Accepted Manuscript Title: Green synthesis of the silver nanoparticles mediated by pullulan and 6-carboxypullulan Author: Sergiu Coseri Alina Spatareanu Liviu Sacarescu Cristina Rimbu Daniela Suteu Stefan Spirk Valeria Harabagiu PII: DOI: Reference:

S0144-8617(14)00584-0 http://dx.doi.org/doi:10.1016/j.carbpol.2014.06.008 CARP 8973

To appear in: Received date: Revised date: Accepted date:

9-12-2013 19-5-2014 5-6-2014

Please cite this article as: Coseri, S., Spatareanu, A., Sacarescu, L., Rimbu, C., Suteu, D., Spirk, S., and Harabagiu, V.,Green synthesis of the silver nanoparticles mediated by pullulan and 6-carboxypullulan, Carbohydrate Polymers (2014), http://dx.doi.org/10.1016/j.carbpol.2014.06.008 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Green synthesis of the silver nanoparticles mediated by pullulan and 6-

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carboxypullulan

3 Sergiu Coseri,1* Alina Spatareanu,1 Liviu Sacarescu,1 Cristina Rimbu,2 Daniela Suteu,3 Stefan

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Spirk,4,5* Valeria Harabagiu1

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Voda Alley, 700487, Iasi, Romania

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Petru Poni Institute of Macromolecular Chemistry of Romanian Academy, 41 A, Gr. Ghica

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University of Agricultural Sciences and Veterinary Medicine, Faculty of Veterinary

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Medicine - UASVM Iaşi, Aleea Mihail Sadoveanu nr.8, 700489, Iaşi, Romania

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Environmental Protection, 71A Prof. Dr. Docent D. Mangeron Blvd, 700050 Iasi, Romania

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“Gheorghe Asachi” Technical University of Iasi, Faculty of Chemical Engineering and Graz University of Technology, Institute for Chemistry and Technology of Materials,

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Stremayrgasse 9, 8010 Graz, Austria.

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Ulica 17, 2000 Maribor, Slovenia.

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University of Maribor, Institute for the Engineering of Materials and Design, Smetanova

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Paper dedicated to the 65th anniversary of "Petru Poni" Institute of Macromolecular Chemistry

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of Romanian Academy, Iasi, Romania.

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*Corresponding authors: Sergiu Coseri: E-mail: [email protected] Phone: +40 232 217454 Fax: +40 232 211299

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Keywords: Pullulan, 6-carboxypullulan, TEMPO, Silver nanoparticles, Oxidation

Stefan Spirk: E-mail: [email protected] Phone: +43 316 873 32284 Fax: +43 316 873 32301

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Highlights

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Pullulan was oxidized using TEMPO protocol to introduce carboxyl groups. 1 Page 1 of 26

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Pullulan and carboxylated pullulan were used as reducing and stabilizing agents for AgNPs preparation.

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Oxidized pullulan mediated silver nanoparticles exhibits long term stability.

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Excellent antibacterial activity of pullulan/oxidized pullulan silver nanoparticles was

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achieved.

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ABSTRACT

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Unoxidized and carboxylated pullulan (obtained by pullulan oxidation using TEMPO-sodium

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hypochlorite-sodium bromide system) have been used as mediators for the silver

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nanoparticles formation (AgNPs), under environmentally-friendly conditions: using aqueous

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solutions, room temperature and notably, by using both mediators as reducing and stabilizing

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agents. The formation of AgNPs was first screened by measuring the surface plasmon

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resonance peak in the range of 380-440 nm using UV-vis spectroscopy. The morphology of

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the synthesized silver nanoparticles was determined by TEM, which indicated that the AgNPs

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differ on shape and thickness of the polymer shell by varying the silver nitrate concentration,

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different size and shape of AgNPs was achieved. The presence of elemental silver and the

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crystalline structure of the AgNPs were confirmed by EDX and XRD analyses. Moreover, the

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possible functional groups of pullulan (oxidized pullulan) responsible for the reduction and

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stabilization of AgNPs were evaluated using FT-IR.

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The results showed that both, pullulan and 6-carboxypullulan could be successfully used as

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reducing as well as capping agents for the AgNPs synthesis which shows potential

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antimicrobial activity against gram positive and gram negative bacteria.

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1. Introduction

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In the past decade, research on nanoscale materials has entered into the focus of many

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different areas since they exhibit unique chemical and physical properties (Garciá-Barrasa,

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López-de-Luzuriaga & Monge, 2011), leading to manifold applications in biomedicine, 2 Page 2 of 26

electronics, biosensors, catalysis, and optics for instance (Alayoglu, Nilekar, Mavrikakis &

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Eichhorn, 2008; Aslan, Lakowicz & Geddes, 2005; Sapsford, Pons, Medintz & Mattoussi,

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2006). Of particular interest are nanoparticles, whose properties can be tuned by variation of

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their size, shape and surface functional groups. There is a huge variety of methods for the

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preparation of metal nanoparticles (MNPs) ranging from physical solid-state treatments

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(including milling, grinding and mechanical alloying techniques) (Pimpang, Sutham,

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Mangkorntong, Mangkorntong & Choopun, 2008; Salah et al., 2011), gas-phase synthesis

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(e.g. high –temperature evaporation (Zschechet al., 2006), laser ablation (Giorgetti, Giusti,

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Laza, Marsili & Giammanco, 2007), pyrolysis (Turner et al., 2010), plasma synthesis (Ko et

78

al., 2006) to liquid-phase synthesis. The latter includes a variety of methods such as co-

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precipitation, microemulsifying, microwave irradiation, solvothermal treatments, sol-gel

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syntheses, for instance (Breitwieser et al., 2013; Khan, Al-Thabaiti, Obaid & Al-Youbi, 2011;

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Khorsand Zak, Razali, Abd Majid & Darroudi, 2011; Kowlgi, Lafont, Rappolt & Koper,

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2012; Sharmaa, Sharmab, Kaitha, Rajputa & Kaurb, 2011; Wu et al., 2011; Xu et al., 2007).

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In all of the afore mentioned methods, the chemical reduction of metal salt precursors to the

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elemental metal nanoparticles plays a crucial role. In general, the chemical reduction of metal

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salts requires a reducing agent and a stabilizer one, in order to obtain shape- and size-

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controlled MNPs. In the case of silver, the most widely used precursor is silver nitrate

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(AgNO3). Usually, the generation of Ag nanoparticles involves three steps, namely the

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reduction reaction of silver ions to free silver atoms, nucleation and growth. Since the kinetics

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of the reduction reaction plays a large role for the nucleation and growth stage (Garciá-

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Barrasa et al., 2011) an impressive number of reducing agents (e.g. NaBH4, citric acid,

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ascorbic acid etc) and capping agents (e.g. oleylamine, cetyltriethylammonium bromide,

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poly(vinylpyrrolidone) etc.) have been described in literature (Garciá-Barrasa et al., 2011).

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A new concept has recently been introduced commonly referred as “green” synthesis where

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the used solvents and chemicals are being non-toxic and benign compounds to the

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environment. Wallen et al. (2003) reported as early as 2003, the completely “green” synthesis

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and stabilization of silver nanoparticles, by using water as solvent, β-D-glucose as a reducing

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agent and starch for the stabilization of the resulted nanoparticles. In this way, the authors

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reported the formation of 5 nm AgNPs. Since then, a large number of polysaccharides were

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used as renewable polymers able to stabilize the AgNPs (Huang & Yang, 2004; Ifuku, Tsuji,

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Morimoto, Saimoto & Yano, 2009; Raveendran, Fu & Wallen, 2003; Tran et al., 2010; Twu,

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Chen & Shih, 2008). Very recently, the synthesis of silver nanoparticles with sizes in the 2-40

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nm range by using pullulan as both a reducing and stabilizing agent has been accomplished at

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rather high temperature (121°C). The formation of AgNPs has been demonstrated by the

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appearance of the surface plasmon resonance (SPR) transition in the visible region of 400-450

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nm. Likewise, it was shown that an increase in the silver nitrate concentration leads to a

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substantial increase in the SPR band (Kanmani & Taik Lim, 2013). Besides applications in

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nanotechnology, silver has been used for ages in the treatment of infections and wounds.

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Recently, upcoming resistances of pathogens against antibiotics (particular in medical care)

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led to an increase of silver incorporation into medical materials and currently a variety of

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applications are on the market (AQUACEL®, SILVERCEL™).

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In the present study, pullulan has been oxidized by

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tetramethylpiperidin-1-yl radical (TEMPO)-sodium hypochlorite-sodium bromide system, to

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convert primary OH groups of pullulan to carboxylic functions. The goal of this

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functionalization was the introduction of carboxylic groups into the pullulan backbone. As a

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consequence, the negatively charged carboxylic groups allow for a better stabilization of the

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silver nanoparticles due to the electrosteric stabilization. Subsequently, the syntheses of silver

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nanoparticles are described in the presence of the pullulan or oxidized pullulan as both

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reducing and stabilizing agents using water as solvent at room temperature. Finally, the silver

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nanoparticles will be tested against their activity to act as antimicrobial agents towards S.

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Aureus and E. Coli strains.

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2. Materials and methods

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employing the 2,2,6,6-

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2.1. Materials

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Pullulan (Mw= 150kDa) purchased from TCI Europe was dried under vacuum at 100°C

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overnight prior to use 2,2,6,6-tetramethylpiperidin-1-yl (TEMPO) 99% Sigma-Aldrich,

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sodium hypochlorite (NaOCl, 9% chlorine, Chemical Company Romania) and sodium

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bromide (99% Alfa Aesar) were used as received. Silver nitrate (≥ 99.0%) was purchased

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from Sigma-Aldrich and used without further purification. All solutions were prepared using

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ultra-filtered high purity deionized water.

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2.2. Pullulan oxidation

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Pullulan (0.25 g) was dissolved in deionized water (40 mL) at room temperature. After

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complete dissolution, TEMPO (5 mg) and NaBr (25 mg) were added under rigorous stirring. 4 Page 4 of 26

A 9% sodium hypochlorite solution (2.1 mL) was adjusted to pH 10 using 2M HCl, and the

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resulting solution was slowly added to the pullulan solution. The pH was monitored by a pH

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meter and maintained at 10 by addition 0.2 M sodium hydroxide solution. After 4h, the

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reaction was stopped by quenching the unreacted NaOCl with methanol (2.5 mL), and then

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acidified to a pH around 6.8. In the next step, the reaction mixture was precipitated using

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ethanol and subjected to centrifugation. After centrifugation, the solid was redissolved in

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water, and the resulted solution was desalted and the oligomers were removed by dialysis.

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Dialysis was performed against water in a 4 L beaker using dialysis tubing from

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polyethersulfone (cut-off: 10,000 g cm-1) for 4 days, replacing the water twice each 24 h. The

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oxidized pullulan was then recovered by freeze-drying.

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2.3. FTIR Measurements

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Infrared absorption spectra of pullulan and oxidized pullulan samples, as well as of the

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AgNPs, were recorded using a Bruker Vertex 70 spectrometer at a scan range from 4000 to

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650 cm-1, at a resolution of 2 cm-1 and 32 scans. The dried samples was powdered by grinding

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with KBr pellets and pressed into a mold. 2.4. NMR Determinations

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dissolving 10 mg or 50 mg respectively, in 0.7 mL of D2O. The obtained solution was filtered

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through a pipette containing glass wool into a standard 5 mm NMR tube. The NMR spectra

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were obtained on a Bruker Avance DRX 400 MHz Spectrometer, equipped with a 5 mm QNP

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direct detection probe and z-gradients.

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H-NMR and13C-NMR analyses of original and oxidized pullulan were performed by

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2.5. Experimental procedure of the silver nanoparticles (AgNPs) preparation

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Four sets of experiments for the AgNPs preparation were performed, by varying the silver

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nitrate (AgNO3) concentration (0.01, 0.05, 0.1 and 0.5 M respectively) and also by changing

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the pullulan with 6-carboxy pullulan as reducing agent as well as stabilizer used for the silver

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nanoparticles. In a typical run, 150 mL aqueous solution of biopolymer (0.5 wt%) was placed

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in a 500 mL three necks flask (protected against light by aluminium foil), and 9 mL fresh

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solution of AgNO3 was added drop wise, keeping vigorous stirring at room temperature. After 5 Page 5 of 26

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six hours, the solutions turned pale yellow, and the mixture was centrifuged. The supernatant

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was dialysed against water for two days, and then, the solid was recovered by freeze-drying.

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2.6. Characterization of AgNPs

175 The AgNPs prepared by using pullulan or 6-carboxypullulan were characterized by using

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ultraviolet-visible (UV-vis) spectroscopy, FTIR, transmission electron microscopy (TEM), X-

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ray diffraction (XRD). The optical absorption properties of AgNPs samples were measured

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using a SPECORD 200 Analytik Jena spectrometer over the range of 300 – 700 nm. Zeta

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potential (ζ) was measured by using a dynamic light scattering technique (Zeta sizer model

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Nano ZS, Malvern Instruments, UK) with red laser 633 nm (He/Ne). The average particle size

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was measured also based on the DLS analysis.

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Zeta Potential (ζ) was calculated from the electrophoretic mobility (μ) determined at 25°C.

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For kα >> 1 (k - Debye-Hűckel parameter and α - particle radius), the Smoluchowski

185

relationship was used, Eq. (1):

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ξ = ημ / ε

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where η is the viscosity, ε - dielectric constant.

(1)

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The apparent hydrodynamic diameter (DH) of pullulan, oxidized pullulan and their

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silver nanoparticles aqueous solutions was determined through dynamic light scattering

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measurements by using Zetasizer ZS apparatus during the same experiment of zeta potential

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evaluation. The system uses a non-invasive back scatter (NIBS) technology, which reduces

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the multiple scattering effects. This Mie method is applied over the whole measuring range,

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situated in the range of 0.6 nm to 6 μm. The Eq. (2) was used to obtain the Z-average

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distribution of the apparent hydrodynamic diameter (DH) of the aggregates:

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(2)

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where DH is the hydrodynamic diameter, k is the Boltzman constant, T is the temperature, η is

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the viscosity, D is the diffusion coefficient. It is worth to note that this mean size

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(hydrodynamic diameter) is intensity mean, being calculated from a signal intensity and is not

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a mass or number mean. The results presented good reproducibility with the deviation of the

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average diameter within 5%. The temperature was maintained constant at 25oC (±0.1o C) with

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a Peltier device. 6 Page 6 of 26

TEM images were acquired with a Hitachi High-Tech HT7700 electron microscope

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(Hitachi High-Technologies Corporation, Tokyo, Japan), by using a drop of AgNPs aqueous

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solution directly placed onto a carbon-coated copper grid and allow to dry prior the TEM

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analysis. No staining is required for the sample preparation for this kind of instrument, due to

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the new high resolution objective lens which is incorporated into this apparatus (http://hitachi-

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hta.com/sites/default/files/literature/HT7700-EXALENS-HTD-E207.pdf). The particle size

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distribution was evaluated using the UTHSCSA Image Tool Version 3.00 program

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(UTHSCSA Dental Diagnostic Science, San Antonio, TX, USA). The Energy dispersive X-

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ray analysis (EDX) was performed on the Quanta 200 (FEI) electron microscope equipped

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with EDX system. The crystal structure of the AgNPs was determined using an X-ray

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diffractor, (D8 Advance Bruker), with data acquisition taken at 0.02o s-1 by the reflection

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method. The operated voltage was 30 kV and the current was 36 mA.

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2.7. Determination of antibacterial activity of AgNPs

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The silver nanoparticles were tested using the agar well diffusion method. All the glassware,

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media and reagents used were sterilized in an autoclave at 121oC for 30 min. Staphylococus

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aureus (ATCC 25923) Escherichia coli (ATCC 25922) were used as a model test strains for

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Gram-pozitive and Gram-negative bacteria respectively. These bacterial strains were

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aseptically inoculated overnight, at 37oC into tryptic soy broth and brain heart infusion broth.

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Cells from different pathogens (1 mL) were added to tryptic soy agar and brain heart infusion

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broth media (100 mL) prior to plating and wells were made using an agar well borer. AgNPs

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(5 μg) were then added to the center well, and the plates were incubated at 37oC for 24 h.

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Zone of inhibition were determined by measuring the diameter of the bacterial growth

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inhibition zone. Three independent experiments were carried out with each strain.

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3. Results and discussion

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3.1. Synthesis and characterization of 6-carboxy pullulan

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Pullulan has been selectively oxidized by using TEMPO/sodium hypochlorite/sodium

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bromide system at room temperature for 4h to convert the primary OH groups to carboxylic

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moieties. Under these conditions, more than 90% of the primary OH groups are converted to

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COOH (Spatareanu et al., 2014). Only catalytic amounts of TEMPO have been used, and the

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oxidant has been regenerated by -OCl/ HOCl at pH 10. During oxidation, TEMPO is

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converted to the nitrosonium ion, which is the actual oxidant, see Fig. 1. ClO-

Cl-

Br

BrO-

+-

+

H

O

O O OH

4

O HO

6

3

N

N

O

O

2

O

OH

HO 6 O

OH

O 1

n

OH

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O HO HO

OH 5

OH

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OH HO HO

O

O HO

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HO

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OH H

4

O HO

5

3

O

2

OH

1

n

OH

6-Carboxypullulan

Pullulan

Fig.1. Simplified scheme of TEMPO-mediated oxidation of pullulan.

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spectrum (a) is characteristic for this polysaccharide and fits well to already report 13C NMR

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data in literature (Paris & Stuart, 1999). In the case of pullulan, the two signals at 69.26 ppm

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and 63.19 ppm can be assigned to the C6 atom (6g1→4) and the primary C6 atom (4g-unit),

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which is capable to be oxidized. All signals can be unambiguously assigned (C1: 100.68-

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102.99, C4: 80.56 ppm C2,3,5: 72.26 to 76.21 ppm). After 4 hours of oxidation (sample

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OxPu4), a decrease of the C6 (4g-unit) is observed, which is accompanied by the appearance

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of a new peak at 175.62 ppm, corresponding to a quaternary C atom of a COOH group. In

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order to assess the degree of oxidation (DO), proton NMR spectra have been acquired. The

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NMR technique is a valuable tool to assess the degree of substitution for pullulan

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functionalized products. Thus, Paris et al. reported back in 1995 the evaluation of the degree

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of oxidation of pullulan by using TEMPO as oxidizing agent (Paris & Stuart, 1999). The

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degree of esterification of water-soluble pullulan with cinnamic acid was also determined by

254

using 1H-NMR (Kaya et al., 2009). Very recently, Edgar et al., used 1H-NMR technique to

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calculate the degree of substitution values of a series of 6-carboxypullulan ethers obtained by

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reaction of 6-carboxypullulan Na salt with tetrabutylammonium salt, follow by etherification

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with alkyl bromides and iodobutane (Pereira, Mahoney & Edgar, 2014). The integration of the

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peaks in the range between 3.5-4.4 ppm and 5.35-5.45 ppm in the 1H-NMR spectrum yield a

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DO of ca. 90% (Spatareanu et al., 2014).

C-NMR spectra of the original and oxidized pullulan are shown in Fig. 2. The

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102.99 102.55 100.68 80.56 80.19 76.21 74.49 69.26 73.94 73.11 72.26 63.19

(a) OH

2

1

OH

OH

6

O 5

O HO

3

4

2

OH

6g

1

6

O 5

O

3

HO

2

1

OH

O

*

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O 5

3

4

4g

150 100 Chemical Shift (ppm)

50

0

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71.59

200

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HO HO

6

cr

O 4

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70.67 69.22 65.58 57.51

te

d

98.33 97.90

175.62

M

76.53

(b)

150 100 Chemical Shift (ppm)

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0

Fig. 2. 13C-NMR spectra of pullulan (a), and OxPu4 (b).

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3.2. Synthesis and characterization of AgNPs

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The formation of AgNPs by the reduction of silver ions to silver metal species was achieved

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in the presence of aqueous solutions of pullulan or oxidized pullulan at room temperature over

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a period of 6h. Four sets of experiments were carried out for each biopolymer (pullulan or

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oxidized pullulan) whereby the silver nitrate concentration was chosen to be in the final

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solution, 0.01, 0.1, 0.05 and 0.5 M, respectively. In the course of the preparation of the 9 Page 9 of 26

AgNPs, the initial colorless solution turned yellowish-brown as a result of the formation of

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silver nanoparticles. In the first step, the silver ions are reduced by the available reducing end

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groups of the pullulan (or oxidized pullulan). The resulted atoms form clusters that

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subsequently act as nucleation centers as soon as their concentration reaches a certain point.

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These nucleation centers in turn grow and form larger aggregates until all small aggregates

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are consumed. The surface ions are subsequently reduced, the aggregation process does not

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cease until larger particles are formed. However, the interaction of these particles with the

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polymer matrix would prevent further coalescence (Goia, 2004). An illustration of the silver

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nanoparticles coated by pullulan and oxidized pullulan is shown in Fig. 3. *

OH

O O

HO HO

OH O HO

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OH O OH

O O

HO

OH O

*

AgNPs COOH

O HO HO

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O OH O HO

COOH

O OH

an

AgNPs *

O O

HO

OH O

*

Fig.3. Illustration of the silver nanoparticles formation mediated by pullulan or oxidized

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pullulan.

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The intrinsic structure of pullulan carrying multiple hydroxyl groups confers a protective and

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highly functionalized shell for nanoparticles. The supramolecular associations created by

283

hydrogen bonds are able to coordinate to Ag providing stability for a longer period of time

284

(steric stabilization). Moreover, in the case of 6-carboxypullulan the introduction of COOH

285

groups will determine electrostatic repulsion between the negatively charged core-shell

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nanoparticles, an effect commonly referred to as electrosteric stabilization. The difference

287

between those samples in terms of stability can be clearly seen by the zeta potential of the

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AgNPs. The silver nanoparticles encapsulated by oxidized pullulan exhibit higher negative

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zeta potential, indicating rather high stability. In contrast, the AgNPs surrounded by

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unmodified pullulan lead to rather neutral particles with a slightly negative zeta potential. An

291

overview is summarized in Table 1.

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292 293 294 295

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Table 1. Sample codes and zeta potential (ζ), determined at 25oC, and pH=6.6, for the AgNPs

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synthesized using pullulan and oxidized pullulan as reducing/stabilizing agents AgNO3 conc. (M) 0.01 0.05 0.1 0.5 0.01 0.05 0.1 0.5

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ζ (mV) -0.35 ± 0.1 -3.46 ± 0.2 -2.2 ± 0.2 -4.3 ± 0.3 -25.6 ± 0.3 -37.4 ± 0.5 -32.2 ± 0.4 -34.4 ± 0.6

ip t

Reducing /Stabilizing agent Pullulan Pullulan Pullulan Pullulan Oxidized Pullulan Oxidized Pullulan Oxidized Pullulan Oxidized Pullulan

cr

Sample code AgPu 0.01 AgPu 0.05 AgPu 0.1 Ag Pu 0.5 AgOxPu 0.01 AgOxPu 0.05 AgOxPu 0.1 AgOxPu 0.5

The effect of the reducing/stabilizing agent on the AgNPs formation is illustrated by recording

300

the UV-vis spectra, Fig. 4. It is well known that silver nanoparticles absorb radiation in the

301

visible region of the electromagnetic spectrum (380-450 nm) due to the SPR transition

302

(Bankura et al., 2012; Pandey, Goswami & Nanda, 2012). The UV-vis absorption spectra of

303

the silver nanoparticles are dependent on many factors, such as the size and shape of the

304

nanoparticles, the silver precursor concentration, as well as the structure of the polymer used

305

for the stabilizing the nanoparticle. As expected, the differences on the macromolecular

306

backbone structures of pullulan and oxidized pullulan (obtained through the replacement of

307

90% of CH2-OH groups from native pullulan by COOH moieties), used as stabilizer in this

308

study will induce variations of the absorption maximum, i.e. 420 nm for oxidized pullulan vs.

309

424 nm for native pullulan). These differences originates in opposite nature of the two

310

biopolymers: one uncharged polysaccharide (pullulan) and another one, highly negatively

311

charged (oxidized pullulan), which essentially trigger different reducing and stabilizing

312

activity. The lower concentrations of silver nitrate added to the pullulan/oxidized pullulan

313

solutions, will shift the surface Plasmon resonance band toward lower wavelengths, (see

314

Supplementary content associated with this paper) which indicates that the size and size

315

distribution were decreased. The maximum absorption peak however, was observed at highest

316

silver nitrate concentration used in this study (samples AgPu 0.5 and AgOxPu 0.5).

317

Additionally, the UV-vis spectra of the same samples were recorded after six months of

318

storage at room temperature (Fig. 4b). The SPR band of the AgOxPu 0.5 sample red shifted

319

with only 4 nm, whereas the AgPu 0.5 sample exhibit a SPR band red shifted with 13 nm.

320

These observations suggest that oxidized pullulan could be a better stabilizing agent for

321

AgNPs than pullulan, when the agglomeration is more pronounced.

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1,8

a

420 nm

1,4

424 nm

1,2

AgOxPu 0.5

1,0 300

350

400

450

500

550

600

650

cr

AgPu 0.5

ip t

Absorbance

1,6

700

us

λ (nm)

2,0

an

322 b

M

424 nm

d

1,0 437 nm 0,5

350

400

450

500

550

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0,0 300

te

Absorbance

1,5

600

AgOxPu 0.5 AgPu 0.5

650

700

λ (nm)

323 324

Fig.4. The UV-vis spectra of the silver nanoparticles during synthesis (a), and after six

325

months storage at room temperature (b).

326 327

The differences in the UV-vis absorption are related also with to the difference in the size of

328

the corresponding nanoparticles. The TEM images (Fig. 5) clearly reveal that the size of the

329

nanoparticles depend on the polysaccharide used for reduction. As expected, in case of the use

330

of unmodified pullulan as stabilizer, the silver nanoparticles form larger aggregates (50-55

331

nm) than those stabilized by the oxidized pullulan (8to 25 nm). The shape of the particles is

332

spherical and in all cases the polysaccharide shell can be visualized by TEM. A rather large 12 Page 12 of 26

nanoparticle with a thick shell is depicted in Figure 5a. In the second picture, Fig. 5b, the

334

AgNPs have been interestingly obtained, in a higher extent as “twins” nanoparticles

335

structures. For the oxidized pullulan – silver nanoparticles, the obtained structures are much

336

smaller but the polysaccharide shell is still present (Fig. 5c, d). For the pullulan stabilized

337

silver nanoparticles, the mean size of the particles increased by increasing the silver nitrate

338

concentration, ranging from 30 nm in the case of sample AgPu 0.01 to 55 nm for the sample

339

having the highest silver nitrate concentration (AgPu 0.5). Similarly, the mean size of the

340

AgOxPu 0.01 nanoparticles as determined by TEM is only 8 nm, increasing gradually once

341

the AgNO3 is enlarged: 15 nm for the sample AgOxPu 0.05, 20 nm for the sample AgOxPu

342

0.1 and 25 nm for the sample AgOxPu 0.5 respectively.

343

Fig. 5. TEM images of the AgPu 0.1 (a), AgPu 0.5 (b), AgOxPu 0.1 (c), and AgOxPu 0.5(d)

344

samples (insets are shown individualized nanoparticles).

345

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13 Page 13 of 26

ip t cr us an M

Fig.6. Mean particle size diameter (nm) as obtained by TEM and DLS measurements for the

348

pullulan and oxidized pullulan mediated silver nanoparticles.

te

349

d

346 347

Dynamic light scattering analyses of the colloidal solutions of the synthesized nanoparticles

351

samples were carried out, showing reasonable PDI values, ranging from 0.106 in the case of

352

AgPu 0.5 sample, to 0.55 for the AgOxPu 0.01 sample, which denote a narrow polydispersity.

353

The diameter observed by DLS measurements varied from 233 nm in the case of pullulan-

354

mediated silver nanoparticles, to 80 nm for those prepared by using oxidized pullulan,

355

(sample AgOxPu 0.01) Fig. 6. These results clearly differ on those obtained by using TEM

356

measurements. The differences are mainly due to the processes involved in the preparation of

357

the samples. TEM technique uses the dry state of the sample to determine its particle size,

358

whereas DLS method implies the measurement of the particle size in the hydrated state.

359

Therefore we can assume that the size obtained by TEM is the actual size of the nanoparticle,

360

since the diameter measured by the light scattering method is a hydrodynamic diameter,

361

which appears always overestimate due to the strong interactions between solvent and

362

nanoparticles constituents, especially hydrophilic biopolimers such as pullulan or its oxidized

363

form (Gao et al., 2008). The hydrodynamic diameter as determined by DLS measurements

364

increased also by increasing the silver precursor concentration, Fig. 6. Besides the factors

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14 Page 14 of 26

linked by samples preparation for TEM and DLS analyses, discussed above, we can’t rule out

366

the aggregation phenomena appeared at high loading AgNO3 for the AgNPs preparation,

367

which are less probable to occur for the samples obtained in diluted solutions of AgNO3 (0.01

368

and 0.05 M AgNO3).

369

The pullulan and oxidized pullulan mediated silver nanoparticles were further analyzed by

370

means of X-ray diffraction (XRD), in order to reveal the crystalline nature of those, Fig. 7. It

371

can be seen, that the crystalline structure of silver nanoparticles is confirmed, since the typical

372

XRD pattern of the AgNPs with four distinctive diffraction peaks in the 2 theta region are

373

present: (111), (200), (220), and (311). These peaks are consistent with a face centered cubic

374

(fcc) phase of Ag0 standard (JCPDS Card no. 04-0783). The plane (111) presents the highest

375

intensity as compared with the other planes, and the most interesting feature showed in Fig.

376

7a, is the difference occurred in the XRD patterns between the pullulan and 6-carboxypullulan

377

used as mediators for the AgNPs, which suggest also differences on the silver capping by

378

these two mediators (i.e. via hydroxyl groups in the case of pullulan and via both hydroxyl

379

and carboxyl groups in the case of oxidized pullulan). Nevertheless, the entire XRD patterns

380

are characteristic for the AgNPs and are in concordance with the previous published results in

381

this field (Kanmani & Taik Lim, 2013; Bankura et al., 2012). However, in the sample

382

AgOxPu 0.1, two additional peaks were observed with the Bragg peaks representative of fcc

383

silver nanocrystals. These sharp peaks, at 2-theta values at ca. 55 and 58 could have resulted

384

from crystallization of the reducing and stabilizing agent in the AgNPs, as have previously

385

been observed by Bankura et al. (2012) and Roopan et al. (2013) in the case of using dextran

386

and Cocos nucifera coir waste extract for preparation of AgNPs.

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15 Page 15 of 26

(111) (111)

a

(200) (220)

(311)

AgPu 0.1

ip t

(200) AgPu 0.5

(220)

(311)

cr

AgOxPu 0.1

30

40

50

60

70

2 Theta

80

Ac ce p

388

te

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an

387

us

AgOxPu 0.5

389

Fig.7. X-ray diffraction pattern of pullulan/oxidized pullulan mediated silver nanoparticles (a)

390

and EDX spectra of AgPu 0.5 sample (b) and AgOxPu 0.5 sample (c).

391 392

The analysis of the energy dispersive X-ray (XRD) spectroscopy can offer valuable

393

information regarding the quantitative and qualitative presence of the elemental silver

394

involved in the synthesis of the nanoparticles. Fig. 7b and 7c pointed out the presence of

395

elemental silver in XRD spectra of AgPu 0.5 and AgOxPu 0.5 samples, confirmed by the

396

strong peak at ~3 keV. Bankura et al. (2012) have also reported the presence of ~3 keV peak

397

signal in the silver region for the dextran-stabilized silver nanoparticles. This is a clear

398

indication of the silver nanoparticles formation in the reaction medium. Moreover, the

399

presence of C, and O elements also proves that the nanocomposite consist on polysaccharide

400

(pullulan or oxidized pullulan) and silver nanoparticles.

16 Page 16 of 26

The FTIR spectra of pullulan, oxidized pullulan and pullulan/oxidized pullulan nanoparticles

402

are presented in the Fig. 8 and also in the Supplementary data associated with this paper.

403

Unoxidized pullulan shows typical FTIR spectrum for this kind of polysaccharide: a broad

404

adsorption between 3000 – 3600 cm-1 region due to OH stretching vibration, centered around

405

3423 cm-1, a band at 2926 cm-1 corresponding to the C-H stretching vibration, adsorption

406

bands at 1458 cm-1 assigned to symmetric CH2 bending vibration and 930 cm-1 assigned to C-

407

O-C stretching at α-(1→4)-glycosidic linkages, a marker sign of “amorphous” region

408

(Ciolacu, Ciolacu & Popa, 2011). Moreover, the absorption peak at 1653 cm-1 indicated the

409

presence of the carbonyl (C=O) stretching frequency (Kanmani & Taik Lim, 2013). After

410

oxidation, some changes in the FTIR spectra appears, the most notable being the presence of

411

one sharp and intense band at 1608 cm-1, assigned to the C=O asymmetrical stretching of the

412

free carboxylate groups introduced into the pullulan structure, whereas the adsorption band at

413

1418 cm-1 represents the C-O symmetric stretching of dissociated carboxyl groups (Saito et

414

al., 2009).

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17 Page 17 of 26

AgPu 0.5 AgPu 0.1

3500

3000

2500

2000

1500

1000

500

Wavenumber (cm-1)

415

an

AgOxPu 0.5

us

4000

cr

ip t

Pullulan

M

AgOxPu 0.1

4000

3500

3000

2500

te

d

OxPu4

2000

1500

1000

500

416 417 418

Ac ce p

Wavenumber (cm-1)

Fig. 8. FTIR spectra of pullulan, oxidized pullulan, and their silver nanoparticles.

419

After incorporating the silver nanoparticles into the pullulan, the FTIR spectrum of resulted

420

product, differ on that of native pullulan; the OH stretching vibration band (centered at 3423

421

cm-1 in pullulan) shifted to 3438 cm-1 in the case of pullulan nanoparticles, Fig. 8, as well as

422

the band from 1653 cm-1 was shifted to lower wave numbers. The strong interaction between

423

pullulan and silver nanoparticles could be better evidenced by calculating the FT-IR peak area

424

ratio (ν 3423 / ν 1653 cm-1) for pullulan and FT-IR peak area ratios (ν 3438 / ν 1636 (1639)

425

cm-1) for the AgPu samples. This ratio increases from 2.24 in native pullulan, to 3.11 in AgPu

426

0.01 sample, 3.23 in AgPu 0.05 sample, 3.27 in AgPu 0.1 sample, and 3.33 in AgPu 0.5

427

sample respectively. The IR absorption for νs C=O (1418 cm-1) and νas C=O (1608 cm-1) of

428

deprotonated carboxylic acid group are observed in the spectra of oxidized pullulan-mediated 18 Page 18 of 26

silver nanoparticles, but their intensities are visibly decreased. These findings are supporting

430

the binding of carboxylic groups with Ag nanoparticles through free carboxylate group

431

(Shankar, Ahmad & Sastry, 2003). Similarly, the FT-IR peak area ratio (ν 3420 / ν 1608 cm-1)

432

for oxidized pullulan and FT-IR peak area ratios (νΟΗ/ νasC=O) for the AgOxPu samples

433

were calculated. Again, the ratio increases from 1.08 in oxidized pullulan, to 1.10 in AgOxPu

434

0.01 sample, 1.22 in AgOxPu 0.05 sample, and 1.77 for both AgOxPu 0.1 and AgOxPu 0.5

435

samples. These results strongly suggest that the νasC=O peak intensities decreases due to their

436

interaction with silver. Interestingly, in the spectra of oxidized pullulan nanoparticles a new

437

adsorption band as a shoulder, around 1736 cm-1, appears due to carboxylate groups which are

438

involved in the attachment of silver atoms. Also the specific absorption peak at 1384 cm-1 of

439

silver nanoparticles can be seen on the FTIR spectra.

441

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440

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3.3.Antimicrobial activity against microorganism

442

Freshly prepared silver nanoparticles were further employed to test their antimicrobial activity

444

against Gram-positive S. aureus ATCC 29213 and Gram-negative E. coli ATCC 25922. Both

445

test organisms have multiresistant strains (MRSA, VREC), which often cause problems in the

446

course of medical interventions. Although those multiresistant pathogens are not more

447

virulent than their “common” congeners, a treatment using broadband antibiotics often fails

448

which can lead to amputation of limbs and in severe cases even to death. In contrast, there are

449

hardly any strains known that develop resistances towards silver based products due to

450

different modes of action of silver against these pathogens.

451

In this paper, we use commonly employed ZOI tests in order to elucidate the antimicrobial

452

activity of the silver nanoparticles. After 24 h of incubation at 37oC, growth suppression was

453

observed in plates loaded with 5 μg of silver nanoparticles. The diameter of the zone of

454

inhibition (ZOI) in millimeters was measured. The mean ZOI of the triplicates of diameter are

455

tabulated in Table 2.

456

Table 2. Mean ZOI values against S. aureus and E. coli (mm)

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Sample code AgPu 0.1 Ag Pu 0.5 AgOxPu 0.1 AgOxPu0.5

S. aureus (mm) 8.6 9.7 12.3 14.8

E.coli (mm) 9.4 10.8 9.8 11.4

457 19 Page 19 of 26

The ZOI of around 15 mm and 10 mm respectively was observed for the Gram-positive

459

bacterial strain S. aureus ATCC 29213 for AgOxPu 0.5 and AgPu 0.5. In the case of Gram-

460

negative bacterial strains E. coli ATCC 25922 the detected ZOI were quite similar for the

461

samples studied, with a maximum value of 11.4 for the AgOxPu 0.5. Based on these results, it

462

can be concluded that both pullulan and oxidized pullulan nanoparticles had significant

463

antibacterial action on a large variety of bacteria ranged from Gram-positive to Gram-

464

negative ones. Similar results were found by Priyadharshini et al. (Priyadharshini, Gopinath,

465

Meera Priyadharsshini, MubarakAli & Velusamy, 2013) against Gram positive bacteria B.

466

cerus and Streptococcus pyogenes and Gram negative bacterium E. coli, this behaviour being

467

linked with the tick cell wall of bacteria. Gram positive bacterial strains posses a three-

468

dimensional thick layer of ~ 20–80 nm composed on linear polysaccharide chains cross linked

469

by more short peptides (peptidoglycan layer) which is arduous to be penetrate by AgNPs. In

470

the case of Gram negative bacteria, the peptidoglycan layer is only ~ 7–8 nm thick, being

471

much more accessible for AgNPs (Priyadarshini et al., 2013). This explanation is in

472

accordance with our results for the uncharged pullulan based nanoparticles, Table 2.

473

However, highly negative charged oxidized pulllan based nanoparticles, exhibit a stronger

474

affinity toward Gram positive bacteria, due to their attachment on the positive surface of

475

bacteria, which induces changes on the morphology of the bacteria membrane, by affecting

476

respiration processes and cell division, leading eventually to the perish of the cell, (Gonzales-

477

Campos et al., 2013). It is important to point out the exceptional activity of the oxidized

478

pullulan nanoparticles against Gram-positive bacterial strains compared with other Gram-

479

negative strains in this antibacterial susceptibility assay, which can be explained by the highly

480

negative zeta potential value of the mentioned silver nanoparticles. Negatively charged OxPu

481

nanoparticles can attach to the positively charged bacterial cell surface and interrupt the

482

membrane’s functionality according with the two mechanisms: i) by causing an effusion of

483

the intracellular components, or ii) by inhibiting the transport of nutrients into cells.

484

(Helander, Nurmiaho-Lassila, Ahvenainen, Rhoades & Roller, 2001). Conversely, positive

485

charged chitosan silver nanocomposites were found to be more effective toward Gram

486

negative bacteria strains, due to their positive charge conferred by chitosan, which enhances

487

the binding ability to negative charges on the cell surface (Gonzales-Campos et al., 2013).

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488 489

4. Conclusions

490

A simple, safe, one-step, environmentally friendly method for the preparation of silver

491

nanoparticles in aqueous solutions of pullulan or oxidized pullulan at room temperature is 20 Page 20 of 26

reported. Both used polysaccharides act as a reducing and stabilizing agent for the synthesis

493

of silver nanoparticles. The stability of the prepared nanoparticles depends on the biopolymers

494

structure used for capping silver atoms, and range from three months (for the pullulan

495

nanoparticles) to more than six months for the oxidized pullulan nanoparticles. All the freshly

496

prepared nanoparticles showed excellent antibacterial activity against different types of

497

bacteria, activity which can be correlated with the zeta potential value of the samples. The

498

oxidized pullulan mediated silver nanoparticles, due to their higher negative values of the zeta

499

potential, acquire an increased stability due to repulsion among the particles, as well as

500

exhibit a strong antibacterial activity against Gram positive bacteria strains.

cr

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us

501 Acknowledgements

503

The authors gratefully acknowledge to the Acad. Professor Bogdan C. Simionescu for

504

providing the all laboratory facilities and fruitful discussions.

an

502

505 Appendinx A. Supplementary data

507

Supplementary data associated with this article can be found, in the online version, at…

509

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antiproliferative activities of

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antibacterial and

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Synthesis, characterization,

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Fig. 1. Simplified scheme of TEMPO-mediated oxidation of pullulan. Fig. 2. 13C-NMR spectra of pullulan (a), and OxPu4 (b).

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Fig. 3. Illustration of the silver nanoparticles formation mediated by pullulan or oxidized pullulan.

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months storage at room temperature (b).

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Fig.4. The UV-vis spectra of the silver nanoparticles during synthesis (a), and after six

cr

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Fig. 5. TEM images of the AgPu 0.1 (a), AgPu 0.5 (b), AgOxPu 0.1 (c), and AgOxPu 0.5(d)

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samples (insets are shown individualized nanoparticles).

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Fig.6. Mean particle size diameter (nm) as obtained by TEM and DLS measurements for the

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pullulan and oxidized pullulan mediated silver nanoparticles.

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Fig. 7. X-ray diffraction pattern of pullulan/oxidized pullulan mediated silver nanoparticles

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(a) and EDX spectra of AgPu 0.5 sample (b) and AgOxPu 0.5 sample (c).

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d

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Fig. 8. FTIR spectra of pullulan, oxidized pullulan, and their silver nanoparticles.

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