Antibacterial ability of immobilized silver nanoparticles in agar-agar films co-doped with magnesium ions

Carbohydrate Polymers 224 (2019) 115187

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Antibacterial ability of immobilized silver nanoparticles in agar-agar films co-doped with magnesium ions

T

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Slađana Davidovića, Vesna Lazićb, , Miona Miljkovića, Milan Gordićb, Milica Sekulićb, Milena Marinović-Cincovićb, Ishara S. Ratnayakec, S. Phillip Ahrenkielc, Jovan M. Nedeljkovićb a

Department of Biochemical Engineering and Biotechnology, Faculty of Technology and Metallurgy, University of Belgrade, Karnegijeva 4, 11120, Belgrade, Serbia Vinča Institute of Nuclear Sciences, University of Belgrade, P.O. Box 522, 11001, Belgrade, Serbia c South Dakota School of Mines and Technology, 501 E. Saint Joseph Street, Rapid City, SD, 57701, USA b

A R T I C LE I N FO

A B S T R A C T

Keywords: Agar-agar Silver nanoparticles Nanocomposite films Antimicrobial activity

The antibacterial ability of in situ prepared nanometer-sized silver particles, immobilized in agar-agar films, was studied as a function of the concentration of co-dopant, magnesium ions. Content of inorganic components in hybrid films was determined using inductively coupled plasma optic emission spectroscopy, and found to be low (< 2 wt.-%). Morphology of prepared hybrid films, studied by transmission electron microscopy, revealed the presence of non-agglomerated and randomly distributed 10–20 nm silver nanoparticles (Ag NPs) within the agaragar matrices. Fourier-transform infrared spectroscopy indicated the distinct chemical interaction between Ag NPs and polymer chains. Thermogravimetric analysis, as well as the determination of tensile strength, Young’s modulus, and elongation at break showed improvement of thermal stability and mechanical properties of agaragar matrices upon the incorporation of Ag NPs due to high compatibility between the hydrophilic organic component and inorganic components. The complete microbial reduction of Gram-positive bacteria Staphylococcus aureuswas observed for all agar-silver films, while satisfactory results were observed for Gramnegative bacteria Pseudomonas aeruginosa (≥99.6%). The release of Ag+ ions is suppressed by the increase of the concentration of Mg2+ ions and it was found to be significantly smaller (≤0.24 ppm) than the harmful ecological level (1 ppm).

1. Introduction Silver nanoparticles (Ag NPs) are widely used as an antimicrobial agent due to their high efficiency against microbial species (Gram-negative and Gram-positive bacteria, fungi, and viruses). The antimicrobial activity of Ag NPs strongly depends on their morphological (shape and size) and surface (the presence of ligands) properties, as well as, environment (pH, availability of oxygen, etc.) (Marambio-Jones & Hoek, 2010; Pal, Tak, & Song, 2007; Panaček et al., 2006; Rai, Yadav, & Gade, 2009). Various reducing agents with a wide range of reducing capabilities such as borohydrides (Jana, Gearheart, & Murphy, 2001; Vukovic & Nedeljkovic, 1993; Zhang et al., 2019), citrate (Bastús, Merkoçi, Piella, & Puntes, 2014), aldehydes (Sarkar, Jana, Samanta, & Mostafa, 2007), sugars (Vigneshwaran, Nachane, Balasubramanya, & Varadarajan, 2006), and alcohols (Pal, Shah, & Devi, 2009), including polyols (Kim, Jeong, & Moon, 2006) have been used to reduce Ag+ ions to metallic Ag NPs of desired morphology. In many applications, it is more convenient to use immobilized instead of free-standing Ag NPs.

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Up to now, a variety of inorganic and organic materials (silica, zeolite, fiberglass, carbon materials, paper, different types of polymers including textile fibers, etc.) have been used to either host or support Ag NPs in order to obtain nanocomposites with antimicrobial activity (Balogh, Swanson, Tomalia, Hagnauer, & McManus, 2001; Dankovich & Gray, 2011; Diagne et al., 2012; Furno et al., 2004; Guo et al., 2017; Ilić et al., 2010; Ilić, Šaponjić, Vodnik, Molina et al., 2009; Ilić, Šaponjić, Vodnik, Potkonjak et al., 2009; Krishnan et al., 2015; Lazic et al., 2012; Lazić et al., 2018; Lv et al., 2009; Marini et al., 2007; Mthombeni, Mpenyana-Monyatsi, Onyango, & Momba, 2012; Muthulakshmi, Rajini, Varada Rajalu, Siengchin, & Kathiresan, 2017; Oyanedel-Craver & Smith, 2008; Panacek et al., 2009; Radetić et al., 2008; Shome, Dutta, Maiti, & Das, 2011; Travan et al., 2009; Vi, Rajesh Kumar, Rout, & Liu, 2018; Vukoje et al., 2017, 2014; Zhang et al., 2008). Although the antimicrobial performance of Ag NPs is beneficial, its toxic action extends to mammalian cells (Ahamed, Alsalhi, & Siddiqui, 2010). However, a few reports indicated that the toxic effect of Ag NPs becomes less pronounced for organisms of increased complexity, i.e., the toxic action

Corresponding author. E-mail address: [email protected] (V. Lazić).

https://doi.org/10.1016/j.carbpol.2019.115187 Received 20 June 2019; Received in revised form 2 August 2019; Accepted 8 August 2019 Available online 09 August 2019 0144-8617/ © 2019 Elsevier Ltd. All rights reserved.

Carbohydrate Polymers 224 (2019) 115187

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of Ag NPs diminishes in degree from viruses to bacteria, fungi, and finally mammalian cells (Rai et al., 2009; Vazquez-Muñoz et al., 2017). Also, the larger silver particles (> 20 nm) have negligible toxicity towards mammalian cells, as well as silver particles caped with bio-active molecules such as polysaccharides, polyphenols, etc (Ahamed et al., 2008; Bondarenko et al., 2013; Braydich-Stolle, Hussain, Schlager, & Hofmann, 2005). Agar-agar is chemically inert and non-toxic hydrophilic polysaccharide extracted from the Gelidiaceae and Gracilariaceae families of seaweeds and mainly composed of alternating repeating units of Dgalactose and 3,6-anhydro-ß-galactopyranose (Tako, Higa, Medoruma, & Nakasone, 1999). Since conventional polymers have an undesired impact on the environment, bio-based materials emerged as an alternative in the area of food packaging; they are renewable, abundant, inexpensive, environmentally friendly and biodegradable. Also, biobased materials can host a wide range of additives including antimicrobial agents, and, because of that, the additional, antimicrobial function of free-standing biopolymer nanocomposite films providing extended shelf-life of food is recognized as a promising direction in the packaging industry. So far, several research groups successfully synthesized Ag NPs embedded in agar-agar matrix and investigated optical, microstructural, thermal, mechanical, and, the most important, antibacterial properties of obtained inorganic-organic hybrids (Arfat, Ahmed, & Jacob, 2017; Basumatary et al., 2018; Ghosh et al., 2010; Liu et al., 2018; Muthuswamy et al., 2007; Rhim, Wang, & Hong, 2013; Shankar & Rhim, 2017). Two different approaches to the preparation of agar-silver nanocomposite films can be distinguished. The first approach is based on separate preparation of silver colloids and consequent dissolution of agar-agar and plasticizer into the silver sol (Rhim et al., 2013). The second approach relays on the in-situ preparation of Ag NPs in agar-agar solution using either ethanol (Ghosh et al., 2010; Muthuswamy et al., 2007) or lignin as reducing agents (Arfat et al., 2017; Shankar & Rhim, 2017); of course, lignin has a dual function, and in addition, serves as a capping agent. The focus of the present study is the influence of the co-dopant, magnesium ions, on the antimicrobial ability of in-situ prepared agarsilver nanocomposite films. Interestingly, the synthesis of Ag NPs does not require the use of any external reducing/stabilizing agents. So, agar-agar has a dual function, and serves, at the same time, as a reducing agent and polymer matrix that accommodate Ag NPs. This synthetic approach is novel and preliminary results have been presented in our previous report (Davidović, Miljković, Radovanović, Dimitrijević, & Nešić, 2017). The thorough characterization of synthesized free-standing agar-silver nanocomposite films was performed including transmission electron microscopy, reflection, and infrared spectroscopy, thermogravimetric analysis, and evaluation of mechanical properties (tensile strength, Young's modulus, and elongation at break). The antibacterial ability of the agar-silver nanocomposite films was tested against Gram-positive bacteria Staphylococcus aureusand Gram-negative bacteria Pseudomonas aeruginosa using the colony count method. An attempt was made to correlate obtained results with the influence of co-dopant on the release of Ag+ ions in the surrounding media.

Table 1 The concentration of Ag and Mg2+ ions in pristine Aa film and nanocomposite agar-silver films (mg/g), and their corresponding photo images. Sample

Concentration of Ag (mg/g)

Concentration of Mg (mg/g)

Aa

0.0

0.17

Aa/Ag-1/Mg1

0.30

0.17

Aa/Ag-1/Mg10

0.30

1.76

Aa/Ag-5/Mg100

1.56

17.4

Photos

by 1 M NaOH. Finally, the desired amount of AgNO3 solution was added, and the solution was kept under constant stirring in dark for an additional 3 h at 60 °C. The formation of colloidal silver is indicated by the appearance of a yellow-brown color. Agar-silver nanocomposite films were cast drying silver colloids (9 mL) in silicone modules (55 mm diameter) for 48 h at room temperature. To obtain agar-silver nanocomposite films co-doped with Mg2+ ions, before film casting, proper amounts of concentrated MgCl2 solution were added to the silver colloid. For the sake of clarity, pristine agar-agar film will be labeled in the text as Aa, while agar-silver films co-doped with Mg2+ ions will be named Aa/Ag-1/Mg-1, Aa/Ag-1/Mg-10, and Aa/Ag-5/Mg-100. It is important to emphasize that Mg2+ ions are present in the agar-agar and index 1 corresponds to the initial concentration of Mg2+ ions in pristine Aa film. Other indexes (5 for Ag, as well as 10 and 100 for Mg2+ ions) correspond to the multiplied content of inorganic components in prepared films in comparison to the lowest content of Ag and Mg2+ ions. The content of silver and magnesium in prepared samples are presented in Table 1. 2.2. Characterization of agar-silver films The content of silver and magnesium in agar-silver nanocomposite films co-doped with Mg2+ ions was determined using inductively coupled plasma optic emission spectroscopy (ICP-OES Thermo Scientific iCAP 7400). Before measurements, all samples were digested in Milestone Start D microwave using concentrated HNO3 and H2O2. The release of silver ions from agar-silver films is studied at static conditions after 4 and 24 h of incubation in distilled water (pH 6.5) at room temperature. The samples (0.01 g) were placed into the flasks containing 10 mL of water. After the incubation period, agar-silver films were separated from the aqueous medium and the concentration of the released Ag+ ions was measured by the ICP-OES technique. The obtained results are the average values of 3 measurements; the standard deviation was within 5%. Microstructural characterization of the agar-silver nanocomposite films was performed on a transmission electron microscope (TEM) JEOL JEM-2100 LaB6. For that purpose, nanocomposite films were embedded in an epoxy resin (Epofix, Electron Microscopy Sciences) and cured overnight at 40 °C. Samples were then microtomed at room temperature to a thickness of around 70 nm, using an RMC PowerTome-XL ultramicrotome and Diatome diamond knife with a water-filled boat. The microtomed sections were transferred from the water surface to copper

2. Experimental 2.1. Preparation of agar-silver films The one-pot synthesis of agar-silver nanocomposite films is based on reducing the capability of deprotonated hydroxyl groups in agar-agar, similar to the procedure developed for the preparation of colloidal Ag NPs capped with dextran (Bankura et al., 2012; Davidović, Lazić et al., 2017). Briefly, 1.5 g of agar-agar powder was suspended in 100 mL of distilled water and autoclaved at 120 °C until its complete dissolution (about 20 min). Then 0.45 g of glycerol (plasticizer) was added, and the mixture was stirred at 60 °C for 1 h. After that, the pH was adjusted to 9 2

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grids using a Perfect Loop. Optical properties of synthesized nanocomposite films were studied by diffuse reflectance spectroscopy (Shimadzu UV–vis UV-2600 spectrophotometer equipped with an integrated sphere ISR-2600 Plus). The coordination of Ag NPs to the agar-agar matrix was examined using Fourier-transform infrared (FTIR) spectroscopy (Nicolet™ iS™ 10 FT-IR Spectrometer (Thermo Fisher Scientific) in attenuated total reflectance mode). The thermal stability of samples was studied using a simultaneous non-isothermal thermogravimetric analysis (TGA) and differential thermogravimetric (DTG) analysis using the SETARAM Setsys Evolution 1750 (SETARAM Instrumentation 7) instrument. The measurements were conducted at a heating rate of 10 °C min−1 in a dynamic argon atmosphere (flow rate 20 cm3 min−1) in the temperature range from room to 600 °C. Tensile strength (TS), Young's modulus (EM), and elongation at break (EAB) of the pristine agar-agar and agar-silver films were evaluated from stress-strain curves obtained using the InstronM1185 universal testing machine. The crosshead speed was 2 mm min−1 for all tested samples. Before testing, the films were conditioned in an environmental chamber at 25 °C and 50% of relative humidity for 24 h. For each formulation, the measurements of six samples (mold 5.0 × 30.0 × 0.05 mm) were performed. The obtained values of Young's modulus were within ± 10%, while the tensile stress and the elongation at break fluctuated in the range of ± 15%.

Fig. 1. The FTIR spectra of pristine Aa film (a), as well as, Aa/Ag-1/Mg-1 (b), Aa/Ag-1/Mg-10 (c), and Aa/Ag-5/Mg-100 (d) nanocomposite films.

the first nanocomposite film (Aa/Ag-1/Mg-1). To estimate the influence of the co-dopant on the properties of agar-silver films, the concentration of Mg2+ ions is increased 10 times, while the content of Ag NPs was kept constant (Aa/Ag-1/Mg-10). Finally, in the last sample (Aa/Ag-5/ Mg-100), concentrations of both inorganic components are additionally increased, as well as their ratio. It should be noted that the total amount of inorganic material in the film with the highest concentration of silver and magnesium (Aa/Ag-5/Mg-100) does not exceed 2 wt.-%, while in all other samples is significantly lower (< 0.2 wt.-%). To understand the interaction between Ag NPs and agar-agar matrix, the FTIR measurements were performed in the range of 4000500 cm−1 (Fig. 1). The FTIR spectrum of the pristine agar-agar film (curve a) was compared with FTIR spectra of agar-silver nanocomposite films loaded with different amounts of silver and Mg2+ ions (curves b, c, and d). The following main bands of the pristine Aa film were found: broadband in the range 3000-3700 cm−1, as well as bands at 2949, 2886, 1650, 1370, 1029, 925, and 884 cm−1. The FTIR spectrum is in agreement with the data reported in the literature by many research groups (Arfat et al., 2017; Hsieh et al., 2010; Kanmani & Rhim, 2014; Liu et al., 2018; Shankar & Rhim, 2017; Tako et al., 1999). Briefly, broadband in the range 3000-3700 cm−1 belongs to stretching vibration of −OH groups, while doublet at 2949 and 2886 cm−1 indicates symmetric and asymmetric stretches of a hydrogen atom in the −CH2 groups, respectively. The peak at 1650 cm−1 can originate from the stretching vibration of the conjugated peptide bond, as well as C]O stretching vibration (Socrates, 2001). The band at 1370 cm−1 can be assigned to ester sulfate groups. The bands at 1029 and 925 cm−1 are due to the CeO stretching, while the band at 884 cm−1 represent the CeH stretching of residual carbons of β-galactose. Peaks observed in the FTIR spectra of the agar-silver nanocomposite films are relatively similar to those of the pristine Aa film. However, there are some changes: doublet at 2949 and 2886 cm−1 is shifted to 2918 and 2835 cm−1 with a significant increase in intensity, the peak at 1650 cm−1 became less intensive, and appearance of two new peaks at 1558 and 1527 cm−1 can be noticed. We speculate that the decrease of the band at 1650 cm−1 and appearance of new bands is the consequence of the formation of metal-oxygen bonds (Ag‒O‒C) with covalent character (Socrates, 2001) that accompany electron-transfer reaction between keto groups and Ag+ ions in the course of formation of Ag NPs. At least, we can say that the changes in FTIR spectra induced by the addition of a new inorganic component indicate the occurrence of distinctive chemical interaction. The morphology of the agar-silver nanocomposite films is studied using TEM measurements. The low-magnification TEM image of Aa/Ag1/Mg-1 film is shown in Fig. 2A. In-situ synthesized non-agglomerated

2.3. Antimicrobial ability The time-dependent antibacterial ability of the agar-silver nanocomposite films was evaluated against Gram-positive bacteria Staphylococcus aureus(ATCC 25923) and Gram-negative bacteria Pseudomonas aeruginosa (ATCC 2739) by counting viable cells. Briefly, the quantitative antimicrobial test was performed as follows: 10 mg pieces of film samples were placed in 10 mL saline solution containing ∼104-105 CFU/mL of the tested bacteria and incubated at 37 °C. The 0.1 mL aliquots were taken after 4 and 24 h. The number of viable cells was determined after 24 h of growth in tryptone soy agar. The percentage of reduction (R%) was calculated according to Eq. (1): R(%) = 100×(C0 – C)/C0

(1)

where C0 is the number (CFU/mL) of microorganisms in control (pristine Aa film) and C is the number (CFU/mL) of microorganism colonies of the agar-silver nanocomposite films. The stated values are averages from three sets of independent measurements. The variation of obtained values was within 5%. 3. Results and discussions The new two-steps method for synthesis of agar-silver nanocomposite films co-doped with Mg2+ ions is based on dual functions of agar-agar. In the first synthetic step, agar-agar was used to reduce silver ions to metallic silver. The adjustment of pH at 9.0 lead to deprotonation of hydroxyl groups (Feng, Bagia, & Mpourmpakis, 2013) and consequent formation of keto groups with enhanced reducing ability due to the presence of lone electron pair. The advantage of an electrontransfer reaction between keto groups and Ag+ ions has been used for the preparation of colloidal Ag NPs capped with dextran (Bankura et al., 2012; Davidović, Lazić et al., 2017), but never for the synthesis of Ag NPs in agar-agar solutions. In the second step, the synthesized Ag NPs are incorporated in agar-agar matrix by removal of the solvent. The proper amount of co-dopant, Mg2+, was introduced before film casting. The prepared films have a sequential increase of silver and magnesium concentrations; the content of silver and magnesium, determined by ICP-OES technique, is presented in Table 1. Since in the pristine agaragar film (Aa) Mg2+ ions are present, only Ag NPs are incorporated in 3

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Fig. 2. (A) Typical TEM images of the Aa/Ag-1/Mg-1 nanocomposite film at low- and high-magnification (inset). (B) The Kubelka-Munk transformation of diffuse reflection data for the pristine Aa (a) film and Aa/Ag-1/Mg-1 (b) nanocomposite film; inset: photo-image of Aa/Ag-1/Mg-1 nanocomposite film.

Ag NPs are randomly distributed in the agar-agar matrix. Additional high-magnification TEM imaging (inset to Fig. 2A) revealed the presence of fairly uniform nearly spherical 10–20 nm in size Ag NPs. The increase of the concentration of Mg2+ ions did not induce agglomeration of Ag NPs since their content in the agar-agar matrix is low. The low-magnification TEM images of Aa/Ag-1/Mg-10 and Aa/Ag-5/Mg100 are presented in Supporting Information 1, and they are similar to the image shown in the main text. The Kubelka-Munk (K-M) transformation of diffuse reflection data for the pristine agar-agar film (Aa) and agar-silver nanocomposite film (Aa/Ag-1/Mg-1) are presented in Fig. 2B (curves a and b, respectively). The inset to Fig. 2B shows a photo-image of the Aa/Ag-1/Mg-1 nanocomposite film. While the K-M transform of the agar-agar film is rather featureless, the distinct surface plasmon resonance band at 420 nm can be observed in agar-silver nanocomposite film, indicating that embedded Ag particles in agar-agar matrix are in the nanometer size range. It should be emphasized that spectral features, i.e.position, and intensity of surface plasmon resonance band of Aa/Ag-1/Mg-10 and Aa/Ag-5/Mg-100 nanocomposite films (see Supporting information 2) are almost the same compared to the Aa/Ag-1/Mg-1 sample. Thus, all synthesized agar-silver nanocomposite films have the optical properties consistent with the presence of non-agglomerated Ag NPs embedded in the polymer matrix as observed by TEM analysis. The thermal stability of pristine agar-agar film (Aa) and agar-silver nanocomposite films co-doped with Mg2+ ions (Aa/Ag-1/Mg-1, Aa/Ag1/Mg-10, and Aa/Ag-5/Mg-100) was investigated using a simultaneous TGA and DTG analysis; the DTG data are presented in Fig. 3. The DTG curves indicate that the thermal degradation of all studied films occurs in three distinct steps. The first step of thermal degradation at ∼100 °C is attributed to the evaporation of moisture. The next two steps (peaks at ∼240 and ∼300 °C) correspond to the main thermal degradation process induced by the decomposition of agar-agar and plasticizer (glycerol). Their patterns are dependent on the content of the inorganic components in organic-inorganic nanocomposites. The thermal stability of the films is increasing with the increase of the content of the inorganic components, i.e., the peak at 300 °C is increasing on the expanse of the peak at 240 °C. This effect is not pronounced for the nanocomposite films with low content of inorganic phase (Aa/Ag-1/Mg-1 and Aa/Ag-1/Mg-10). However, the significant improvement of the thermal stability was observed for the Aa/Ag-5/Mg-100 sample with the highest concentration of Ag NPs and Mg2+ ions. Knowing that in the Aa/Ag-5/Mg-100 nanocomposite film weight percentage of magnesium is more than ten times larger than the weight percentage of silver (see Table 1), the improved thermal stability can be attributed to the presence of a high concentration of Mg2+ ions. It should be mentioned that the residual char content at 600 °C for all films was within 20 and 25 wt.-%. Similar char content, as well as the DTG pattern, was

Fig. 3. The DTG curves of pristine Aa film (a), as well as, Aa/Ag-1/Mg-1 (b), Aa/Ag-1/Mg-10 (c), and Aa/Ag-5/Mg-100 (d) nanocomposite films obtained under a dynamic argon atmosphere (flow rate 20 cm3 min−1) and the heating rate of 10 °C min−1.

recently reported in the literature by Basumatary et al. (2018), Kanmani and Rhim (2014) for the pristine agar-agar film. The influence of the composition of agar-silver nanocomposite films on the mechanical properties was compared with the pristine agar-agar film. The measurements of the tensile strength (TS), Young's modulus (EM), and elongation at break (EAB) were used to characterize mechanical properties of synthesized samples, and obtained data are presented in Table 2. The thickness of all prepared films was kept constant (53 ± 7 μm). The values for TS, EM, and EAB for pristine agar-agar (Aa) film were found to be 19.4 MPa, 0.40 GPa, and 12%, respectively. Taking into account possible differences in origin and conditioning of samples before measurements, the values for TS and EM for pristine agar-agar are close to the reported data in the literature (Wu, Geng, Chang, Yu, & Ma, 2009). The incorporation of small amount of Ag NPs in agar-agar matrix (0.03 wt.-% of silver in Aa/Ag-1/Mg-1; Table 2 Tensile strength (TS, MPa), Young's modulus (EM, GPa), and elongation at break (EAB, %) of pristine Aa film and agar-silver nanocomposite films.

4

Sample

TS (MPa)

EM (GPa)

EAB (%)

Aa Aa/Ag-1/Mg-1 Aa/Ag-1/Mg-10 Aa/Ag-5/Mg-100

19.4 58 65 51

0.40 2.04 1.96 1.34

12 13 20 17

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see Table 1) induced significant increase in TS and EM (58 MPa and 2.04 GPa, respectively), while the value for EAB (13%) is within the experimental error with the value obtained for Aa sample. Further increase of the content of inorganic components (Aa/Ag-1/Mg-10 and Aa/Ag-5/Mg-100) led to a slight increase of EAB values (20 and 17%, respectively), while the values for TS and EM remained close to the values obtained for Aa/Ag-1/Mg-1 nanocomposite film. The results show that the strength, stiffness, and flexibility of all agar-silver films co-doped with Mg2+ ions are improved in comparison to the pristine agar-agar film. This finding indicates high compatibility between the organic and inorganic phase in nanocomposite films, supported, also, by TEM data showing well-dispersed Ag NPs within the polymer matrix. The observed mechanical properties of nanocomposite agar-silver films are similar to the mechanical properties of agar-lignin-silver nanocomposite films reported by Shankar et al (Shankar & Rhim, 2017). The antibacterial efficiency of Aa/Ag-1/Mg-1, Aa/Ag-1/Mg-10, and Aa/Ag-5/Mg-100 nanocomposite films was tested against Gram-positive bacteria S. aureus and Gram-negative bacteria P. aeruginosa as a function of contact time (4 and 24 h). The concentration of nanocomposite films was kept constant in all experiments (1.0 mg/mL). The data concerning the counted number of colonies and the percentage of microbial reductions are collected in Table 3. Based on the presented data some general features can be recognized. First, the reduction of viable bacterial cells is, as expected, higher for the longer contact time. Second, the complete reduction of S. aureus (99.9%) was reached after 24 h of contact for all synthesized agar-silver nanocomposite films. Third, in the case of P. aeruginosa, the satisfactory values of microbial reductions (99.6, 99.8, and 99.9%) were found after 24 h for Aa/Ag-1/ Mg-1, Aa/Ag-1/Mg-10, and Aa/Ag-5/Mg-100 nanocomposite films, respectively. Fourth, at shorter time of contact (4 h), the difference in biological response of S. aureus and P. aeruginosa can be observed. It is well-known that due to the reduced membrane permeability built up of a bilayer of lipids, P. aeruginosa possesses low vulnerability toward antibiotics (Ghai & Ghai, 2017; Morita, Tomida, & Kawamura, 2014) and silver (Salomoni, Léo, Montemor, Rinaldi, & Rodrigues, 2017). Three different toxic mechanisms, responsible for the antibacterial action of Ag NPs, can be recognized in the literature. First is the disruption of bacterial membranes by Ag NPs itself (Xiu, Mao, & Alvarez, 2011). Second is the change of metabolic activity of cells due to the formation of reactive oxygen species (Morones et al., 2005). The third is the damage of bacterial cell walls due to the interaction of released Ag+ ions with amino and carboxyl groups of peptidoglycans from cell walls (Lok et al., 2007; Xiu, Zhang, Puppala, Colvin, & Alvarez, 2012). Having in mind that Ag NPs are immobilized within the polymer matrix, the release of Ag+ ions is the most likely behind the effects of nanocomposite films on bacteria. However, due to the co-occurrence of Ag NPs and free Ag+ ions during the exposure period, it is difficult to

Table 4 The concentration (ppm) and weight-percentage of released Ag+ ions normalized to the content of silver in agar-silver nanocomposite films as a function of contact time with saline solutions. Sample

Aa/Ag-1/Mg-1 Aa/Ag-1/Mg10 Aa/Ag-5/Mg100

CFU/mL

The weight-percent of released Ag+ ions

Time (h)

Time (h)

4

24

4

24

0.15 0.08

0.24 0.21

50.0 26.7

80.0 70.0

0.05

0.20

3.2

12.8

discriminate their contributions. Since, in this particular case, Ag NPs are embedded into the polymer matrix, their ability to interact with bacteria is significantly diminished, and, it is our belief that released Ag+ ions are mainly responsible for the toxic action of agar-silver nanocomposite films. Of course, proper adjustment of the concentration of released Ag+ ions is necessary for the usage of Ag NPs as a bactericidal agent, since Ag+ ions retain its cytotoxicity and ecotoxicity even at concentrations as low as 1 mg/L (Panacek et al., 2009). An attempt was made to correlate the antimicrobial efficiency of agar-silver nanocomposite films with the concentration of released Ag+ ions. The concentration of released Ag+ ions, as well as weight-percentages of released Ag+ ions normalized to the content of silver in agar-silver nanocomposite films (see Table 1) after 4 and 24 h of contact with saline solution, are shown in Table 4. It should be noticed that the increase of concentration of co-dopant Mg2+ ions suppresses the release of Ag+ ions. The lower release of Ag+ ions is even observed for the sample with the five-fold higher content of Ag NPs (Aa/Ag-5/Mg-100) than others (Aa/Ag-1/Mg-1, Aa/Ag-1/Mg-10), but with the highest concentration of Mg2+ ions. This effect is more pronounced at shorter contact time (4 h), while, when contact time is longer (24 h), concentrations of released Ag+ ions are becoming close to each other. Also, the concentration of released Ag+ ions ≥0.20 ppm was found to be sufficient to induce the complete reduction of S. aureus (99.9%) and satisfactory reduction of P. aeruginosa (≥99.6%). This concentration of Ag+ ions is significantly below the harmful ecological level (1 ppm) (Panacek et al., 2009). It should be emphasized that nanocomposite agar-silver films (Aa/Ag-1/Mg-1 and Aa/Ag-1/Mg-10) are designed in such way that concentrations of Ag+ ions after complete dissolution of metallic silver should be bellow undesired level under experimental conditions used in the antibacterial tests. For the sample with the highest content of Ag NPs (Aa/Ag-5/Mg-100), the weight-percentages of released Ag+ ions normalized to the content of metallic silver in nanocomposite is small (12.8%) after 24 h. This result indicates that nanocomposite films with the high content of Ag NPs can be used under long-run-working conditions.

Table 3 The antibacterial ability of agar-silver nanocomposite films against S. aureus and P. aeruginosa as a function of time; the number of bacterial colonies (CFU/ mL) and the percentage of microbial reduction (R, %). Sample*

Concentration of released Ag+ ions (ppm)

4. Conclusion

R (%)

4h

24 h

4h

24 h

S. aureus** Aa/Ag-1/Mg-1 Aa/Ag-1/Mg-10 Aa/Ag-5/Mg-100

5.2 × 104 1.7 × 103 9.0 × 104

300 0 2

81.4 89.4 86.0

99.9 99.9 99.9

P. aeruginosa** Aa/Ag-1/Mg-1 Aa/Ag-1/Mg-10 Aa/Ag-5/Mg-100

4.0 × 103 2.1 × 103 2.3 × 103

2.7 × 103 1.2 × 103 800

70.5 79.0 77.0

99.6 99.8 99.9

To summarize, the novel synthetic procedure is developed for the preparation of the agar-silver nanocomposite films co-doped with magnesium ions. The satisfactory antibacterial performance against Gram-positive bacteria S. aureus and Gram-negative bacteria P. aeruginosa was observed for all synthesized samples, although the content of metallic silver in hybrids does not exceed 0.2 wt.-%. The increase in the content of co-dopant (Mg2+ ions) in nanocomposite films leads to a decrease in the release of silver ions. This effect provides an easy way to adjust the concentration of released silver ions (< 0.25 mg/L) in surrounding media significantly below harmful ecotoxicity level (1 mg/L), but to keep it sufficient to induce toxic action against bacteria. The simplicity of synthetic approach, the antimicrobial ability of

* Concentration of nanocomposite films was 1.0 mg/mL. ** Initial numbers of S. aureus and P. aeruginosa were 5.0 × 105 and 1.0 × 104 CFU/mL, respectively. 5

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immobilized Ag NPs in a polymer matrix, as well as improved thermomechanical properties of hybrid films are promising starting point for further studies oriented to potential applications such as bio-medical and food packing. Of course, a prerequisite for any such applications includes experiments with mammalian cells to assess the possible toxicity of the nanocomposite.

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