silver nanocomposite hydrogels and their antimicrobial properties

    In Situ Synthesis of High Swell Ratio Polyacrylic Acid/Silver Nanocomposite Hydrogels and Their Antimicrobial Properties Yi-Syuan Wei, Ko-Shao Chen, Lii-Tzu Wu PII: DOI: Reference:

S0162-0134(16)30233-1 doi: 10.1016/j.jinorgbio.2016.08.007 JIB 10062

To appear in:

Journal of Inorganic Biochemistry

Received date: Revised date: Accepted date:

24 March 2016 5 August 2016 22 August 2016

Please cite this article as: Yi-Syuan Wei, Ko-Shao Chen, Lii-Tzu Wu, In Situ Synthesis of High Swell Ratio Polyacrylic Acid/Silver Nanocomposite Hydrogels and Their Antimicrobial Properties, Journal of Inorganic Biochemistry (2016), doi: 10.1016/j.jinorgbio.2016.08.007

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ACCEPTED MANUSCRIPT In Situ Synthesis of High Swell Ratio Polyacrylic Acid/Silver Nanocomposite Hydrogels

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and Their Antimicrobial Properties

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Department of Materials Engineering, Tatung University, Taipei, Taiwan The Institute of Medical Science and Department of Microbiology, China Medical

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Yi-Syuan Wei1, Ko-Shao Chen*1, Lii-Tzu Wu2

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University Hospital; Taiwan.

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Professor Ko-Shao Chen

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*Corresponding Author

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Department of Materials Engineering, Tatung University, Taipei, Taiwan 10461

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TEL: 886-2-25925252 ext3411 Re-ext.416 FAX: 886-2-25936897 E-mail: [email protected]

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ACCEPTED MANUSCRIPT Highlights

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An easy, fast, and highly moldable process to produce nanocomposite hydrogels. The said hydrogels contain acrylic acid monomer only without cross-linker. Silver ion acts as catalyst in initiating persulfate and as a cross-linking agent. The proposed method yields superabsorbent hydrogels. The silver nanocomposite hydrogels have highly antibacterial capability.

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1. 2. 3. 4. 5.

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ACCEPTED MANUSCRIPT Abstract Silver nanocomposites embedded within a polymer matrix have attracted attention in

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recent years. Ionic polymer hydrogels comprise networks of chemically or physically

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cross-linked polymers that swell considerably in an appropriate solvent. In this study, we used a solution of the carboxylic monomer acrylic acid and silver nitrate to prepare

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nanocomposite hydrogels through ultraviolet (UV)-light irradiation. Silver-impregnated

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biomaterial composed of acrylic acid contains only a monomer and no cross-linker. The formation of hydrogels and reduction of silver nanoparticles were affected by the preparation

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parameters, that is, the monomer concentration and silver nitrate concentration. The

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morphology, structure, and size of the silver nanocomposite hydrogels were evaluated

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through field emission scanning electron microscopy and UV-visible absorption. The antimicrobial activity of the samples was tested against fourstandard strains Candida

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albicans, Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli; and five clinical bacterial isolates Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, Acinetobacter baumannii, Klebsiella pneumonia. The silver nanocomposite hydrogels exhibited an interconnected porous structure and could absorb 400 to 550 grams of deionized water per gram of dried hydrogel. Moreover, these hydrogels produced a strong antibacterial effect, which can be useful in developing new superabsorbent antimicrobial pharmaceutical products. Keywords: Ag nanoparticles; composite hydrogel; antimicrobial; drug-resistance bacteria 3

ACCEPTED MANUSCRIPT 1.

Introduction Hydrogels are three-dimensional cross-linked hydrophilic polymers that can absorb

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considerable quantities of water (10g/g) [1] or saline or physiological solutions compared

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with general absorbing materials. Because of their high swelling ratio (SR) and biocompatibility, hydrogels have several applications in biomedical engineering, agriculture,

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biosensors, drug delivery, and adsorption of heavy metals. Polyacrylic acid (PAAc) with

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carboxylic acid functional groups can stabilize compounds with metal ions. The carboxylic acid loses protons and becomes negatively charged in an alkaline or neutral pH solution;

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water-absorbent materials.

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therefore, hydrogels exhibit a swelling state, and PAAc is suitable for fabricating

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Zheng et al. [2] used an organic solvent and a cross-linker to synthesize a poly(ethylene glycol) and PAAc interpenetrating network hydrogel for tissue engineering, observing that

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58% of rabbits retained clear corneas for more than 6 months. Franklin et al. [3] synthesized pH-responsive citric acid–glycerol–acrylic acid biopolymeric hydrogels at 140 °C for 2 h. Furthermore, Amin et al. [4] exposed a cross-linking agent to electron-beam radiation to synthesize thermo- and pH-responsive bacterial cellulose/acrylic acid hydrogels for drug delivery. Currently, PAAc gel is used in physical supplies and diapers, and it provides added value in applications such as biomedical materials [5], contact lenses [6], drug delivery [7],and biosensing materials [8]. However, hydrogel preparation methods often involve using an organic solvent, high 4

ACCEPTED MANUSCRIPT temperature, long reaction time, or complicated steps to confer certain properties (such as temperature or pH sensitivity) upon the hydrogels. To improve the properties of hydrogels,

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metal ions, in the form of micro- or nanoparticles, such as gold [9], silver [10, 11], iron [12],

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TiO2 [13], and carbon nanotubes [14], can be added. Nanometal composite hydrogels have been prepared through free-radical polymerization and graft copolymerization [15],

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cross-linking [16], and radiation and gamma irradiation [17].

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Silver ions and nanoparticles are the most effective because they exhibit potent antimicrobial efficacy against bacteria, fungi, and viruses. Silver can bind to microbial DNA,

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generate reactive oxygen species, inhibit bacterial replication, and bind to the thiol groups of

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the metabolic enzymes of the bacterial electron transport chain, causing their alteration and

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inactivation [18, 19]. Silver ions and nanoparticle polymer composites have been widely used as antibacterial agents in wound dressings, scaffolds, water purification systems, and

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medical devices [20].

Nanosilver production methods such as gas condensation [21] and electrochemical [22] and salt reduction [23] are widely used. Nanosilver/polymer composites, such as poly (acryloyl phenylalanine-co-N'-isopropylacrylamide) [24] and benzotriazole maleimide/ furan-modified gelatin [25], have a relatively uniform silver particle size and exhibit excellent chemical and physical properties as well as antibacterial properties. Xuet al. [26] synthesized solutions of Ag+ ions in acrylic acid monomer by using gamma-ray irradiation. Mohan

et

al.

[27]

used

N-isopropylacrylamide, 5

sodium

acrylate,

and

ACCEPTED MANUSCRIPT N,N’-methylenebisacrylamide (cross-linker) to prepare hydrogels and then immersed them in silver ion solutions for the in situ reduction of silver nitrate in the presence of sodium

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borohydride. Vimala et al. [28] first reduced a silver nanoparticle solution and then added

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chitosan-polyvinyl alcohol (PVA) and glutaraldehyde (cross-linker)to prepare chitosan-PVA silver nanoparticle films. Furthermore, Dong et al. [29] used cellulose nanofibrils in

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chelating silver ions to prepare a hydrogel, aerogel, and film. These methods can produce

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highly stable and uniformly distributed silver nanoparticles in hydrogels. However, these methods require high energy or a long time to complete polymerization and many tedious

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steps for preparing composite hydrogels.

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The objective of this study was to use an easy, fast, and highly shape process to produce

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nanocomposite hydrogels with excellent antibacterial ability by combining PAAc and silver. Silver ions play two crucial roles as catalysts in persulfate initiation and cross-linking agents

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[30]. It is very important, that there were no references to combine these two roles and to study the inhibitory effect of multidrug resistant bacteria of this polyacrylic acid/silver hydrogel. In this unique one-step process for preparing composite hydrogels, PAAc can self-assemble in water to form a hydrogel. By adjusting the reducing agent concentration, we controlled the reduction rate and size of silver nanoparticles and tested the antimicrobial properties of the silver nanocomposite hydrogels. 2.

Experimental Methods

2.1 Hydrogel Formation 6

ACCEPTED MANUSCRIPT To synthesize silver composite hydrogels, a freshly prepared 8–16 wt % solution of acrylic acid (Wako Pure Chemical Co., USA) was mixed with 10 mM silver nitrate solution

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(Showa Denko) in water. Ammonium persulfate (Wako Pure Chemical Co., USA.) was then

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added as an initiator and the solution was stirred sufficiently to dissolve it. The solution was then placed into a box with an ultraviolet (UV) lamp (1000 W) and exposed to UV

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irradiation (wavelength 275 nm, time 6 min) to induce the in situ polymerization of acrylic

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acid to form a hydrogel (silver ion composite hydrogel). The hydrogel was immersed in NaOH at various concentrations (0.25–10N) for 24 h to processing the reduction of Ag+ salt

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into the silver nanoparticles (silver nanocomposite hydrogels) and then washed three times

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by using deionized water (titration with hydrochloric acid in a pH range of approximately

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7–8). The washed residue was then dried at 30 °C in an oven. Table 1 lists the feed

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compositions of the samples. Table 1 Feed compositions of silver composite hydrogels

Sample code

AAc

AgNO3

NaOH

(wt %)

(mM)

(N)

8A 10S

8

10

-

8A 10S 0.5N

8

10

0.5

8A 10S 1N

8

10

1

8A 10S 5N

8

10

5

8A 10S 10N

8

10

10

16A 10S

16

10

-

16A 10S 1N

16

10

1

16A 10S 10N

16

10

10

2.2 Characterization 7

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Optical properties.

The hydrogels were characterized using a JASCO V670 UV-visible spectrophotometer

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X-ray diffraction.

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with a scan range of 300–800 nm.

X-ray diffraction (XRD) was used to identify the silver nanoparticles in the hydrogel.

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nm) operating at 40 kV and 40 mA.

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The XRD analyses were performed using the MAV Science M21X (Cu radiation, λ = 0.1546

Inductively coupled plasma-optical emission spectrometry.

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The silver nanoparticles in the hydrogels were digested with nitric acid for 4 h, and this

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mixture was then added to 5N NaOH three times for 4 h. After dilution of the solution, the

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silver ions were determined through inductively coupled plasma-optical emission spectrometry (ICP-OES) (PerkinElmer Optima-2000DV). Field emission scanning electron microscopy.

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2.2.4

Freeze-dried hydrogels were produced by placing the equilibrium swelling ratio hydrogels in the -20℃ freezer for 24 h, then moved it to the freeze dryer for preserving the structure of holes; however, air-dried hydrogels were generated by drying hydrogels slowly under 30℃ constant temperature oven for naturally contract. The porous structures of freeze-dried hydrogels and silver nanoparticle distributions of air-dried hydrogels were examined through field emission scanning electron microscopy (FESEM) by using the Hitachi S-8020 at an accelerating voltage of 10 kV. Sputter-coated palladium was not used to 8

ACCEPTED MANUSCRIPT reduce charging in the sample before SEM. 2.2.5 Transmission electron microscopy and energy-dispersive X-ray spectroscopy.

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The silver nanoparticles were characterized through transmission electron microscopy

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(TEM) by using the FEI Tecnai G2 F20 operating at 200 kV. The hydrogel was dissociated in an aqueous solution through stirring intensely overnight followed by centrifugation

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(centrifuged at 3000 rpm for 3 min) to obtain a clear solution. A droplet of the diluted

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aqueous dispersion was cast on the grid and remained on the grid for 3 min. The additional fluid was then removed using the edge of the filter paper, and the grid was air-dried. An

Swelling studies.

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a voltage of 20 kV.

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energy-dispersive X-ray spectroscopy (EDX) detector was connected with a TEM detector at

The swelling kinetics of the gels was measured at 25 °C. After water was wiped from

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the surface by using filter paper, the SRs of the hydrogels were recorded during swelling at regular intervals (Eq. 1). SR = (Ws−Wd)/Wd

(1)

where Ws is the weight of the swelling gel at different time points, and Wd is the weight of the dry gel. 2.2.7

Mechanical properties.

The dried hydrogels were immersed in deionized water at 25 °C for 8 h to reach equilibrium. The diameter and thickness of the gels were recorded. The mechanical strength 9

ACCEPTED MANUSCRIPT of the hydrogels was measured through a uniaxial compression experiment by using a Lloyd LRX universal tester (J.J Lloyd, Poole, UK). The shear modulus and cross-linking density

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(ρx) were calculated using the following equations:

(2)

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τ=F/A=G（λ-λ-2） ρx=Gυ2 -1/3/(RT)

(3)

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where τ is the compression stress; F is the compression load; A is the cross-sectional

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area of the swollen gels; R is the ideal gas constant; T is the absolute temperature; and λ is the compression strain(L/L0), where L is the thickness of the wet gel after compression, and

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L0 is the thickness of the dried gel. At low strain, a plot of shear stress versus –(λ−λ−2)

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yielded a straight line, whose slope was the shear modulus (G).

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2.3 Microbial Experimentation

The bacterial strains used in this study including standard strains Candida albicans

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CCRC21538, Staphylococcus aureus ATCC 25923, Pseudomonas aeruginosa ATCC 27853, and Escherichia coli ATCC 25922; and clinical bacterial isolates S. aureus 13 (Sta13,methicillin-resistant (coagulase-negative

Staphylococcus

staphylococci,

CNS),

aureus

(MRSA)),

Pseudomonas

S.

aeruginosa

epidermidis 7

(Pa7,

extended-spectrum β-lactamases (ESBL)), Acinetobacter baumannii 22 (Ab22, imipenem resistance), Klebsiella pneumonia 95 (KP 95). Mueller–Hinton and Luria-Bertani (LB, Becton Dickinson, Franklin Lakes, NJ, USA) were the media used for bacterial growth. Yeasts Molds (YM, Becton Dickinson, Franklin Lakes, NJ, USA) were the media used for 10

ACCEPTED MANUSCRIPT yeast growth. All strains were incubated in atmosphere at 37°C for 24 h. 2.3.1

Growth inhibition test:

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The antimicrobial activity of the hydrogels was tested according to growth inhibition of

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all strains used in this study. The microbial strains were first cultured within the hydrogels in 2 mL of media on sterile 12-well plates. The inoculum was then monitored for changes in

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absorbance at 600 nm (OD600) after 24 h. Strains grown only in media were used as the

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positive control. Each measurement was performed in triplicate. Diffusion method for evaluating antimicrobial properties:

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The antimicrobial activity of the hydrogels was tested using a modified disc diffusion

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assay, as previously described. Briefly, bacterial strains and yeast grown in broth media were

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swabbed on the surface of agar plates. To the plates, 8A 10S 0.5N hydrogel was added. Following incubation at 37 °C overnight, the diameters of the clear zones around the

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hydrogels, called “zones of inhibition,” were recorded. Time-dependent kinetic study:

S. aureus were selected for time-dependent kinetic studies. Organisms were grown in Mueller–Hinton agar for 4 h (log phase of growth) and were further diluted in 20 mL of the same medium to yield a concentration of approximately 1 × 108 CFU/mL. Wells containing hydrogels at the aforementioned concentrations were tested for each strain. Aliquots (0.1 mL of broth) were removed from each well and serial dilutions were plated onto LB plates after 0, 2, 4, 8, 16, 20, and 24 h of incubation. Colony counts were obtained after 24 h of 11

ACCEPTED MANUSCRIPT incubation at 37 °C. Aliquots (0.1 mL of broth) were removed from each well after 0, 2, 4, 8, 16, 20, and 24 h of incubation, and serial dilutions were measured at an optical density of

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Mode of interaction of silver nanoparticles in bacterial and fungal cell

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600 nm (OD600).

membranes:

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The mode of interaction of silver nanoparticles in bacterial cell membranes was

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observed through FESEM (Hitachi S-8020, Japan). First, 3 vol% glutaraldehyde in PBS was added to the bacterial medium at 4 °C for incubation (reaction) for 4 h. The glutaraldehyde

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solution was removed, and the medium was washed three times with PBS (the bacterial

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medium was centrifuged at 8000 rpm for 5 min). The precipitate was then collected on filter

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paper. Step dehydration with 25%, 50%, 70%, 95%, and 99% ethanol was performed on the filter paper for 10 min at each ethanol concentration. The filter paper was then dried and

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sputter-coated with a thin film of platinum for imaging. Results

3.1 Preparing Silver Ion Composite Hydrogels A polymer comprises repeated monomers, which form a chain structure with considerable molecular weight. By adding a cross-linking agent to the monomers, the chain structure polymerizes into a network structure. As shown in Fig. 1 (1), after UV irradiation for 6 min, the sample was still liquid. As shown in Fig. 1 (2), after the addition of silver ions to the sample in an 8 wt% acrylic acid solution and UV irradiation for 6 min, a gel formed. 12

ACCEPTED MANUSCRIPT As shown in Figs. S1, when the silver ion concentration was higher than 2.5 mM, the hydrogel formed completely. Moreover, when the concentration was lower than 1.25 mM,

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the hydrogel formed only partly. Finally, when the concentration was lower than 1 mM, only

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fluid gels formed. Therefore, silver ions play an important role of fabricating silver-nanoparticle composite hydrogels. Because silver is a transition metal which possesses

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empty orbitals to attribute to carboxylate metal ion complex formation [29].

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Figure 1 The photos of polyacrylic acid hydrogel with or without silver ions.

3.2 Optical Properties of Hydrogel The 8A 10S hydrogel and 0.5 N NaOH were used to form the silver nanocomposite hydrogel. As shown in Fig. 2 (a), the top and side views depict the 8A 10S and 8A 10S 0.5N hydrogels, respectively. When the silver particle size decreased to the nanoscale, the color of the transparent hydrogel changed to brown. Fig. 2 (b) shows FESEM micrographs of the 8A 10S 0.5N hydrogel when air-dried. The silver particles of the 8A 10S 0.5N hydrogel were uniformly distributed. 13

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Figure 2 (a) Top and side views of the 8A 10S and 8A 10S 0.5N hydrogels; (b) FESEM

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micrographs of the 8A 10S 0.5N hydrogel; (c) optical absorption spectra of the hydrogel; and

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(d) XRD pattern of the hydrogel.

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Silver nanoparticles exhibit unique and tunable optical properties because of their surface plasmon resonance (SPR), which depends on the shape, size, and distribution of the nanoparticles [31]. Fig. 2 (c) illustrates the optical absorption spectra of the 8A 10S and 8A 10S 0.5N hydrogels. The absorption peak was centered at 432 nm for the typical SPR absorption of metallic silver nanoparticles [32]. The 8A 10S hydrogel with no silver exhibited minimal UV and visible absorption. Fig. 2 (d) shows the XRD pattern of the silver nanoparticles in the hydrogel networks. The peaks of the silver nanocomposite hydrogel at 38.1°, 44.3°, 64.7°, 76.5°, and 81.5° were assigned to reflections on the (111), (200), (220), 14

ACCEPTED MANUSCRIPT (311), and (222) planes of the face-centered cubic silver nanoparticles, respectively [33], confirming that silver nanoparticles existed in the hydrogel nanocomposites.

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3.3 Size Control and Morphology of Silver Nanoparticles

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As shown in Fig. 3(a), the 8A 10S hydrogel was added to 0–10 N NaOH solution. After 24 h, the NaOH solution was removed, and digital images of the top and side views of the

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hydrogel were captured. Evidently, increasing the NaOH concentration reduced the hydrogel

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size, and the color changed from light orange to deep brown. According to the SPR principle, could surmise that increasing the concentration of NaOH increases the silver nanoparticle

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size. The schematic of changes in the volume of the hydrogel after reduction was shown in

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Fig. 3 (b).

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(a)

(b)

Figure 3 (a) NaOH concentration dependent for 8A 10S silver nanocomposite hydrogels; and (b) schematic of changes in the volume of the hydrogel after reduction. 15

ACCEPTED MANUSCRIPT Notably, different silver nanoparticle sizes were obtained with different reducing agents for the 8A 10S hydrogel. The silver nanoparticle sizes in the 8A 10S 0.5N, 8A 10S 1N, 8A

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10S 5N, and 8A 10S 10N hydrogels were 5.9 ± 2.5, 6.1 ± 1.7, 12.5 ± 4.7, and 22.8 ± 8.8 nm,

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respectively (in Fig.4). The EDX spectra collected from the samples imaged using TEM revealed the presence of Ag signal peaks, implying that the nanoparticles were silver (in Fig.

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4 (c)). The silver concentrations of the 8A 10S 0.5N, 8A 10S 1N, and 8A 10S 5N hydrogels

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obtained through ICP-OES (data not shown) were 64.4 ± 0.7, 59.8 ± 1.5, and 36.4 ± 0.4 ng in

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each piece, respectively.

(b)

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(a)

(c)

(d)

Figure 4 TEM and EDX images of silver nanoparticles in (a) 8A 10S 0.5N, (b) 8A 10S 1N, (c) 8A 10S 5N, and (d) 8A 10S 10N hydrogels.

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ACCEPTED MANUSCRIPT 3.4 Properties of Hydrogels As shown in Fig. 5, the SR of the hydrogels formed was determined using different

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concentrations of NaOH, with the concentrations of silver nitrate and AAc being fixed, to

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investigate the effect of this variation on the network structure of the hydrogels. The differences in the volume after water absorption are illustrated in the inset of Fig. 5. The SR

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increased with the addition of NaOH in the hydrogel. The SRs of the silver nanocomposite

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hydrogels in water were 337.8 ± 76.9, 527.6 ± 27.6, and 560.9 ± 22.4 when NaOH

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concentrations of 0.5 N, 5 N, and 10 N were used, respectively.

Figure 5 NaOH effects swelling ratio of the silver nanocomposite hydrogel.

Equilibrium swelling was used to demonstrate that the structural properties and cross-linking density of the network of silver composite hydrogels can be controlled by reducing the amount of silver. The effective cross-linking density (ρ) depended on the SR and shear modulus at room temperature (Fig 6). The SR increased and ρ values decreased 17

ACCEPTED MANUSCRIPT with the addition of NaOH to the hydrogel. The gel strength can be estimated using the shear modulus (G) measured in the uniaxial compression experiment. The results indicated that the

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G values decreased with the addition of NaOH to the silver ion composite hydrogel,

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evidencing that the silver ions were reduced to silver nanoparticles in the hydrogels.

280

120 80

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40

1N

10N

0.3

0N

1N

10N

0.0

16A 10S

8A 10S

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0N

0.6



0.9

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160



200

G (Pa)

1.2

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240

0

1.5

Mechanical strength Crosslink density

Figure 6 Acrylic acid and NaOH both effects mechanical properties and crosslink

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density of the silver nanocomposite hydrogel.

Fig. 7 (a)–(f) shows FE-SEM micrographs of air-dried 8A 10S, 8A 10S 0.5N, and 8A 10S 5N silver nanocomposite hydrogels. In Fig. 7 (a) - (c), the air-dried 8A 10S hydrogel had a clear and flat surface. However, it was uniformly covered with silver nanoparticles when treated with NaOH. Fig. 7 (d)–(f) shows cross-sectional micrographs of freeze-dried silver nanocomposite hydrogels treated with or without NaOH. The hydrogels contained interior porous structures and open networks of extended honeycomb-like structures. 18

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Figure 7 FE-SEM micrographs of (a) air-dried and (d) freeze-dried 8A 10S; (b) air-dried

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and (e) freeze-dried 8A 10S 0.5N; and (c) air-dried and (f) freeze-dried 8A 10S 5N silver

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nanocomposite hydrogels.

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3.5 Antibacterial Properties of Silver Nanocomposite Hydrogels

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Many studies discovered that PAAc copolymers possess an antimicrobial effect [34, 35]; however, when it was transfer into sodium polyacrylate or the carboxylic acid group was

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cross-linked, the antibacterial effect might be eliminated. The antimicrobial effect of different silver composite hydrogels on microorganisms in LB or YM broth was examined by measuring the cell concentration in a suspension at OD600 (Fig. 8). The results indicated that all hydrogels exhibited effective inhibition activity in E. coli and S. aureus. However, the inhibition effect on the yeast was poorer than that on the bacterial strains. For C. albicans, only the 8A 10S 0.5N and 8A 10S 1N hydrogels exhibited effective inhibition.

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Figure 8 NaOH effects antimicrobial of the 8A 10S hydrogel

For investigating the antibacterial activity of the 8A 10S 0.5N hydrogel, E. coli, S.

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aureus, and C. albicans were subjected to a disc diffusion assay. After incubation at 37 °C overnight, an inhibition zone was observed in each of the samples. Fig. 9 shows the results of the disc diffusion assays for E. coli, S. aureus, and C. albicans, and Table 2 presents the widths of the inhibition zones around the samples. The results indicated that all the samples had inhibition zones greater than 1 mm, and the antibacterial ability can be considered to be at a “good” level according to the standard SNV 195920-1992 [36].

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Figure 9 Inhibition zones of microorganism produced using diffusion test by 8A 10S 0.5N hydrogel.

diffusion tests

E. coli

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Microorganism

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Table 2 Inhibition area of bacterial growth calculated around the samples through agar

Inhibition area 3.6 ± 0.1mm 2.0 ± 0.3 mm

Can. albicans

1.9 ± 0.2 mm

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S. aureus

The time-dependent growth kinetics of S. aureus treated with silver composite

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hydrogels was determined, as shown in Fig. 10 (a). The results showed that for S. aureus, introducing silver nanoparticles had a more marked effect on the growth kinetics compared with the negative control; the silver nanoparticles effectively inhibited bacterial growth. After incubation for 24 h, bacteria were treated with glutaraldehyde to prepare FESEM samples. FESEM was used to evaluate the surface morphology of both native S. aureus (Fig. 10 (b)) and that treated with the 8A 10S 0.5N hydrogel (Fig. 10 (c)) in the LB medium. The treated bacterial surfaces were covered with silver nanoparticles, and the morphology

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considerably changed and showed major damage.

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Figure 10 Time-dependent effect of silver nanocomposite hydrogels on (a) S. aureusand FESEM images of S. aureus (b) treated with the control and (c) treated with the 8A 10S 0.5N

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hydrogel.

In the present study we investigated antimicrobial properties using type strains and clinical (multidrug-resistant from patients) isolates. The phenotypic of drug resistance isolates using in this study described as in material and methods. The in vitro antibacterial properties of 8A 10S 0.5N and 8N 10S 1N hydrogels were evaluated comparatively against standard strains and clinical isolates (in Fig.11). The results indicated that all hydrogels exhibited effective inhibition activity in standard strains and clinical isolates. No matter 22

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positive bacteria or negative bacteria, it had good inhibitory effect.

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Figure 11 Antimicrobial effects of silver nanocomposite hydrogels of standard strains

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4.

and clinical isolates.

Discussion In

this

study,

we

have

explored

the possibility of

imparting anti-drug-

resistance-bacterial properties to high-swell-ratio polyacrylic acid hydrogels by incorporating in situ silver nanoparticles. Fig. 1 verified that silver ions are crucial in hydrogel formation for the following two reasons. First, the silver ions acted as catalysts in persulfate initiation [37]; they accelerated polymerization, and the acrylic acid then became a polymer. Second, the cross-linking agents of the hydrogel were the silver ions, and the carboxylic group and 23

ACCEPTED MANUSCRIPT silver ions combined to form the O–Ag–O bond of Ag4O2 [38]. In Fig. 2, using UV light to irradiate the hydrogels for transforming into silver ion

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composite hydrogel; however, no further reduction of silver nanoparticle had been

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discovered. It could be examined by the actual condition of hydrogels which presented transparent color, and further, analysis did not reveal any SPR peak related to silver

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nanoparticle. When hydrogels was reduced by NaOH (reducing agent), the color of

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hydrogels became yellow and the surface was covered by silver nanoparticles evenly (Fig. 7).

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In Fig. 3 the 8A 10S hydrogel was added to 0–10 N NaOH solution. After 24 h, the

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hydrogel size, and the color changed from light orange to deep brown with increasing the

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NaOH concentration. The hydrogel volume decreases when the NaOH concentration is increased. Because under the same volume of reaction solution, the increase of NaOH

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content might relatively cause the content of water reduce (Fig. 3(b)). The decrease of water content might lead to the swelling rate decline and the size of hydrogel deflation. In Fig. 4, with increasing the NaOH concentration, the silver particles size were increasing from 5.9 ± 2.5 to 22.8 ± 8.8 nm, it could control the silver particles size by different NaOH concentration. Combine Fig. 3 and Fig. 4, the NaOH concentration increased, the silver nanoparticle size increased, and the hydrogel color changed from light orange to deep brown. This phenomenon implied that increasing the concentration of NaOH increases the reaction rate but the reduction space was restricted (space limitation of hydrogel volume). Therefore, 24

ACCEPTED MANUSCRIPT the reduction of silver particles might trend to growth, and then the particle size increase. In Fig. 5, the increase in the SR can be attributed to the following two factors. First, the

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sodium polyacrylate hydrogel had a high concentration of sodium ions; therefore, water

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could diffuse in the hydrogel network to balance the internal osmosis. Second, the concentration of NaOH increased with nanosilver reduction, causing the cross-linking

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density of silver ions and PAAc to decrease. Therefore, the SR increased with the addition of

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NaOH to the hydrogel. The SR increased and ρ values decreased with the addition of NaOH to the hydrogel (Fig. 6). Because the silver ions were used as cross-linking agents, the

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amount of reduction increased with the concentration of NaOH, and the cross-linking density

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decreased because of the increase in the SR [39]. And the G values decreased with the

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addition of NaOH to the silver ion composite hydrogel, evidencing that the silver ions were reduced to silver nanoparticles in the hydrogels. Fig.5 and Fig. 6 indicates that increasing the

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concentration of NaOH causes the cross-linking density to decrease; thus, the shear modulus decreases, resulting in an increased SR. The morphology of air-dried and freeze-dried silver nanocomposite hydrogels (Fig 7). It was uniformly covered with silver nanoparticles when treated with NaOH. Moreover, the hydrogels contained interior porous structures and open networks of extended honeycomb-like structures. The pores were small and compact in the 8A 10S hydrogel; however, they were large in the 8A 10S 0.5N and 8A 10S 5N hydrogels. Thus, we confirmed (Fig. 5 to Fig. 7) that the reduction of nanosilver results in cross-linking density change. 25

ACCEPTED MANUSCRIPT Most of the clinical isolates have emerged as a predominant cause of healthcare-associated infection in the Taiwan and worldwide [40]. Multidrug-resistant

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organisms cause clinical failures in the treatment of infections and public health crises. The

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ability of the composite material to inhibit bacterial growth was tested using two different methods, the disc diffusion method and the solution method (Fig. 8 to 11). Both methods

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showed a similar trend with respect to growth inhibition, and thus reaffirmed the observed

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effects. In Fig. 8, the 8A 10S 0.5N and 8A 10S 1N hydrogels exhibited effective inhibition. This may be because the silver particle size of the 8A 10S 1N hydrogel was smaller than that

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of the 8A 10S 5N hydrogel. Moreover, the silver concentration of the 8A 10S 1N hydrogel

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(59.8 ± 1.5 ng) was slightly higher than that of the 8A 10S 5N (36.4 ± 0.4 ng) hydrogel.

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The results of disc diffusion assays for E. coli, S. aureus, and C. albicans, that all the samples had “good” antibacterial ability (Fig. 9). Although the antibacterial tests confirmed

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that the level of antibacterial ability was good, differences were observed in the antibacterial activity of the bacterial strains tested. In particular, E. coli exhibited slightly higher antimicrobial activity than that of the other strains. Compared with the cell wall of the gram-positive bacteria (S. aureus), the cell wall of the gram-negative bacteria (E. coli) has a thinner peptidoglycan layer, which enables easier penetration of silver nanoparticles into the bacterial structure [41]. In Fig. 10(c), the treated S. aureus surfaces were covered with silver nanoparticles, and the morphology showed major damage, which was characterized by the formation of pores in the cell walls [42]. 26

ACCEPTED MANUSCRIPT In Fig. 11, all hydrogels exhibited effective inhibition activity in standard strains and clinical isolates. Taking into account that Sta13 (MRSA), Pa7 (ESBL), Ab22 (imipenem

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resistance) and KP95 are very hard to treat and thus can lead to severe health problems upon

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infection, the development of efficient antibacterial compounds is a very important issue [40, 43]. Based on the obtained bacterial inhibition data (Fig. 8 to Fig. 11), the 8A 10S 0.5N and

Conclusions

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5.

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8N 10S 1N hydrogels could effectively reduce in the treatment of wound infections.

In this study, a fast and novel method was used to prepare silver ion composite

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hydrogels and silver nanocomposite hydrogels. The method yields superabsorbent hydrogels,

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increasing the SR by up to 55,000%. The volume of the hydrogel increased with the

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reduction of the hydrogel as the NaOH concentration decreased. The hydrogels had a porous structure, and the surface was uniformly covered with silver nanoparticles. With an

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increasing concentration of NaOH, the silver nanoparticle sizes increased from 6 to 22 nm. Antibacterial tests were conducted using three bacterial, one yeast strains and five clinical isolates. The silver nanocomposite hydrogels have high antibacterial ability against microbial strains. The complete inhibition of bacteria and yeast proliferation induced by the silver composite hydrogels can be useful in developing new superabsorbent antimicrobial pharmaceutical products and could offer new valuable new opportunities in the treatment of wound infections. Acknowledgments 27

ACCEPTED MANUSCRIPT The authors are grateful for the financial support provided by the Ministry of Science and Technology (MOST) through grant No. MOST 104-2221-E-036-025.

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Graphical abstract

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In situ synthesis of high swell ratio, sized controlled within polyacrylic acid/silver nanocomposite hydrogels and had good antimicrobial properties.

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