nanomaterials Article

Quaternized Carboxymethyl Chitosan-Based Silver Nanoparticles Hybrid: Microwave-Assisted Synthesis, Characterization and Antibacterial Activity Siqi Huang 1,2 , Jing Wang 1,2 , Yang Zhang 1,2, *, Zhiming Yu 1,2, * and Chusheng Qi 1,2 1 2

*

Beijing Key Laboratory of Wood Science and Engineering, Beijing Forestry University, Beijing 100083, China; [email protected] (S.H.); [email protected] (J.W.); [email protected] (C.Q.) MOE Key Laboratory of Wooden Material Science and Application, Beijing Forestry University, Beijing 100083, China Correspondence: [email protected] (Y.Z.); [email protected] (Z.Y.); Tel.: +86-10-6233-8295 (Y.Z.); +86-10-6233-6907 (Z.Y.)

Academic Editor: Thomas Nann Received: 3 May 2016; Accepted: 1 June 2016; Published: 17 June 2016

Abstract: A facile, efficient, and eco-friendly approach for the preparation of uniform silver nanoparticles (Ag NPs) was developed. The synthesis was conducted in an aqueous medium exposed to microwave irradiation for 8 min, using laboratory-prepared, water-soluble quaternized carboxymethyl chitosan (QCMC) as a chemical reducer and stabilizer and silver nitrate as the silver source. The structure of the prepared QCMC was characterized using Fourier transform infrared (FT-IR) and 1 H nuclear magnetic resonance (NMR). The formation, size distribution, and dispersion of the Ag NPs in the QCMC matrix were determined using X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), ultraviolet-visible (UV-Vis), transmission electron microscopy (TEM), and field emission scanning electron microscope (FESEM) analysis, and the thermal stability and antibacterial properties of the synthesized QCMC-based Ag NPs composite (QCMC-Ag) were also explored. The results revealed that (1) QCMC was successfully prepared by grafting quaternary ammonium groups onto carboxymethyl chitosan (CMC) chains under microwave irradiation in water for 90 min and this substitution appeared to have occurred at -NH2 sites on C2 position of the pyranoid ring; (2) uniform and stable spherical Ag NPs could be synthesized when QCMC was used as the reducing and stabilizing agent; (3) Ag NPs were well dispersed in the QCMC matrix with a narrow size distribiution in the range of 17–31 nm without aggregation; and (4) due to the presence of Ag NPs, the thermal stability and antibacterial activity of QCMC-Ag were dramatically improved relative to QCMC. Keywords: quaternized carboxymethyl chitosan; uniform silver nanoparticles; microwave synthesis; thermal stability; antibacterial activity

1. Introduction Silver nanoparticles (Ag NPs) have recently received extensive attention from researchers and developers due to their attractive optical, electronic, and catalytic properties and excellent antimicrobial activity [1–3]. Ag NPs exhibit strong antibacterial activity toward a broad range of microorganisms [4], but simultaneously possess a remarkably low human toxicity. These properties have caused these new materials to be recognized as one of the most powerful antimicrobial agents of the 21st century, resulting in their wide use in the biomedical field, food packaging, coating, clothing, textile, and other applications [5,6]. Numerous methods have been developed to prepare Ag NPs, including chemical reduction, thermal decomposition, UV irradiation reduction, photoreduction, electrolytic process, gamma Nanomaterials 2016, 6, 118; doi:10.3390/nano6060118

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irradiation, and others [7]. In these methods, the physical approaches require highly sophisticated instruments and specific conditions [8], while chemical reduction offers the superiority of simple processibility making it widely used. However, conventional chemical reduction methods require reducing agents including sodium borohydride (NaBH4 ), hydrazine hydrate (HHA), or other organic compounds in addition to stabilizing agents such as triphenylphosphine, polyvinylpyrrolidone, and sodium citrate. Many of these reagents have potential environmental toxicity and biological risks [9,10]. The increasing awareness of environmental protection has led to the development of an eco-friendly approach for the synthesis of Ag NPs using judicious choices of reducing and stabilizing agents and solvents. From the perspective of green synthesis, the biopolymer chitosan has attracted extensive research interest for the preparation of Ag NPs. Chitosan is the second most abundant natural polysaccharide with β-1,4-linked N-acetyl-D-glucosamine and D-glucosamine units [11,12], which possess largely free amino and hydroxyl groups offering a template for growing nanometals. Wei et al. synthesized Ag NPs by reducing cationic silver using polycationic chitosan, but this process was time-consuming and the resulting Ag NPs varied in size [2]. In effort to prepare uniform Ag NPs rapidly, An et al. used carboxymethyl chitosan (CMC) as a stabilizer, but an additional reducing agent NaBH4 was also required [13]. Quaternized carboxymethyl chitosan (QCMC) is a novel amphoteric polymer synthesized by grafting carboxymethyl groups and quaternary ammonium groups onto chitosan chains [14]. Favored for its properties of good water solubility, antimicrobial effect, biodegradability, biocompatibility, high moisture-absorption and moisture-retention, QCMC is widely applied in antioxidant, drug encapsulation, tissue engineering, as well as the cosmetics and antibacterial fields [15,16]. What’s more, QCMC is considered a promising green reducing and stabilizing agent for the preparation of nanometals. It has been deduced that the large number of hydroxyl groups, carboxyl groups and quaternary ammonium groups on the chains of QCMC makes it remarkable as a reducing agent, and it also shows great stabilizing performance due to the surfactant-like structure [17,18]. The use of microwave heating for chemical synthesis, pioneered by Gedye, is faster and simpler than similar thermal transfer methods and it is especially appropriate for nanometal synthesis, because it provides uniform heating around the nanoparticles and can assist in their ripening without aggregation [19,20]. Therefore, the combination of microwave heating with a green reducing and stabilizing agent can open a new pathway for the synthesis of Ag NPs. In the present study, a rapid, simple and totally green method was developed to synthesize uniform, stable Ag NPs using laboratory-prepared quaternized carboxymethyl chitosan (QCMC) as a reducing and stabilizing agent. The structure of the prepared QCMC was characterized and the formation, size distribution, and dispersion of the Ag NPs in the QCMC matrix were investigated in detail. From another point of view, the presence of Ag NPs may also enhance the thermal stability and antimicrobial activity of the QCMC, resulting in outstanding hybrid performance. However, to the best of our knowledge, there have been relatively few reports in the literature of the properties of QCMC-based Ag NPs composite (QCMC-Ag). Thus, the thermal stability and antibacterial capability of the synthesized QCMC-Ag were also explored. 2. Materials and Methods 2.1. Materials CMC was obtained from Zhejiang Biochemical Co. (Taizhou, China) and was used as received. The molecular weight of the CMC was 100 kDa, the degree of substitution of the carboxymethyl groups was 0.91, and the degree of deacetylation was 90.2%. The 2,3-epoxypropyltrimethyl ammonium chloride (ETA) was provided by Shanghai Dibo Chemical Technology Co., Ltd. (Shanghai, China). Silver nitrate was purchased from Guangdong Guanghua Science and Technology Co., Ltd. (Guangdong, China). An XH-100A microwave synthesis system was obtained from Beijing Xiang-Hu

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from BeijingCo., Xiang-Hu Technology Co., Ltd. 1(Beijing, China). Figure 1 shows chemical Technology Ltd. (Beijing, China). Figure shows the chemical structure of the CMC [21,22] structure and ETA. of CMC and ETA. All other and solvents were of used analytical grade and were used as All other[21,22] reagents and solvents werereagents of analytical grade and were as received. received.

(a)

(b)

Figure 1. 1. Chemical of (a) (a) carboxymethyl carboxymethyl chitosan chitosan (CMC) (CMC) and and (b) (b) 2,3-epoxypropyltrimethyl 2,3-epoxypropyltrimethyl Figure Chemical structure structure of ammonium chloride (ETA). ammonium chloride (ETA).

2.2. Preparation of QCMC 2.2. Preparation of QCMC The QCMC was prepared based on a reference with modification [16]. The synthesis was The QCMC was prepared based on a reference with modification [16]. The synthesis was conducted in the microwave unit using ETA as the quaternized modification agent. Five grams of conducted in the microwave unit using ETA as the quaternized modification agent. Five grams CMC powder were dissolved in distilled water with ultrasonic stirring for 30 min and then placed in of CMC powder were dissolved in distilled water with ultrasonic stirring for 30 min and then placed in the microwave synthesis equipment. The ETA was dissolved in 50 mL of distilled water and the microwave synthesis equipment. The ETA was dissolved in 50 mL of distilled water and dropped dropped slowly into the CMC solution under microwave irradiation for 1000 W and reacted at 80 °C slowly into the CMC solution under microwave irradiation for 1000 W and reacted at 80 ˝ C for 90 min. for 90 min. The mass ratio of the ETA to CMC was six to one. Next, the reaction mixture was The mass ratio of the ETA to CMC was six to one. Next, the reaction mixture was precipitated and precipitated and washed three times using pure ethanol. After dialyzing the mixture against washed three times using pure ethanol. After dialyzing the mixture against deionized water for deionized water for 7 days, the resultant product was freeze-dried at −60 °C and ground to a powder 7 days, the resultant product was freeze-dried at ´60 ˝ C and ground to a powder in a glass mortar. in a glass mortar. The molecular weight of QCMC determined using GPC was 98 kDa and the degree The molecular weight of QCMC determined using GPC was 98 kDa and the degree of substitution of of substitution of the quaternary ammonium groups was 0.89 which was determined by the quaternary ammonium groups was 0.89 which was determined by potentiometry [23]. potentiometry [23]. 2.3. Preparation of the QCMC-Based Ag NPs Composite 2.3. Preparation of the QCMC-Based Ag NPs Composite The QCMC powder was dissolved in distilled water to prepare a 2 mg/mL solution after The QCMCfor powder wasSilver dissolved in was distilled water into 10 prepare 2 mg/mL solution after ultrasonication 10 min. nitrate dissolved mL ofadistilled water to obtain ultrasonication for 10 min. Silver nitrate was dissolved in 10 mL of distilled water to obtain a 0.1 a 0.1 mmol/mL solution, and then was added drop-wise to the QCMC aqueous solution with magnetic mmol/mL solution, and then was added drop-wise to the QCMC aqueous solution with magnetic stirring at room temperature. Next, the mixture was heated via microwave irradiation at 1000 W at ˝ C for stirring at8room temperature. Next, waswas heated via microwave at 1000water W at 70 min. A QCMC-based Ag the NPsmixture composite obtained by dialysisirradiation against distilled 70 °C for 8 min. A QCMC-based Ag NPs composite was obtained by dialysis against distilled water until silver ions could not be detected in the dialysate using 0.1mol/L HCl solution. The product ˝ C. The ratio untilthen silverlyophilized ions could at not´60 be detected in theofdialysate using 0.1mol/L HCl solution. The product was was the QCMC to AgNO 3 was 500 mg: 1 mmol. The final then lyophilized at −60 °C. ratio of the QCMC to AgNO3 was 500 mg: 1 mmol. The final composite was designated as The QCMC-Ag. composite was designated as QCMC-Ag. 2.4. Characterization 2.4. Characterization Waters-1515 gel permeation chromatograph (GPC, Waters, MI, USA) was used to evaluate the Waters-1515 permeation (GPC, Waters,(FT-IR) MI, USA) waswere used obtained to evaluate the molecular weightgel of the products.chromatograph Fourier transform infrared spectra using 1 weight the products. Fourier transform infrared (FT-IR) H spectra obtained using a amolecular Vertex 70v FT-IR of spectrophotometer (Bruker, Karlsruhe, Germany). NMR were spectra were recorded Vertex 70v FT-IR spectrophotometer (Bruker, Karlsruhe, Germany). NMR spectra recorded on an ASCEND 400 spectrometer (Bruker, Karlsruhe, Germany). X-ray1H diffraction (XRD)were patterns were on an ASCEND spectrometer Karlsruhe, Germany). X-ray diffraction (XRD) intensity patterns measured using a400 D8 advance X-ray(Bruker, diffractometer (Bruker, Karlsruhe, Germany); the relative wererecorded measured a D8 advance Karlsruhe, Germany); the relative was inusing the scattering rangeX-ray (2θ) ofdiffractometer 5˝ –90˝ . X-ray(Bruker, photoelectron spectroscopy (XPS) spectra intensity was recorded in the scattering range (2θ) of 5–90°. X-ray photoelectron spectroscopy were obtained using PHI 5000CESCA System surface analysis equipment (PHI Co., Chanhassen,(XPS) MN, spectra were obtained System surface analysis Co., USA) with Mg-K source PHI (hγ =5000CESCA 1253.6 eV). Ultraviolet-Visible (UV-Vis) equipment spectra were(PHI recorded α X-ray using Chanhassen, MN, USA)nm with Mg-K α X-ray UV-Vis source (hγ = 1253.6 eV). Ultraviolet-Visible in the range of 300–600 using UV2310II spectrophotometer (INESA, Shanghai,(UV-Vis) China). spectra weretransmission recorded inelectron the range of 300–600 nm using UV2310II spectrophotometer JEOL-2100F microscopy (TEM, JEOL, Tokyo, Japan)UV-Vis was used to investigate the (INESA, Shanghai, JEOL-2100F electron microscopy (TEM, JEOL, Tokyo, Japan) microstructure andChina). size distribution of transmission the Ag NPs. The size distribution was statistically analyzed was300 used to investigate thenanoparticles microstructure andthe sizepublic distribution of Image the AgJ.NPs. Themorphology size distribution for randomly selected using software Surface was was statistically analyzedfield for emission 300 randomly selected nanoparticles using the public software Image J. observed via an SU8010 scanning electron microscope (FESEM, Hitachi, Japan). Surface morphology was observed via an SU8010 field emission scanning electron microscope (FESEM, Hitachi, Japan).

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2.5. Thermal Stability Experiment Thermal stability was evaluated by thermogravimetric analysis (TGA) using a TGAQ50 thermal analyzer (TA, New Castle, DE, USA). TGA and derivative thermogravimetry (DTG) thermograms Nanomaterials 2016, 6, 118 of 13 were obtained in the temperature range of 30 to 700 ˝ C under nitrogen and with a ramp rate4 of 10 ˝ C/min. 2.5. Thermal Stability Experiment 2.6. Antibacterial Assay Thermal stability was evaluated by thermogravimetric analysis (TGA) using a TGAQ50 thermal

To study the antibacterial property of the QCMC-based Ag NPs composite, gram-positive analyzer (TA, New Castle, DE, USA). TGA and derivative thermogravimetry (DTG) thermograms Staphylococcus aureus in (provided by therange Chinese Academy of Sciences) served model were obtained the temperature of 30 to 700 °C under nitrogen and with aasramp rate pathogenic of 10 °C/min. bacteria. The antibacterial test was performed according to our previous report [24]. Antibacterial solutions were diluted to appropriate concentrations in phosphate buffer (pH 7.2), and 10 mL of each sample 2.6. Antibacterial Assay was added to sterile petri dishes containing 10 mL of nutrient agar solution. Staphylococcus aureus the antibacterial property of 7the QCMC-based Ag NPs suspension composite, gram-positive was adjustedTobystudy sterile distilled water to 10 cfu/mL. A bacterial of 2 µL was then Staphylococcus aureus (provided by the Chinese Academy of Sciences) served as model pathogenic inoculated on the prepared nutrient medium-containing antibacterial suspension and incubated at bacteria. The antibacterial test was performed according to our previous report [24]. Antibacterial 37 ˝ C. The minimum inhibition concentration (MIC) in values were measured a mL 24 hofculture. solutions were diluted to appropriate concentrations phosphate buffer (pH 7.2),after and 10 each was added sterile petri dishes containing 10 mL of analyzed nutrient agar solution. Staphylococcus The sample concentration of to silver released from QCMC-Ag was using a Thermo Scientific iCAP was adjusted by sterile distilled water toemission 107 cfu/mL.spectrometer A bacterial suspension of 2 μL was then 6500 Duoaureus inductively coupled plasma-optical (ICP-OES, TMO, Cambridge, inoculated on the prepared nutrient medium-containing antibacterial suspension and incubated at UK). To measure the silver release, the QCMC-Ag (approximately 0.05 g) was immersed in 10 mL of 37 °C. The minimum inhibition concentration (MIC) values were measured after a 24 h culture. phosphate buffer (pH 7.2) and 37 ˝ C from (the same procedure carried outaduring test). The concentration of kept silverat released QCMC-Ag was analyzed using Thermoantibacterial Scientific After 24 hiCAP of release, above sample centrifugated forspectrometer 40 min at (ICP-OES, the speedTMO, of 4800 rpm, then 6500 Duothe inductively coupled was plasma-optical emission South Wales, UK). To measure silverout release, QCMC-Ag 0.05solutions g) was immersed 10 HNO3 . 1 mL of the supernatant wasthe taken andthe diluted with(approximately concentrated of HClinand mL of phosphate buffer (pH 7.2) kept at 37 coupled °C (the same procedure carried out during The diluted supernatant was used forand inductively plasma optical emission spectroscopy antibacterial test). After 24 h of release, the above sample was centrifugated for 40 min at the speed (ICP-OES) analysis. of 4800 rpm, then 1 mL of the supernatant was taken out and diluted with concentrated solutions of HCl and HNO3. The diluted supernatant was used for inductively coupled plasma optical emission

3. Results and Discussion spectroscopy (ICP-OES) analysis.

3.1. Structure Characterization of QCMC 3. Results and Discussion The 3.1. FT-IR spectra of CMC and QCMC are shown in Figure 2. In the characteristic peaks of CMC Structure Characterization of QCMC (Figure 2a), the absorption peaks at 1602 and 1407 cm´1 correspond to the asymmetrical and symmetrical The FT-IR spectra of CMC and QCMC are shown in Figure 2. In the characteristic peaks of CMC stretching(Figure vibrations of absorption COO´ group. spectrum shown to in Figure 2b, the characteristic 2a), the peaks In at the 1602QCMC and 1407 cm−1 correspond the asymmetrical and ´1 that belonged to − peaks of symmetrical CMC are stretching present and a new adsorption appeared at 1483 vibrations of COO group. Inband the QCMC spectrum showncm in Figure 2b, the −1 that characteristic of CMC are present and a salt, new adsorption band at 1483 cmC6-substituted the methyl groups ofpeaks the quaternary ammonium confirming theappeared existence of the belonged to the methyl groups of the quaternary ammonium salt, confirming the existence of the carboxymethyl groups and quaternary ammonium groups on the QCMC chains. In addition, compared C6-substituted carboxymethyl groups and quaternary ammonium groups on the QCMC chains. In to CMC, addition, the peakcompared at around 1133 cm´1 , which belonged to the C–O stretching band of the secondary to CMC, the peak at around 1133 cm−1, which belonged to the C–O stretching hydroxylband group, is unchanged in QCMC, verifying that in quaternization didthat notquaternization occur at thedid C3 position. of the secondary hydroxyl group, is unchanged QCMC, verifying not occur at the C3 the position. These results imply that and the quaternization of CMC and occur the substitution These results imply that quaternization of CMC the substitution may at -NH2 sites on mayofoccur at -NH2 sites on C2 position of the pyranoid whichstudies is consistent with previous C2 position the pyranoid ring, which is consistent with ring, previous [25–27]. studies [25–27].

Figure 2. Fourier transform infrared (FT-IR) spectra of (a) carboxymethyl chitosan (CMC) and (b) Quaternized carboxymethyl chitosan (QCMC).

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Figure Figure 2. 2. Fourier Fourier transform transform infrared infrared (FT-IR) (FT-IR) spectra spectra of of (a) (a) carboxymethyl carboxymethyl chitosan chitosan (CMC) (CMC) and and (b) (b)

Quaternized chitosan Nanomaterials 2016, 6,carboxymethyl 118 Quaternized carboxymethyl chitosan (QCMC). (QCMC).

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1 To NMR spectrum To verify verify the the QCMC QCMC structure, structure, the the 1H H spectrum was was investigated investigated as as shown shown in in Figure Figure 3. 3. In In 1 HNMR To verify the QCMC structure, the NMR spectrum was investigated as shown in Figure 3. the spectrum of QCMC, the peaks at δ = 4.44, 2.42, 3.51, 3.64, 3.58 and 3.85 ppm correspond to H–1, the spectrum of QCMC, the peaks at δ = 4.44, 2.42, 3.51, 3.64, 3.58 and 3.85 ppm correspond to H–1, In the spectrum of QCMC, the peaks at δ = 4.44, 2.42, 3.51, 3.64, 3.58 and 3.85 ppm correspond to H–2, H–2, H–3, H–3, H–4, H–4, H–5 H–5 and and H–6. H–6. The The signal signal at at δδ == 4.06 4.06 ppm ppm can can be be attributed attributed to to the the protons protons of of the the H–1, H–2, H–3,carboxymethyl H–4, H–5 and H–6. TheThe signal at intensive δ = 4.06 ppm canatbe attributed to thebeprotons of the C6-substituted groups. most signal δ = 3.10 ppm can attributed C6-substituted carboxymethyl groups. The most intensive signal at δ = 3.10 ppm can be attributed to to C6-substituted carboxymethyl groups. The most intensive signal at δwith = 3.10 ppm can beCai attributed to the the methyl methyl protons protons of of the the quaternary quaternary ammonium ammonium salt, salt, which which agrees agrees with the the report report by by Cai et et al. al. [25]. [25]. the methyl protons of the quaternary ammonium salt, which agrees with the report by Cai et al. [25]. The The chemical chemical shifts shifts at at 2.65, 2.65, 4.19 4.19 and and 3.28 3.28 ppm ppm belong belong to to H–A, H–A, H–B H–B and and H–C H–C of of the the C2-substituted C2-substituted The chemical shifts at 2.65, 4.19 The and113.28 ppmresults belong toin H–A, H–B and H–C of the studies C2-substituted quaternary ammonium groups. H NMR are agreement with previous quaternary ammonium groups. The 1H NMR results are in agreement with previous studies [16,26], [16,26], quaternary ammonium groups. The H NMR results are in agreement with previous studies [16,26], which which further further demonstrated demonstrated that that QCMC QCMC was was successfully successfully synthesized synthesized under under microwave microwave irradiation irradiation which further possible demonstrated that QCMC was successfully synthesized under microwave irradiation in in in water. water. The The possible mechanism mechanism for for the the preparation preparation of of QCMC QCMC is is illustrated illustrated in in Figure Figure 4. 4. water. The possible mechanism for the preparation of QCMC is illustrated in Figure 4.

11 Figure H nuclear nuclear magnetic magnetic resonance resonance (NMR) Figure 3. 3. 1H H nuclear magnetic resonance (NMR) spectrum spectrum of of QCMC. QCMC.

Figure Figure 4. 4. The The reaction reaction illustration illustration of of QCMC. QCMC.

3.2. Ag NPs NPs Composite Composite 3.2. Characterization Characterization of of the the QCMC-Based QCMC-Based Ag 3.2.1. Analysis 3.2.1. XRD XRD analysis analysis The The XRD XRD method method is is widely widely used used to to examine examine the the crystallographic crystallographic nature nature of of aa material material to to confirm confirm the formation of of nanoparticles [28]. The XRD patterns CMC, QCMC and QCMC-Ag are the formation of nanoparticles [28]. The XRD patterns of CMC, QCMC and QCMC-Ag are shown shown in in Figure 5. In the XRD pattern of QCMC-Ag (Figure 5c), the diffraction peaks at 2θ = 38.01, 44.14, 64.47, diffraction peaks at 2θ = 38.01, 44.14, 64.47, Figure 5. In the XRD pattern of QCMC-Ag (Figure 5c), the diffraction peaks at 2θ = 38.01, 44.14, 64.47, 77.21, and 81.39 are due reflections from (111), (200), (220), (331), planes of from (111), (200), (220), (331), (222)(222) planes of metallic silversilver with 77.21, and and 81.39 81.39are aredue duetoto toreflections reflections from (111), (200), (220), (331), (222) planes of metallic metallic silver with face centred-cubic lattice structure (JCPDS 04-0783), which indicate that pure well-crystallized awith faceaacentred-cubic lattice structure (JCPDS 04-0783), which indicate that pure well-crystallized silver face centred-cubic lattice structure (JCPDS 04-0783), which indicate that pure well-crystallized silver was the facile and green In experiment, no other was obtained via thevia proposed facile and green In the reported experiment, no other silver was obtained obtained via the proposed proposed facile andmethod. green method. method. In the the reported reported experiment, noagents other agents were added to the reaction system implying that QCMC solution played a vital role in were added to the reaction system implying that QCMC solution played a vital role in the synthesis agents were added to the reaction system implying that QCMC solution played a vital role in the the synthesis of the crystals. QCMC possesses many hydroxyl, and quaternary of the silver QCMC possesses hydroxyl, and quaternary synthesis of crystals. the silver silver crystals. QCMCmany possesses manycarboxymethyl, hydroxyl, carboxymethyl, carboxymethyl, andammonium quaternary ammonium groups, have aa relatively good reducing capability groups, which have awhich relatively reducing capability [1,29]. ammonium groups, which havegood relatively good reducing capability [1,29]. [1,29]. In 5a), two major characteristic the XRD pattern of the CMC (Figure 5a), two major In the XRD pattern of the CMC (Figure 5a), two major characteristic characteristic crystalline crystalline peaks peaks were were ˝ ˝ observed near 9.49° and 19.98°, which indicate crystallization. The crystalline peaks shifted towards 9.49 19.98 , observed near 9.49° and 19.98°, which indicate crystallization. The crystalline peaks shifted towards a lower angle and became weaker in the QCMC, while they disappeared entirely in the XRD pattern of the QCMC-Ag. Therefore, we deduced that the crystal structure of the original polymer remained in

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a lower angle and became weaker in the QCMC, while they disappeared entirely in the XRD pattern aoflower angle and became weaker in the QCMC, they disappeared the XRD pattern the QCMC-Ag. Therefore, we deduced that thewhile crystal structure of the entirely originalin polymer remained the QCMC but was destroyed in the QCMC-Ag. This result can be explained by the embedding of the of the QCMC-Ag. Therefore, we deduced that the crystal structure of the original polymer remained in the QCMC but was destroyed in the QCMC-Ag. This result can be explained by the embedding of silver particles in the QCMC polymer network, which disrupted the regularity of the polymer chains in QCMC but was the QCMC-Ag. This result can be explained by theofembedding of thethe silver particles in destroyed the QCMCinpolymer network, which disrupted the regularity the polymer and the intramolecular hydrogen bonding in polymer molecules, resulting in significant changes in the silver particles in the QCMC polymer network, which disrupted the regularity of the polymer chains and the intramolecular hydrogen bonding in polymer molecules, resulting in significant the polymer’s crystalline structure. chains thepolymer’s intramolecular hydrogen bonding in polymer molecules, resulting in significant changesand in the crystalline structure. changes in the polymer’s crystalline structure.

Figure (b) QCMC QCMC and and (c) (c) QCMC-Ag. QCMC-Ag. Figure 5. 5. X-ray X-ray diffraction diffraction (XRD) (XRD) patterns patterns of of (a) (a) CMC, CMC, (b) Figure 5. X-ray diffraction (XRD) patterns of (a) CMC, (b) QCMC and (c) QCMC-Ag.

3.2.2. 3.2.2. XPS XPS Analysis Analysis 3.2.2. XPS Analysis XPS product to to further determine the the chemical composition and XPS analysis analysis was wasconducted conductedononthe the product further determine chemical composition XPS analysis was conducted on the product to further determine the chemical composition and the states of the prepared material [28,30]. QCMC-Ag and valence the valence states of the prepared material [28,30].AAsurvey-scan survey-scanXPS XPSspectrum spectrum of of QCMC-Ag the valence states ofthat thethe prepared material [28,30]. A survey-scan XPS spectrum of QCMC-Ag (Figure 6I) revealed sample was composed of 70.53% atomic weight carbon, 25.13% (Figure 6I) revealed that the sample was composed of 70.53% atomic weight carbon, 25.13% oxygen, oxygen, (Figure 6I) revealed that the sample was chlorine. composedThe of 70.53% atomic weight carbon, 25.13% 2.85% nitrogen, 1.16% silver, and0.33% 0.33% high-resolution XPS result silver isoxygen, shown 2.85% nitrogen, 1.16% silver, and chlorine. The high-resolution XPS result forfor silver is shown in 2.85% nitrogen, 1.16% silver, and 0.33% chlorine. The high-resolution XPS result for silver is shown in Figure 6II. The XPS signals for Ag 3d show two clear peaks attributed to Ag 3d 5/2 (368.3 eV) orbit Figure 6II. The XPS signals for Ag 3d show two clear peaks attributed to Ag 3d5/2 (368.3 eV) orbit and in Figure 6II. The XPS signals for Ag 3d show two clear peaks attributed to Ag 3d5/2 (368.3 eV) orbit 0 and Ag 3d 3/2 (374.3 eV) orbit. Both of these peaks at 368.3 and 374.3 eV are associated with Ag0 Ag 3d 3/2 (374.3 eV) orbit. Both of these peaks at 368.3 and 374.3 eV are associated with Ag [31–34], and Ag 3d 3/2 (374.3 eV) orbit. Both of these peaks at 368.3 and 374.3 eV are associated with Ag 0 [31–34], indicating that thewas sample was composed a single phasewhich of silver, is in good indicating that the sample composed of a singleofphase of silver, is inwhich good accordance [31–34], indicating that the sample was composed of a single phase of silver, which is in it good accordance with the XRD results shown in Figure 5. Based of the XPS can with the XRD results shown in Figure 5. Based on the resultson of the the results XPS analysis, it cananalysis, be concluded accordance with the XRD results shown in Figure 5. Based on the results of the XPS analysis, it can be concluded that pure metallic silver was synthesized as a result of the reaction, further confirming that pure metallic silver was synthesized as a result of the reaction, further confirming the complete be concluded that pure 0metallic silver was synthesized as reducing a result ofagent. the reaction, further confirming the complete reduction Ag+ to Ag0 using as the + to reduction of Ag Ag of using QCMC as theQCMC reducing agent. the complete reduction of Ag+ to Ag0 using QCMC as the reducing agent.

Figure 6. X-ray photoelectron spectroscopy (XPS) spectra of as-prepared QCMC-Ag. (I): Survey-scan Figure 6. 6. X-ray X-ray photoelectron spectroscopy spectroscopy (XPS) spectra spectra of of as-prepared as-prepared QCMC-Ag. QCMC-Ag. (I): (I): Survey-scan Survey-scan Figure photoelectron (XPS) spectrum. (II): High-resolution Ag 3d spectrum. spectrum. (II): (II): High-resolution High-resolution Ag Ag 3d 3d spectrum. spectrum. spectrum.

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3.2.3. UV-Vis Spectroscopy Analysis Analysis 3.2.3. UV-Vis Spectroscopy UV-Visspectroscopy spectroscopyisis a simple sensitive technique forcharacterization the characterization of Ag NPs UV-Vis a simple andand sensitive technique for the of Ag NPs thanks thanks to its singular excitation of surface plasmon resonance (SPR) in the UV-Visible region [18]. to its singular excitation of surface plasmon resonance (SPR) in the UV-Visible region [18]. The formation The size formation and size Ag revealed NPs canusing be easily revealed using a UV-Vis and distribution of Agdistribution NPs can beofeasily a UV-Vis spectrophotometer [35]. spectrophotometer [35]. As shown in Figure 7a, original QCMC exhibited no peaks range of As shown in Figure 7a, original QCMC exhibited no peaks in the range of 300–600 nm, in butthe QCMC-Ag 300–6007b) nm,displayed but QCMC-Ag (Figure 7b)absorption displayedband the unique bandverifying for the Ag NPs (Figure the unique SPR for theSPR Ag absorption NPs at 405 nm, that Agat+ + 0 0 and 405 nm, verifying Ag was reduced and formed Ag NPs the in the QCMC. In addition,peak the was reduced to Agthat formed Ag NPs to in Ag the QCMC. In addition, sharp SPR absorption sharp SPR absorption peak indicates a very narrow distribution in the particle size [36,37]. The shape indicates a very narrow distribution in the particle size [36,37]. The shape of the SPR absorption band of the SPR absorption band was symmetrical andthe quite narrow, that the Ag NPs were was symmetrical and quite narrow, suggesting that Ag NPs weresuggesting spherical and monodispersed [38]. spherical and monodispersed [38].

Figure spectra of of (a) (a) QCMC QCMC and and (b) (b) QCMC-Ag. QCMC-Ag. Figure 7. 7. Ultraviolet-visible Ultraviolet-visible (UV-Vis) (UV-Vis) spectra

3.2.4. Morphology Analysis (TEM (TEM and and FESEM) FESEM) 3.2.4. Morphology Analysis An inspection inspectionofofthe the TEM image in Figure 8a shows spherical Agwith NPssmall withsize small and An TEM image in Figure 8a shows spherical Ag NPs andsize uniform uniform shape were dispersed evenly in the QCMC matrix. The size distribution of the Ag NPs was shape were dispersed evenly in the QCMC matrix. The size distribution of the Ag NPs was centered centered in the rangenm of and 17–31 and the mean wasnm, 24.71 ± 2.80 nm, confirmed which further in the range of 17–31 thenm mean diameter wasdiameter 24.71 ˘ 2.80 which further the confirmed the findings of the UV-Vis results. The inverse fast Fourier transform (IFFT) image of a findings of the UV-Vis results. The inverse fast Fourier transform (IFFT) image of a single particle single particle (Figure 8c) and corresponding selected area (SAED) electronpattern diffraction (SAED) pattern (Figure 8c) and corresponding selected area electron diffraction (Figure 8d) confirmed (Figure 8d) confirmed the crystalline of the Ag particular, the 8c IFFT imagelattice in Figure 8c the crystalline nature of the Ag NPs. nature In particular, theNPs. IFFT In image in Figure showed fringe showed lattice fringe spacing that was determined to be 0.237 nm. This was indexed to the d-spacing spacing that was determined to be 0.237 nm. This was indexed to the d-spacing of the crystalline of the crystalline silver (111) plane (JCPDS 04-0783). The SAED pattern in Figure 8d showed periodic silver (111) plane (JCPDS 04-0783). The SAED pattern in Figure 8d showed periodic diffraction spots, diffraction spots, indicating that the Ag NPs were monocrystalline. indicating that the Ag NPs were monocrystalline. The surface morphology of QCMC-Ag aNPs composite was further examined using FESEM. b Figure 9 displays the FESEM micrographs of the QCMC and QCMC-Ag, in which thousands of silver particles can be seen distributed homogeneously on the surface of the QCMC (Figure 9b,d). The diameter of the particles was similar and almost no agglomeration was observed, which directly demonstrated that QCMC is also an ideal growth template for Ag NPs. The QCMC could reduce Ag+ to Ag0 due to the functional hydroxyl, carboxymethyl and quaternary ammonium groups in the polymer. During the redox reaction, some of these active groups in the QCMC were oxidized to new groups, but whole polymer 200 nmchains remained. The remaining QCMC and the oxidized polymer chains 200 nm acted as an excellent stabilizing agent adsorbing that wrapped the formed Ag NPs tightly to avoid aggregation. Thus, it is not surprising that the Ag NPs were well-dispersed in the QCMC matrix and showed a narrow size distribiution, in agreement with the UV-Vis results and TEM images.

centered in the range of 17–31 nm and the mean diameter was 24.71 ± 2.80 nm, which further confirmed the findings of the UV-Vis results. The inverse fast Fourier transform (IFFT) image of a single particle (Figure 8c) and corresponding selected area electron diffraction (SAED) pattern (Figure 8d) confirmed the crystalline nature of the Ag NPs. In particular, the IFFT image in Figure 8c Nanomaterials 2016, 6, 118 8 of 13 showed lattice fringe spacing that was determined to be 0.237 nm. This was indexed to the d-spacing Nanomaterials 2016, 6, 118 8 of 13 of the crystalline silver (111) plane (JCPDS 04-0783). The SAED pattern in Figure 8d showed periodic c d diffraction spots, indicating that the Ag NPs were monocrystalline. b

a

1 nm 10 1/nm

1 nm 1 nm

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200 nm

Figure 8. (a) Transmission electron microscopy (TEM) image of QCMC-Ag, (b) size distribution 200 nm histogram of Ag NPs, (c) inverse fast Fourier transform (IFFT) image and (d) selected area electron diffraction (SAED) pattern. c d

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The surface morphology of QCMC-Ag NPs composite was further examined using FESEM. Figure 9 displays the FESEM micrographs of the QCMC and QCMC-Ag, in which thousands of silver particles can be seen distributed homogeneously on the surface of the QCMC (Figure 9b,d). The diameter of the particles was similar and almost no agglomeration was observed, which directly demonstrated that QCMC is also an ideal growth template for Ag NPs. The QCMC could reduce Ag+ to Ag0 due to the functional hydroxyl, carboxymethyl and quaternary ammonium groups in the nm 1 nm reaction, some of these active 1groups polymer. During the redox in the QCMC were oxidized to new 10 1/nm 1 nm groups, but whole polymer chains remained. The remaining QCMC and the oxidized polymer chains acted as Transmission an excellent stabilizing agent adsorbing wrapped the formed Ag NPs tightly to Figure 8. electron microscopy (TEM) that image of (b) distribution Figure 8. (a) image of QCMC-Ag; QCMC-Ag, (b) size size distribution avoid aggregation. Thus, it is not surprising that the Ag NPs were well-dispersed in the QCMC histogram of Ag NPs; NPs, (c) (c) inverse inverse fast fast Fourier Fourier transform transform (IFFT) image and (d) (d) selected selected area area electron electron matrix and showed a narrow size distribiution, in agreement with the UV-Vis results and TEM diffraction diffraction (SAED) (SAED) pattern. pattern. images.

The surface morphology of QCMC-Ag NPs composite was further examined using FESEM. Figure 9 displays the FESEM micrographs of the QCMC and QCMC-Ag, in which thousands of silver particles can be seen distributed homogeneously on the surface of the QCMC (Figure 9b,d). The diameter of the particles was similar and almost no agglomeration was observed, which directly demonstrated that QCMC is also an ideal growth template for Ag NPs. The QCMC could reduce Ag+ to Ag0 due to the functional hydroxyl, carboxymethyl and quaternary ammonium groups in the polymer. During the redox reaction, some of these active groups in the QCMC were oxidized to new groups, but whole polymer chains remained. The remaining QCMC and the oxidized polymer (a) agent adsorbing that wrapped (b) chains acted as an excellent stabilizing the formed Ag NPs tightly to avoid aggregation. Thus, it is not surprising that the Ag NPs were well-dispersed in the QCMC matrix and showed a narrow size distribiution, in agreement with the UV-Vis results and TEM images.

(c)

(d)

Figure Fieldemission emission scanning microscope (FESEM) micrographs of (a,c) and (b,d) Figure 9. 9.Field scanningelectron electron microscope (FESEM) micrographs ofQCMC (a,c) QCMC and QCMC-Ag. (b,d) QCMC-Ag.

(a)

(b)

(c)

(d)

3.3. Thermal Stability Analysis 3.3. Thermal Stability Analysis The TGA and DTG curves of CMC, QCMC and QCMC-Ag are shown in Figure 10. The thermal The TGA and curves of CMC, QCMCand andQCMC-Ag QCMC-Ag aresimilar, shown consisting in Figure 10. Thestages. thermal decomposition ofDTG the two chitosan derivatives were of two decomposition of the two chitosan derivatives and QCMC-Ag were similar, consisting of two stages. The first stage occurred below 100 °C, and is associated with the loss of adsorded and bound water. ˝ The first stage occurred below 100 C, and is associated with the loss of adsorded and bound water. The second stage (200–400 ˝ C) corresponds to the degradation of the molecular chains of the chitosan

Figure 9. Field emission scanning electron microscope (FESEM) micrographs of (a,c) QCMC and (b,d)

water loss that occurred below 100 °C for CMC, and an 8.2% loss for the QCMC. This indicates that the QCMC had higher water retention capacity than the CMC due to the presence of the quaternary ammonium groups. Additionally it is noteworthy that the QCMC was more thermally unstable than the neat CMC; only 19% of the QCMC remained up to 700 °C. By contrast, the CMC showed high Nanomaterials 2016, 6, 118 13 thermal stability, with a residual weight at 700 °C of 39% and Tmax (the temperature at which the9 of rate of weight loss reaches a maximum) at 269 °C. These results demonstrate that the quaternary ammonium groups grafted on the CMC chains significantly decreased the thermal stability. The Tmax derivatives [39]. These results suggest that the entire polymer chain was present in the QCMC-Ag of the QCMC-Ag increased by 9 °C compared to the QCMC and 50% of the QCMC-Ag mass so that the Ag NPs remained stable without aggregating, which is in agreement with the other remained at 700 °C, indicating the QCMC-Ag exhibited better thermal stability than the QCMC due experimental results reported here. to the high thermal stability of Ag NPs.

Figure 10. (TG)(TG) curvescurves of (a) CMC, (b) QCMC QCMC-Ag, 10.Thermogravimetric Thermogravimetric of (a) CMC, and (b) (c) QCMC and (I): (c)thermogravimetric QCMC-Ag, (I): analysis (TGA) curves; (II): derivative thermogravimetry (DTG) curves. thermogravimetric analysis (TGA) curves; (II): derivative thermogravimetry (DTG) curves.

3.4. Antibacterial Activity The TGA curves in Figure 10I show that there was about 4.7% weight loss, corresponding to waterThe lossinhibitory that occurred below 100 ˝ CQCMC, for CMC, 8.2% loss for the QCMC. This indicates that effects of CMC, Agand NPsanand QCMC-Ag against Staphylococcus aureus the QCMC had higher water capacity thanAsthe CMC in due to the ofhas the aquaternary were measured based on the retention MIC determinations. shown Table 1, presence pure CMC relatively ammonium groups. Additionally it is noteworthy that the QCMC was more thermally unstable weak inhibition for bacteria, the untreated QCMC showed a slight enhanced inhibitory effect, than and ˝ C. By contrast, the CMC showed high the only 19%aof the QCMC remained up to 700 pureneat Ag CMC; NPs displayed relatively strong antibacterial property. Notably, the QCMC-Ag exhibited thermal with activity. a residual weight 700 ˝ Cthe of 39% and Tmaxof(the temperature at which the superior stability, antimicrobial Figure 11 at displays morphology Staphylococcus aureus colonies ˝ C. These results demonstrate that the quaternary rate of weight loss reaches a maximum) at 269 after treatment with CMC, QCMC, Ag NPs and QCMC-Ag at a concentration of 0.005%, compared ammonium groups grafted in onFigure the CMC significantly decreasedinhibited the thermal stability. Theaureus Tmax to the control. As shown 11,chains the QCMC-Ag completely Staphylococcus ˝ of the QCMC-Ag increased 9 Cconcentration compared to the QCMC CMC, and 50% of theand QCMC-Ag remained growth. By contrast, at the by same of 0.005%, QCMC Ag NPsmass did not affect ˝ C, indicating the QCMC-Ag exhibited better thermal stability than the QCMC due to the high at 700 the growth of Staphylococcus aureus. thermal stability of Ag NPs. Table 1. Minimum inhibition concentrations (MICs) of CMC, QCMC, Ag NPs and QCMC-Ag; the concentration of silver released from Ag NPs and QCMC-Ag.

3.4. Antibacterial Activity

The inhibitory effects of CMC, QCMC, Ag NPs and QCMC-Ag against Staphylococcus aureus Sample code MICs (%, w/v, n =3) Ag release after 24 h (µg/mL) were measured based As shown in Table 1, -pure CMC has a relatively Blank on the MIC determinations. -1 weak inhibition for bacteria, the untreated QCMC showed a slight enhanced inhibitory effect, and 1 PBS pure Ag NPs displayed a relatively strong antibacterial property. Notably, the QCMC-Ag exhibited CMC 2 superior antimicrobial activity. Figure 11 displays the morphology of Staphylococcus aureus colonies after treatment with CMC, QCMC, Ag NPs and QCMC-Ag at a concentration of 0.005%, compared to the control. As shown in Figure 11, the QCMC-Ag completely inhibited Staphylococcus aureus growth. By contrast, at the same concentration of 0.005%, CMC, QCMC and Ag NPs did not affect the growth of Staphylococcus aureus.

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0.5 0.025 0.005 1

(a)

1.3 0.12

(b)

(d)

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No antibacterial properties.

(c)

(e)

Figure 11. 11. Appearance Appearance of of colonies colonies of of Staphylococcus Staphylococcus aureus aureus after after treatment treatment with with CMC, CMC, QCMC QCMC ,, Ag Ag Figure NPs and QCMC-Ag at a concentration of 0.005%: (a) blank, (b) CMC, (c) QCMC, (d)Ag NPs and (e) NPs and QCMC-Ag at a concentration of 0.005%: (a) blank; (b) CMC; (c) QCMC; (d) Ag NPs and QCMC-Ag. (e) QCMC-Ag.

The mechanism the antibacterial activity of theofAg NPsQCMC, is still Ag a matter of dispute. According Table 1. Minimumofinhibition concentrations (MICs) CMC, NPs and QCMC-Ag; the to Dallas et al., theofthree popular mechanisms proposed are: (a) Ag NPs directly damaging cell concentration silvermost released from Ag NPs and QCMC-Ag. membranes, (b) gradual release of free silver ions, followed by disruption of adenosine triphosphate (ATP) production and deoxyribonucleic acid (DNA) and (c)24Ag NPs and silver ions Sample Code MICs (%, w/v, n = 3) replication, Ag Release after h (µg/mL) generating reactive oxygen species [40]. Lok et al. showed that the oxidation of metallic silver to 1 Blank 1 active silver ions species is required for Ag NPs to be fully efficient, since partially oxidized Ag NPs PBS CMC 2 exhibit antibacterial properties and zero-valent nanoparticles do not [41]. Kumar reported that the 0.5be achieved through the interaction oxidation of metallicQCMC silver to silver ions can of the silver with Ag NPs 0.025 1.3 water molecules [42]. Additionally, electron microscopic analyses showed that Ag NPs can attach to QCMC-Ag 0.005 0.12 the surface of the cell membrane and thus could damage membrane function or penetrate the 1 No antibacterial properties. bacteria via diffusion or endocytosis [43]. Embedding Ag NPs in a QCMC polymer network enables the sustained release of silver ions The mechanism of the antibacterial activity of the Ag NPs is still a matter of dispute. According to and/or silver nanoparticles to exert long-acting and high-efficiency antibacterial activity. The ability Dallas et al., the three most popular mechanisms proposed are: (a) Ag NPs directly damaging cell of the QCMC to absorb and hold water can facilitate oxidation of Ag NPs to active silver ions. In membranes, (b) gradual release of free silver ions, followed by disruption of adenosine triphosphate addition, QCMC is a positively-charged polyelectrolyte and thus can interact vigorously with (ATP) production and deoxyribonucleic acid (DNA) replication, and (c) Ag NPs and silver ions negatively-charged bacteria at the cell surface, change bacterial cell membrane permeability, restrain generating reactive oxygen species [40]. Lok et al. showed that the oxidation of metallic silver to the growth of cells, and even kill cells [44]. Since QCMC-Ag can combine both advantages of QCMC active silver ions species is required for Ag NPs to be fully efficient, since partially oxidized Ag NPs and Ag NPs and exhibit a synergistic antibacterial effect, its antimicrobial properties in this study exhibit antibacterial properties and zero-valent nanoparticles do not [41]. Kumar reported that the were improved compared to pure QCMC and Ag NPs. oxidation of metallic silver to silver ions can be achieved through the interaction of the silver with ICP-OES testing showed that the concentration of the silver released from QCMC-Ag in the test water molecules [42]. Additionally, electron microscopic analyses showed that Ag NPs can attach to solution was extremely low, in the order of 0.12 µ g/mL (Table 1), in comparison with pure Ag NPs the surface of the cell membrane and thus could damage membrane function or penetrate the bacteria (1.3 µ g/mL). The fact that only small amounts of silver led to antimicrobial activity is a great via diffusion or endocytosis [43]. Embedding Ag NPs in a QCMC polymer network enables the sustained release of silver ions and/or silver nanoparticles to exert long-acting and high-efficiency antibacterial activity. The ability of

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the QCMC to absorb and hold water can facilitate oxidation of Ag NPs to active silver ions. In addition, QCMC is a positively-charged polyelectrolyte and thus can interact vigorously with negatively-charged bacteria at the cell surface, change bacterial cell membrane permeability, restrain the growth of cells, and even kill cells [44]. Since QCMC-Ag can combine both advantages of QCMC and Ag NPs and exhibit a synergistic antibacterial effect, its antimicrobial properties in this study were improved compared to pure QCMC and Ag NPs. ICP-OES testing showed that the concentration of the silver released from QCMC-Ag in the test solution was extremely low, in the order of 0.12 µg/mL (Table 1), in comparison with pure Ag NPs (1.3 µg/mL). The fact that only small amounts of silver led to antimicrobial activity is a great advantage, because silver ions and/or Ag NPs may exhibit cytotoxic and genotoxic effects on human cells at high concentrations [45]. 4. Conclusions In this study, Ag NPs were synthesized in a QCMC aqueous solution without an additional reducing and stabilizing agent, using silver nitrate as the silver source. The synthesis was conducted in an aqueous medium in the presence of microwave irradiation for 8 min. The results showed that uniform and stable spherical Ag NPs were successfully obtained via a facile, green method. The prepared Ag NPs were well dispersed in a QCMC matrix with a narrow size distribution and a mean diameter of 24.71 ˘ 2.80 nm. Due to the presence of the Ag NPs, the thermal stability and antibacterial properties of the QCMC-Ag were significantly improved compared to the QCMC. The QCMC-Ag exhibited outstanding hybrid performance with favorable thermal stability, strong antibacterial activity and low human toxicity, which implies that this material might be a promising antimicrobial candidate for application in the biomedical field, food packaging, coating, clothing, textile and other similar areas. Acknowledgments: The authors are grateful for support from the Fundamental Research Funds for the Central Universities (No. 2016ZCQ01 and No. 2016BLRD03). Author Contributions: Siqi Huang, Yang Zhang and ZhimingYu conceived and designed the experiments; Jing Wang performed the experiments; Siqi Huang and Yang Zhang analyzed the data; Siqi Huang wrote the paper; Chusheng Qi contributed materials and provided assistance for English writing. All authors have approved the final version of the manuscript. Conflicts of Interest: The authors declare no conflict of interest.

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