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Antimicrobial activity of Siver nano

Antimicrobial activity of Siver nano

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A versatile strategy to fabricate hydrogel–silver nanocomposites and investigation of their antimicrobial activity
A versatile strategy to fabricate hydrogel–silver nanocomposites and investigation of their antimicrobial activity

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Journal of Colloid and Interface Science 315 (2007) 389–395www.elsevier.com/locate/jcis
A versatile strategy to fabricate hydrogel–silver nanocomposites andinvestigation of their antimicrobial activity
V. Thomas
, Murali Mohan Yallapu
, B. Sreedhar
, S.K. Bajpai
 Department of Chemistry, Polymer Research Laboratory, Govt. Model Science College, Jabalpur, MP 482001, India
 Department of Polymer Science & Technology, Sri Krishnadevaraya University, Anantapur, AP 515003, India
 Inorganic and Physical Chemistry, Indian Institute of Chemical Technology, Tarnaka, Hyderabad, AP 500007, India
Received 16 April 2007; accepted 25 June 2007Available online 17 August 2007
In this study, hydrogel–silver nanocomposites have been synthesized by a unique methodology, which involves formation of silver nanoparti-cles within swollen poly (acrylamide-
-acrylic acid) hydrogels. The formation of silver nanoparticles was confirmed by transmission electronmicroscopy (TEM) and surface plasmon resonance (SPR) which was obtained at 406 nm. The TEM of hydrogel–silver nanocomposites showed al-most uniform distribution of nanoparticles throughout the gel networks. Most of the particles, as revealed from the particle-size distribution curve,were 24–30 nm in size. The X-ray diffraction pattern also confirmed the face centered cubic (fcc) structure of silver nanoparticles. The nanocom-positesdemonstratedexcellentantibacterial effectson
.Theantibacterial activity dependedonsizeofthenanocomposites,amount of silver nanoparticles, and amount of monomer acid present within the hydrogel–silver nanocomposites. It was also found that immersionof plain hydrogel in 20 mg
30 ml AgNO
solution yielded nanocomparticle–hydrogel composites with optimum bactericidal activity.
2007 Elsevier Inc. All rights reserved.
Hydrogel; Silver nanoparticles; Surface plasmon resonance; Nanocomposites;
E. coli
; Transmission electron microscopy; Silver nitrate;Antibacterial activity; Scanning electron microscopy; Colony-forming unit
1. Introduction
The design and developmentof nanoparticles and nanostruc-tural materials have opened a new era for constructing well-designed nanostructures that have been considered as a novelclass of materials for catalytic, optical, electronic, and bio-medical applications[1]. It is widely renowned that nano-sizedmetal particles such as silver, gold, and copper are highly toxicto microorganisms[2–6],exhibiting strong biocidal effects on as manyas 16speciesof bacteriaincluding
[7].Nanoparticles have an extremely large relative surface area,thus increasing their contact with bacteria or fungi, and vastlyimproving their bactericidal and fungicidal effectiveness. Silvernanoparticles show excellent antimicrobial activity by binding
Corresponding author.
 E-mail address:
mnlbpi@rediffmail.com(S.K. Bajpai).
Current address: Department of Biomedical Engineering ND4-20, LernerResearch Institute, Cleveland Clinic Foundation, Cleveland, OH 44195, USA.
both to microbial DNA, preventing bacterial replication, and tothe sulfhydryl groups of the metabolic enzymes of the bacterialelectron transport chain causing their inactivation[8,9].Hence, nanosilverparticleshavebeenappliedtoawiderangeofhealth-care products such as burn dressings, scaffold, skin donor andrecipient sites, water purification systems, and medical devices[10–14].Most recently, it has been demonstrated that silvernanoparticles undergo a size-dependent interaction with HIV-Ivirus via preferential binding to the gp120 glycoprotein knobs,thus inhibiting the virus from binding to the host cells[15].Multipurpose systems are required to exhibit superior an-tibacterial activity toward germs on contact without releas-ing any toxic biocides. Ongoing research efforts, on three-dimensional network hydrogels, suggest that huge free spaceavailable between the cross-linked networks in the swollenstage behaves as nanoreactors for generating the nanoparti-cles[16].These hydrogel nanoreactors offer a platform for nucleation and growth of nanocrystals, which eventually leadto nanoparticle formation. Further, gel–nanoparticle systems
0021-9797/$ – see front matter
2007 Elsevier Inc. All rights reserved.doi:10.1016/j.jcis.2007.06.068
V. Thomas et al. / Journal of Colloid and Interface Science 315 (2007) 389–395
have opened a new skylight for different applications in bio-medical engineering and these approaches are most effectiveand safe because they are compatible with most of biolog-ical molecules, cells, tissues, etc. Current studies have ex-ploited in situ synthesis of metal nanoparticles within theswollen hydrogel networks[17]and the resulting products areleading to new hybrid or composite systems that have ver-satile applications in bioengineering and biomedical fields.The most advanced feature of this novel approach is thatwe can alter the size and morphology of the nanoparticlesby changing the monomer and crosslinker concentrations inthe gel formulations. Well-defined gold nanoparticles embed-ded inside thermosensitive hydrogel matrices were reportedby Wang et al.[18,19].In their work, the templates of gel networks were prepared using a combination of two crosslink-ers, namely
-methylenebisacrylamide (MBA) and
-cystaminebisacrylamide (CBA). CBA is highly responsible incontrolling the morphology of the nanoparticles by control-ling the crosslinking density of the networks. In another study,Zhang et al.[20]produced polymeric microgels which can actas versatile nanoreactors for semiconductor, metal, and mag-netic nanoparticles. Recently, microgel formulations based on
-isopropylacrylamide with nanoparticles have been effec-tively employed for photonic, electronic, and electroanalyticalapplication purposes[21–23].Hydrogels have also been em- ployed for magnetic nanoparticle entrapment and the result-ing ferronanohydrogel composite properties have been stud-ied[24].Gels constructed with methyl methacrylate (MMA) and ethylenedimethacrylate(EDMA) have beenutilizedas tem-plates to control the size and morphology of Pd nanoparticles.The developed microgel–metal nanoclusters are appropriatefor catalysis applications[25].More recently, silver nanopar- ticles of 
35 nm are embedded in a novel hydrogel systembased on poly(vinyl alcohol)/poly(styrene)-
-poly(ethyleneglycol methacrylate) [PVA/PS-PEGMA][26].A recent study demonstrates that hydrogels are capable of producing nanosil-ver particles about
4 nm in size[27]. In these studies, dif-ferent strategies have been followed to prepare hydrogel–silvernanocomposites. These include (1) polymerization of monomerin the presence of initiator-funtionalized metal nanoparticles;(2) shrinking of a swollen gel in acetone followed by itsreswelling in solution of metal nanoparticles and then again itsshrinking in acetone, thus finally resulting in the formation of nanoparticle-loaded gels; (3) surfactant-free emulsion polymer-ization (SFEP) to encapsulate metal nanoparticles within largerspherical hydrogel particles; and (4) mixing a colloidal solutionof metalnanoparticleswith aqueous polymer solution, followedby solvent evaporation. However, the method used in our studyis very simple and economic and does not involve the use of organic solvents. The use of organic solvents and other toxicreagents, harsh synthesis conditions, limit their use in preparinghydrogel/silver nanocomposites for biomedical applications.Contrary to this, we hereby report a universal approach forsynthesizing nanocomposites. In the present work, we havereported a novel approach for synthesizing Ag nanoparticlescontaining poly(acrylamide-
-acrylic acid) hydrogels and in-vestigated their antimicrobial activity against
E. coli
.The antibacterial property of silver has been studied by anumber of microbiologists. For example, Deitch et al.[28]eval-uated the antibacterial activity of silver nylon fabrics on thegrowth of 
S. aureus
C. albicans
to develop these fabricsas wound dressings. In another study, Sondi and Salopek-Sondi[7]evaluated the antimicrobial activity of silver nanoparticlesagainst
E. coli
. The results confirmed that the treated
E. coli
cells were damaged, showing pit formation on bacterial cellwalls. Recently, Jain and co-workers[10]tested the bacter-ial action of silver nanoparticle-coated polyurethane foam andsuggested its use as an antibacterial water filter. In anotherwork, Hu et al.[29]treated cotton fabrics with a suspensionof silver oxide in chitosan and studied its antibacterial actionagainst
S. aureus
. Attempts have also been made to coat urinarycatheterswithsilvercompoundstopreventinfection[30].Apart from silver, polycationic groups like alkylammonium have alsobeen employed to provide antimicrobial surfaces[31].The real significance of this work is that if the surfaceof medical prostheses is grafted with polymeric chains, thenthis proposed novel approach may be used to incorporate Agnanoparticles into the grafted polymer network, followed bycitrate reduction, to yield devices with Ag-impregnated antimi-crobial surfaces.
2. Experimental
2.1. Materials
The two monomers used in this study, namely acrylamide(AAm) and acrylic acid (AAc), and the crosslinking agent
-methylenebisacrylamide were obtained from HiMediaLaboratories, Mumbai, India. The salts silver nitrate (AgNO
),trisodium citrate (SC), and potassium persulfate (KPS) wereobtained from E. Merck, Mumbai, India, and used as received.Standard cultures of the organisms were provided by the De-partment of Biotechnology. Nutrient broth and Nutrient andm-Endo agars were obtained from HiMedia Chemicals, In-dia. Double-distilled water was used throughout the investiga-tions.
2.2. Synthesis of poly(acrylamide-co-acrylic acid) hydrogels
The hydrogel disks were prepared by carrying out free rad-ical aqueous copolymerization of AAm and AAc using MBAas the crosslinker and KPS as the initiator. In brief, in order toprepare a sample (HG2), 14.08 mM AAm, 5.55 mM AAc and0.32 mM crosslinker MBA were dissolved in water and the finalvolume was made 5 ml. Then, 0.11 mM KPS was dissolved andthe whole reaction mixture was transferred in the test tube (in-ternal diameter 1.5 cm) and kept in an electric oven (Tempstar,India) at 60
C for a period of 2 h. After the polymerization wasover, the test tube was broken and the resulting almost trans-parent hydrogels were cut into slices of same thickness. Thehydrogels were equilibrated in the distilled water for a periodof 2 days to remove the unreacted monomers, crosslinker, andinitiator and finally dried in a dust-free vacuum chamber till thegels attained constant weight. A blank poly (AAm) hydrogel,
V. Thomas et al. / Journal of Colloid and Interface Science 315 (2007) 389–395
HG1, was also prepared by employing the same conditions andcomposition, without AAc.
2.3. Synthesis of hydrogel–Ag nanoparticle composites
The hydrogel disk was equilibrated in the distilled waterfor 24 h followed by its immersion in solution of silver ni-trate (10 mg AgNO
in 30 ml distilled water) for another 24 h.Then the disk was taken out and put in trisodium citrate solu-tion (20 mg dissolved in 30 ml water) for another 24 h to reduceAg
ions into silver nanoparticles within the swollen gel. Thedark brown-cum-black color of the disk indicated formation of silver nanoparticles. The disk was dried in an electric oven,grinded, and finally sieved with mesh size No. 350 to yield finehydrogel–Ag nanoparticle composites.
2.4. Characterization
The Fourier transformation infrared (FTIR) spectra of plainand silver nanoparticle-loaded hydrogel samples were recordedon a FTIR spectrophotometer (Shimadzu 8400 S) using KBr.Absorption measurements were carried out on a Systron-ics 2201 UV–visible spectrophotometer for well-dispersedhydrogel–silver nanocomposite solutions (1 mg
1 ml) in thewavelength range 400–800 nm. XRD analyses for hydrogel–silver nanocomposites were performed with a Rikagu diffrac-tometer (Cu radiation,
1546 nm) running at 40 kV and40 mA. The nanocomposite structural and morphological vari-ations were observed by using a JOEL JSM 840A (Japan)scanning electron microscope (SEM). Scanning electron mi-croscope specimens were prepared by placing 2–3 drops of thehydrogel–silver nanocomposite solution on a silicon wafer anddried in air. Transmission electron microscopy (TEM) imagesof the samples were recorded using a Tecnai F 12 TEM instru-ment. TEM samples were prepared by dispersing 2–3 drops of gel–silver nanoparticle solution on a copper grid and dried atroom temperature after removal of excess solution using a filterpaper.
2.5. Microbial experimentation
Microbial experimentation was done to find the effect of sil-ver hydrogel particles on gram-negative bacteria
E. coli
. Forthis purpose approximately 10
colony-forming units (CFU) of 
 E. coli
were cultured on a Nutrient agar plate supplementedwith silver hydrogel particles. Plates, free of silver hydrogelparticles, were used as a control set. The plates were incubatedfor 24 h at 37
C and the numbers of colonies were counted.
3. Results and discussion
3.1. Fabrication of hydrogel–Ag nanoparticles
During the past two decades, a number of different ap-proaches have been employed to obtain better stabilization of nanoparticles using various polymers, block copolymers, star
Fig. 1. Formation of silver nanoparticles within the swollen
-polymeric net-work.
polymers and dendrimers, microgels, hydrogels, and so on. Re-cently, as noted in the Introduction, there is a lot of interestin producing nanoparticles in the hydrogel networks since theyhave enormous valuable applications in bio-related fields. But,most of the hydrogels and microgels employed for this pur-pose must follow either controlled polymerization paths or havewell-organized networks through the gel structure, which mayincrease the cost of the nanoparticle–hydrogel hybrid materi-als. In contrast, we have developed a facile in situ approach of nanoparticle synthesis using a conventional hydrogel. The em-ployed hydrogel networks in this study are random copolymersof acrylamide and acrylic acid repeating units throughout thegel macromolecular chains that are constructed with variouscrosslinking junctions. This macromolecular gel network willserve effectively as nanoreactors for silver nanoparticle prepa-ration.Fig. 1depicts a clear scheme for the formation of silvernanoparticles within the swollen co-polymeric network. Whena fully swollen hydrogeldisk is put in the aqueous AgNO
solu-tion, there occurs an ion exchange between Ag
ions present inthe outer solution and H
ions present within the gel phase. Af-ter equilibrium is attained, the hydrogel disk is transferred intotrisodium citrate solution. This results in a reduction of Ag

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