubes by oxygen glow discharge to reduce bacterial adhesion, Surf. Interface Anal. 35 (3) (2003) 301–309. [20] K. Triandafillu, D.J. Balazs, B.O. Aronsson, P. Descouts, P. Tu Quoc, C. van Delden, H.J. Mathieu, H. Harms, Adhesion of Pseudomonas aeruginosa strains to untreated and oxygen-plasma treated poly(vinyl chloride) (PVC) from endotracheal intubation devices, Biomaterials 24 (8) (2003) 1507–1518. [21] A.C. Abreu, A. Borges, F. Mergulhão, M. Simões, Use of phenyl isothiocyanate for biofilm prevention and control, Int. Biodeterior. Biodegrad. 86 ( (2014) 34–41. [22] R.P. Allaker, The use of nanoparticles to control oral biofilm formation, J. Dent. Res. 89 (11) (2010) 1175–1186. [23] B. Subhadra, D. Kim, K. Woo, S. Surendran, C. Choi, Control of biofilm formation in healthcare: recent advances exploiting quorum-sensing interference strategies and multidrug efflux pump inhibitors, Materials 11 (9) (2018) 1676. [24] S.M. Hudson, C. Smith, Polysaccharides: chitin and chitosan: chemistry and technology of their use as structural materials, in: D.L. Kaplan (Ed.), Biopolymers from Renewable Resources, Springer Berlin Heidelberg, Berlin, Heidelberg 1998, pp. 96–118. [25] E.-R. Kenawy, F.I. Abdel-Hay, M.S. Mohy Eldin, T.M. Tamer, E.M.A.-E. Ibrahim, Novel aminated chitosan-aromatic aldehydes Schiff bases: synthesis, characterization and bio-evaluation, Int. J. Adv. Res. 3 (2) (2015) 563–572. [26] S. Kumar, J. Dutta, P.K. Dutta, Preparation and characterization of N-heterocyclic chitosan derivative based gels for biomedical applications, Int. J. Biol. Macromol. 45 (4) (2009) 330–337. [27] M. Másson, J. Holappa, M. Hjálmarsdóttir, Ö. Rúnarsson, T. Nevalainen, T. Järvinen, Antimicrobial activity of piperazine derivatives of chitosan, Carbohydr. Polym. 74 (3) (2008) 566–571. [28] A. Basu, K.R. Kunduru, E. Abtew, A.J. Domb, Polysaccharide-based conjugates for biomedical applications, Bioconjug. Chem. 26 (8) (2015) 1396–1412. [29] Kashish, S. Bansal, A. Jyoti, K. Mahato, P. Chandra, R. Prakash, Highly sensitive in vitro biosensor for enterotoxigenic Escherichia coli detection based on ssdna anchored on PtNPs-chitosan nanocomposite, Electroanalysis 29 (11) (2017) 2665–2671. [30] A. Baranwal, A. Kumar, A. Priyadharshini, G.S. Oggu, I. Bhatnagar, A. Srivastava, P. Chandra, Chitosan: an undisputed bio-fabrication material for tissue engineering and bio-sensing applications, Int. J. Biol. Macromol. 110 (2018) 110–123. [31] F. Croisier, C. Jérôme, Chitosan-based biomaterials for tissue engineering, Eur. Polym. J. 49 (4) (2013) 780–792. [32] S.M. Ahsan, M. Thomas, K.K. Reddy, S.G. Sooraparaju, A. Asthana, I. Bhatnagar, Chitosan as biomaterial in drug delivery and tissue engineering, Int. J. Biol. Macromol. 110 (2018) 97–109. [33] W.-W. Hu, W.-J. Syu, W.-Y. Chen, R.-C. Ruaan, Y.-C. Cheng, C.-C. Chien, C. Li, C.-A. Chung, C.-W. Tsao, Use of biotinylated chitosan for substrate-mediated gene delivery, Bioconjug. Chem. 23 (8) (2012) 1587–1599. [34] T.C. Dakal, A. Kumar, R.S. Majumdar, V. Yadav, Mechanistic basis of antimicrobial actions of silver nanoparticles, Front. Microbiol. 7 (1831) (2016). [35] G.V. Vimbela, S.M. Ngo, C. Fraze, L. Yang, D.A. Stout, Antibacterial properties and toxicity from metallic nanomaterials, Int. J. Nanomedicine 12 (2017) 3941–3965. [36] M. Bilal, T. Rasheed, H.M.N. Iqbal, C. Li, H. Hu, X. Zhang, Development of silver nanoparticles loaded chitosan-alginate constructs with biomedical potentialities, Int. J. Biol. Macromol. 105 (2017) 393–400. [37] A. Baranwal, K. Mahato, A. Srivastava, P.K. Maurya, P. Chandra, Phytofabricated metallic nanoparticles and their clinical applications, RSC Adv. 6 (107) (2016) 105996–106010. [38] R. Kalaivani, M. Maruthupandy, T. Muneeswaran, A. Hameedha Beevi, M. Anand, C.M. Ramakritinan, A.K. Kumaraguru, Synthesis of chitosan mediated silver
S.T. Gaballah et al. / International Journal of Biological Macromolecules 121 (2019) 707–717
[39]
[40]
[41]
[42]
[43]
[44]
[45]
[46] [47] [48]
nanoparticles (Ag NPs) for potential antimicrobial applications, Front. Lab. Med. 2 (1) (2018) 30–35. S. Kumar-Krishnan, E. Prokhorov, M. Hernández-Iturriaga, J.D. Mota-Morales, M. Vázquez-Lepe, Y. Kovalenko, I.C. Sanchez, G. Luna-Bárcenas, Chitosan/silver nanocomposites: synergistic antibacterial action of silver nanoparticles and silver ions, Eur. Polym. J. 67 (2015) 242–251. L. Biao, S. Tan, Y. Wang, X. Guo, Y. Fu, F. Xu, Y. Zu, Z. Liu, Synthesis, characterization and antibacterial study on the chitosan-functionalized Ag nanoparticles, Mater. Sci. Eng. C 76 (2017) 73–80. A. Baranwal, A. Srivastava, P. Kumar, V.K. Bajpai, P.K. Maurya, P. Chandra, Prospects of nanostructure materials and their composites as antimicrobial agents, Front. Microbiol. 9 (422) (2018). R. Taurino, C. Sciancalepore, L. Collini, M. Bondi, F. Bondioli, Functionalization of PVC by chitosan addition: compound stability and tensile properties, Compos. Part B 149 (2018) 240–247. L. Zhang, W. Cai, W.-Y. Chen, L. Zhang, K. Hu, Y.-Q. Guan, Synthesis of AzPhchitosanbifenthrin-PVC to protect cables against termites, Carbohydr. Polym. 139 (2016) 50–60. P. Dwivedi, S.S. Narvi, R.P. Tewari, A novel Ag/CS-PVC nanomaterial with high antimicrobial properties: a potential self-sterilizing biomaterial Int, J. Sci. Res. 2 (7) (2012) 1–5. M.S. Mohy Eldin, T.M. Tamer, M.A. Abu Saied, E.A. Soliman, N.K. Madi, I. Ragab, I. Fadel, Click grafting of chitosan onto PVC surfaces for biomedical applications, Adv. Polym. Technol. 37 (1) (2018) 38–49, https://doi.org/10.1002/adv.21640. L.C. Leitch, B.E. Baker, L. Brickman, Synthesis of sulphanilylthiourea and related compounds, Can. J. Res. Sec. B: Chem. Sci. 23 (4) (1945) 139. R. Pohloudek-Fabini, M. Schuessler, Pharmazie 22 (1967) 620–624. M. Bellardita, H.A.E. Nazer, V. Loddo, F. Parrino, A.M. Venezia, L. Palmisano, Photoactivity under visible light of metal loaded TiO2 catalysts prepared by low frequency ultrasound treatment, Catal. Today 284 (2017) 92–99.
717
[49] APHA, Standard Methods for the Examination of Water and Wastewaterb, American Public Health Association, Washington, D.C., USA, 2012. [50] W.K. Jung, H.C. Koo, K.W. Kim, S. Shin, S.H. Kim, Y.H. Park, Antibacterial activity and mechanism of action of the silver ion in Staphylococcus aureus and Escherichia coli, Appl. Environ. Microbiol. 74 (7) (2008) 2171–2178. [51] İ. Yalçin, İ. Ören, E. Şener, A. Akin, N. Uçartürk, The synthesis and the structureactivity relationships of some substituted benzoxazoles, oxazolo(4,5-b)pyridines, benzothiazoles and benzimidazoles as antimicrobial agents, Eur. J. Med. Chem. 27 (4) (1992) 401–406. [52] Z. Zhong, B. Aotegen, H. Xu, S. Zhao, Structure and antimicrobial activities of benzoyl phenyl-thiosemicarbazone-chitosans, Int. J. Biol. Macromol. 50 (2012) 1169–1174. [53] Y. Qin, S. Liu, R. Xing, K. Li, H. Yu, P. Li, Synthesis and antifungal evaluation of (1,2,3triazol-4-yl)methyl nicotinate chitosan, Int. J. Biol. Macromol. 61 (2013) 58–62. [54] Ö.V. Rúnarsson, J. Holappa, C. Malainer, H. Steinsson, M. Hjálmarsdóttir, T. Nevalainen, M. Másson, Antibacterial activity of N-quaternary chitosan derivatives: synthesis, characterization and structure activity relationship (SAR) investigations, Eur. Polym. J. 46 (2010) 1251–1267. [55] N. Leopold, B. Lendl, A new method for fast preparation of highly surface-enhanced Raman scattering (SERS) active silver colloids at room temperature by reduction of silver nitrate with hydroxylamine hydrochloride, J. Phys. Chem. B 107 (24) (2003) 5723–5727. [56] M.A. Diab, A.Z. El-Sonbati, I.M. El-dien, D.M.D. Bader, Thermal stability and degradation of chitosan modified with phenylacetic acid, Korean J. Chem. Eng. 30 (10) (2013) 1966–1971. [57] R.C. Goya, S.T.B. Moraisb, O.B.G. Assis, Evaluation of the antimicrobial activity of chitosan and its quaternized derivative on E. coli and S. aureus growth, Braz. J. Pharmacol. 26 (2016) 122–127. [58] S.J. Jeon, M. Oh, W.-S. Yeo, K.N. Galva, K.C. Jeong, Underlying mechanism of antimicrobial activity of chitosan microparticles and implications for the treatment of infectious diseases, PLoS One 9 (3) (2014), e92723.