Fabrication of porous chitosan films impregnated with silver nanoparticles: A facile approach for superior antibacterial application

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Colloids and Surfaces B: Biointerfaces 76 (2010) 248–258

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Colloids and Surfaces B: Biointerfaces

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abrication of porous chitosan films impregnated with silver nanoparticles:facile approach for superior antibacterial application

. Vimalaa, Y. Murali Mohana,1, K. Samba Sivudua, K. Varaprasada, S. Ravindraa, N. Narayana Reddya,

. Padmab, B. Sreedharc, K. MohanaRajua,∗

Synthetic Polymer Laboratory, Department of Polymer Science and Technology, Sri Krishnadevaraya University, Anantapur 515055, A.P., IndiaDepartment of Botany, Sri Krishnadevaraya University, Anantapur 515055, A.P., IndiaInorganic and Physical Chemistry, Indian Institute of Chemical Technology, Tarnaka, Hyderabad 500007, India

r t i c l e i n f o

rticle history:eceived 21 October 2009eceived in revised form 27 October 2009ccepted 29 October 2009vailable online 10 November 2009

eywords:hitosanilver nanoparticlesntibacterial activity

a b s t r a c t

The present investigation involves the synthesis of porous chitosan–silver nanocomposite films in view oftheir increasing areas of application in wound dressing, antibacterial application, and water purification.The entire process consists of three-steps including silver ion-poly(ethylene glycol) matrix preparation,addition of chitosan matrix, and removal of poly(ethylene glycol) from the film matrix. Uniform porousand brown colour chitosan films impregnated with silver nanoparticles (AgNPs) were successfully fab-ricated by this facile approach. Both, poly(ethylene glycol) (PEG) and chitosan (CS) played vital roles inthe reduction of metal ions into nanoparticles (NPs) as well as provided good stability to the formednanoparticles. The developed porous chitosan–silver nanocomposite (PCSSNC) films were characterizedby UV–vis and FTIR spectroscopy, and thermogravimetric analysis for the confirmation of nanoparti-

ound dressingoly(ethylene glycol)ydrogels

cles formation. The morphology of silver nanoparticles in nanocomposite films was tested by opticalmicroscopy, scanning electron microscopy (SEM), and transmission electron microscopy (TEM). Theembedded AgNPs were clearly observed throughout the film in SEM and the extracted AgNPs from theporous chitosan–silver nanocomposite showed ∼12 nm in TEM. Improved mechanical properties wereobserved for porous chitosan–silver nanocomposite than for chitosan blend (CSB) and chitosan–silvernanocomposite (CSSNC) films. Further, the examined antibacterial activity results of these films revealed

er na

that porous chitosan–silv

. Introduction

The use of silver and its salts have attracted much interest dueo their antibacterial and medicinal applications [1–3]. The antimi-robial activity of silver is much higher than other metals, such asercury, copper, lead, chromium and tin [4]. The development of

ntibiotic resistant bacteria stains issue has resulted a new era forhe revival of the long well-known antibacterial properties of silvernd silver ions. The antibacterial mechanism of silver and its ionsnvolves the interaction with the thiol groups of protein moleculesresent inside or outside the cell membrane and binds the bacte-

ial cell membrane which inhibiting the replication capacity of DNAolecule, thereby affecting the cell viability [3,5–8]. As a result, the

mportance of silver-based commercial products including topicalintments, bandages, and gels have increased to improve public

∗ Corresponding author. Tel.: +91 8558 255655; fax: +91 8558 255655.E-mail address: [email protected] (K. MohanaRaju).

1 Present address: Cancer Biology Research Center, Sanford Research/USD, Siouxalls, SD 57105, USA.

927-7765/$ – see front matter © 2009 Elsevier B.V. All rights reserved.oi:10.1016/j.colsurfb.2009.10.044

nocomposite films exhibited superior inhibition.© 2009 Elsevier B.V. All rights reserved.

health care. It was noticed that silver-based materials exhibit weakwashing resistance so that maintenance of an optimal release levelsare difficult. This results in a short life time of antibacterial activity.

Recently, silver-based nanostructure materials have gainedmuch attention to control infections [9]. The use of silver nanopar-ticles (AgNPs) have exhibited improved antibacterial propertiesthan bulk silver due to high surface area and high fraction ofsurface atoms, leading to incorporating more NPs inside the bac-teria and promoting its efficacy in a sustained manner [10,11].The main advantage of AgNPs is that even nanomolar concen-trations are effective than micromolar concentration of silverions [12]. In addition, AgNPs have proven relatively nontoxic tohuman cells [13–15]. The drinking water purification and theantimicrobial dressing materials based on silver impregnated orsurface coated with silver membranes are reported to remove 99%pathogens [16–19]. Many commercial products such as ActicoatTM,

Bactigrass®, Fucidin® (Ülkür, Oncul, Karagoz, Yeniz & Celiköz 2005),SilvaSorb® (AcryMed/Medline, Mundelein, IL, USA), AQUACEL Ag(ConvaTec, Skillman, NJ, USA) SILVERCELL® (Johnson & JohnsonWound Management, Somerville, NJ, USA), PolyMem® Silver (Fer-ris Pharmaceuticals, Burr Ridge, IL, USA) have approved by the US

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cheme 1. (A) Schematic illustration of preparation of porous chitosan–silver nanorous chitosan–silver nanocomposite films, (a–c) different composition of chitosa

ood and drug administration (FDA) or the European Food Safetyuthority (EFSA) for wound dressings and antibacterial applica-

ions [20]. Therefore, further refinement in the design of silveranocomposite products are always looked for bioactive biomateri-ls [21,22]. However, the synthesis of AgNPs by chemical reductionhydrazine hydrate, sodium borohydride, DMF, ethylene glycol,

tc.) often involves absorption of harsh chemicals on the surfacesf NPs raising the toxicity issue. In addition, AgNPs have a tendencyo agglomerate, thus losing the peculiar properties associated withhe nanoscale. Therefore, the synthesis protocols often involve a

ig. 1. (A) Formation of silver nanoparticles in the presence of PEG 6000 ((1% (w/v), 1 mlhe presence of different molecular weights of PEGs at 80 ◦C, and (C) silver nanoparticles

osite (PCSSNC) films. (B) (1) chitosan, (2) chitosan–silver nanocomposite, and (3), 1:1, 2:1, and 5:1, respectively.

stabilization process by reducing the silver ions in the presence ofsurfactant, polymers, and hydrogels [11,21].

Until now, water soluble polymer based biomaterials arecapable of the combined antibacterial properties of AgNPs hav-ing no toxicity [23]. Basing on this, numerous polymers havebeen employed to prepare polymer-silver nanocomposites [11].

Among the various biopolymers, poly-cationic chitosan, which iscomposed of polymeric 1-4-linked 2-amino-2-deoxy-�-d-glucose,units is considered as a potential antimicrobial active materialwhich has been reported to prepare and stabilize the metal

)) within an hour at different temperatures, (B) formation of silver nanoparticles information in the presence of different concentrations of PEG 6000 at 80 ◦C.

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anoparticles [24]. Chitosan (CS) has attracted much atten-ion not only due to its broad spectrum antibacterial activityut also most abundant natural, renewable, biocompatible, andiodegradable polymeric nature with enormous metal complex-tion capacity. Therefore, the present investigation has take up toesign a novel and “green approach” for the synthesis of poroushitosan–AgNPs composite (PCSSNC), using poly(ethylene gly-ol) (PEG) as reducing/porogenator/stabilizing agent [11,25]; andhitosan as reducing/stabilizing agent [11,23,26]. The developedanocomposites were characterized and evaluated for their supe-ior antibacterial applications.

. Materials and methods

.1. Materials

Chitosan (CS) (high MW, >75% deacetylated) was purchasedrom Sigma Chemical, St. Louis, USA. Acetic acid (glacial, 99–100%),ifferent molecular weights of poly(ethylene glycol)s (PEG) (200,00, 400, 4000, and 6000), silver nitrate (AgNO3), and glutaralde-yde (GH) were obtained from Merck (Bombay, India). Mineral saltroth and nutrient agar were obtained from Himedia chemicalsMumbai, India). The Department of Botany (Sri Krishnadevarayaniversity, Anantapur, India) has provided standard cultures of therganisms.

.2. Silver nanoparticles (AgNPs) synthesis

The optimization of AgNPs was performed using differentolecular weights of poly(ethylene glycol)s (PEG)s. In detail,

00 mg of silver nitrate was added to 25 ml of 1% PEG containing

ig. 2. Swelling rate of chitosan, chitosan–silver nanocomposite and chitosan–silver nanoc

Biointerfaces 76 (2010) 248–258

solution. The reduction of silver nitrate into AgNPs formation wasmonitored by using UV–vis spectrophotometer.

2.3. Preparation of chitosan–PEG blend films (CSB) [27]

Chitosan dissolved in 2% acetic acid (75 ml) and the counterpartpolymer (PEG) dissolved in water (25 ml) with different percent-age amounts (1;1, 2:1, and 5:1 g) were mixed in 500 ml beaker andstirred for 1 h. To this solution, 1 ml of 2% glutaraldehyde solution(cross-linking agent) was added under stirring at room tempera-ture (25 ◦C). The reactant solution was transferred immediately intoa Teflon covered glass plate (100 mm × 100 mm × 3 mm, SabeanTraders, Chennai, India) and dried at 80 ◦C in an electric oven for2 h (Baheti Enterprises, Hyderabad, India). The formed cross-linkedchitosan–PEG blend (CSB) films were incubated in water to removeany un-crosslinked CS, PEG, and glutaraldehyde over a period of 5 h.Then these films were dried under room temperature. The thick-ness of the films was ∼500 �m.

2.4. Preparation of chitosan–PEG silver nanocomposite films(CSSNC)

100 mg of AgNO3 was added separately into three beakers con-taining 25 ml of 1% PEG-6000 solution at 80 ◦C. The correspondingsolutions were stirred at this temperature for 1 h to generate AgNPs.The transparent colorless solution started to convert from pale pink

to grey-black indicating the formation of AgNPs. To this AgNP solu-tions, different percentage amounts of chitosan (75 ml of 1%, 2% and5%) dissolved in 2% acetic acid was added and stirrer for 1 h. For allthese solutions 1 ml of 2% glutaraldehyde (cross-linker) was addedunder stirring at room temperature. The solution were then poured

omposite films prepared at (A) 1:1, (B) 2:1, and (C) 5:1, composition of chitosan:PEG.

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reduction capacity of Ag+ ions, good dispersion capacity in CSmatrix, and non-fouling properties [4]. The formation of the PCSSNCwas carried out in three step process as described in the experimen-tal procedure (Scheme 1). In detail, (1) AgNPs were prepared byadding silver nitrate into PEG solution to form PEG solution which

K. Vimala et al. / Colloids and Surfa

nto Teflon covered glass plates and dried in an oven maintaininghe temperature at 80 ◦C and purification process resulted in CSSNClms.

.5. Fabrication of porous chitosan–silver nanocomposite filmsPCSSNC)

Fabrication of PCSSNC films involves the removal of most of theEG from the CSSNC films, which were prepared following the pro-edure mention in Section 2.4. For this, CSSNC films were immersedn hot distilled water (80 ◦C) over a period of 1 h. During this pro-ess, most of the PEGs were extracted into distilled water since PEGs highly soluble in water at this temperature [27] and the resultedSSNC films were left with highly porous structures (PCSSNC) asresented in Scheme 1.

.6. Swelling studies

Pre-weighed CSB, CSSNC, and PCSSNC were equilibrated in50 ml of phosphate buffer (pH 7.4) at 25 ◦C. The water up takeny the films was measured for every 30 min up to equilibrium, byn analytical balance (ARO 640, AdventurerTM, OHAUS, Mumbai,ndia). The swelling ratio (Q) of the films was calculated using anquation, i.e., Q = Ws/Wd; where, Ws is the weight of the swollenlm and Wd is the dry weight of the film.

.7. Characterization

The UV–vis spectroscopic measurements were carried out usinghimadzu 1600 UV spectrometer (Kyoto, Japan) from 250 to00 nm. The Fourier transform infrared (FTIR) spectra of the filmsere recorded with a Thermo Nicolet Nexus 670 spectrophotome-

er (Washington, USA). X-ray diffraction (XRD) patterns of the filmsere recorded with Rigaku D/Max- 2550Pc (Tokyo, Japan) usingu K� radiation generated at 40 kV and 50 mA. Thermal studiesf the films were carried out on a Mettler Toledo 851e thermalystem (Zurich, Switzerland) at a heating rate of 10 ◦C/min, underitrogen atmosphere (flow rate, 10 mL/min), from room tempera-ure to 700 ◦C. The morphology of films was observed through anptical microscopy (Olympus BX 41, Olympus, Center Valley, PA,SA) and scanning electron microscopy (SEM) (JSM-840 scanningicroscope, JEOL, Tokyo, Japan). Transmission electron microscopy

TEM) (Techai F 12 TEM, Tokyo, Japan) was used to record AgNPsize and morphology. For this study, the samples were preparedy placing a drop of AgNPs extracted from CSSNC and PCSSNC onarbon-coated copper grid and subsequently drying in air, beforeransferring them to the microscope operated at an acceleratedoltage of 120 kV.

.8. Mechanical properties

The mechanical properties of the chitosan blend films andanocomposites films were measured by using an INSTRON 3369TM running at a cross head speed of 5 mm/min. The sample filmsere cut into 1 cm × 10 cm gauge length is about 5 cm. The tensilearameters, maximum stress, modulus and % elongation at break,ere measured using a 10 kg load cell.

.9. Antimicrobial activity

The antimicrobial activity of developed CSB, CSSNC, and PCSSNClms against E. coli, bacillus, and K. pneumoniae as model bacteriaas measured by disc diffusion and viable cell count method [26].

or disc diffusion method, the films were cut into a disc shape withmm diameter, sterilized by autoclaving for 30 min at 120 ◦C, and

Biointerfaces 76 (2010) 248–258 251

placed on different cultured agar plates. These plates were incu-bated for 2 days at 37 ◦C in an incubation chamber maintaining with5% CO2 flow and the inhibition zone was then measured. Viablecount method measures the bacterial growth. For this study, 108

colony forming units (CFU) of E. coli were grown in 10 ml nutrientbroth supplemented with the film discs (20 mm dia.). The bacterialviability was checked using their O.D. values by U.V–vis spectrom-eter at 600 nm.

3. Results and discussion

A lot of work was published on polymer and hydrogel sil-ver nanocomposites to employ as antibacterial materials [28–30].However, these systems are able to release the silver nanoparti-cles only in aqueous media from their swollen polymer/hydrogelnetworks, but not under dry condition. Due to this limita-tion, chitosan–silver nanoparticles systems have exhibited limitedantibacterial properties [4,31,32], in addition to their poor mechan-ical properties. Because of greater importance of chitosan–silvernanofilms/membranes in antimicrobial activity, water purificationand wound dressing applications, we have developed a “green pro-cess” for the preparation of porous chitosan–silver nanocomposites(PCSSNC) films, using PEG as a reducing agent as well as porogener-ator. The main reason to choose PEG, is due to it’s water solubility,

Fig. 3. FTIR spectra of (A) chitosan (CSB), (B) chitosan–silver nanocomposite(CSSNC), and (C) porous chitosan–silver nanocomposite (PCSSNC) films.

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orms the PEG stabilized NPs; (2) PEG stabilized AgNPs solutionas mixed with chitosan solution containing glutaraldehyde, and

llowed to form films where the AgNPs were uniformly dispersednd entrapped throughout the chitosan cross-linked polymeric net-orks via amine (–NH2) and hydroxyl (–OH) functional groups

33,34]; and (3) The porosity in these composite films was achievedy removing the excess/unbound PEG from the chains in CS matrixy heating the films in hot water at 80 ◦C (Scheme 1). This approachimed to improve the conventional problems such as slowelease of AgNPs and poor mechanical strength of the compositelms.

.1. Optimization of synthesis of AgNPs using PEG

To evaluate the useful composition of PEG-silver nanoparticleshat can be implied directly in the preparative process of CSSNC andCSSNC, that can result as an effective antibacterial product. Forhis, the formation of AgNPs was examined employing PEGs under

ild conditions in the absence of other reducing agents, by vary-ng the reaction temperature, molecular weight and compositionf PEGs.

The reduction of silver ions (1% (w/v), 1 ml) in combination ofEG 6000 ((1% (w/v), 1 ml)) results in the formation of AgNPs withinn hour after certain reaction temperature (Fig. 1A). The forma-ion of AgNPs formation can be detected simply by change in coloro light brown. In detail, at temperature below 40 ◦C, no changen optical features was observed in the UV–vis spectra correlates

hat no silver nanoparticles are formed. When the temperatureas increased to 60 ◦C resulting in the appearance of a broad sur-

ace Plasmon resonance (SPR) absorption peak around 433 nm,elonging to the dipole resonance of conducting electron on theurface of silver nanoparticles. Interestingly at temperature 80 ◦C,

ig. 4. (A) UV–vis spectra of CSB, CSSNC, and PCSSNC films, (B) XRD patterns of CS, CSSNC

Biointerfaces 76 (2010) 248–258

the intensity of the surface plasmon resonance peak enhanced witha concomitant wavelength shift toward lower wavelength of bandto 416 nm, indicating the formation of smaller silver nanoparticles.On the other hand, as the temperature underwent a red shift andcausing absorption peak to broaden possibly indicates the increasein the particle size and their distribution. This implied that thelarger silver nanoparticles and wide size distribution were probablyformed when the temperature was 60 ◦C. Therefore, an optimumtemperature of 80 ◦C was fixed for the development of AgNPs in ourexperiments.

In the next step, keeping the temperature constant at 80 ◦C,employing 1 ml of 1% (w/v) of silver nitrate and in combination of 1%(w/v) of different molecular weights of PEG, i.e., PEG-200, PEG-300,PEG-400, PEG-2000 and PEG-6000, experiments were conducted tosynthesize AgNPs (Fig. 1B). Surprisingly, lower molecular weightsof PEGs (200–400) were not efficient in reducing the silver ionsinto AgNPs within an hour. A typical small intense SPR peak wasobserved in the UV–vis spectra for PEG-4000 indicates a slightlyimproved efficiency in the reduction of silver ions. But, silver ionrelated peak still exist in the UV–vis spectra demonstrates that amajor portion of ions were not reduced. In the case of PEG-6000, aclear disappearance of silver ions peak and appearance of strongSPR peak at 416 nm dictates its potential use of this molecularweight PEG for the development of AgNPs.

Finally, to fix the optimum concentration of PEG-6000 in theformulations, experiments were conducting by taking differentconcentrations of PEG-6000 (0.1–2%, w/v) in the reaction mixtures

and keeping 1% (w/v) silver ions and at a temperature of 80 ◦C forthe synthesis of silver nanoparticles (Fig. 1C). As the concentrationof PEG-6000 was increased from 0.1 to 1% (w/v), the intensity of SPRabsorption peak (�max, 415 nm) was also gradually increased. Thisindicates a continuous improved reduction capacity of PEG-6000

, and CSSNC films, and (C) thermogravimetric curves of CS, CSSNC, and CSSNC films.

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The formation of silver nanoparticles in the PCSSNC was clearlydemonstrated from its UV–vis spectra at 406 nm (Fig. 4A). Thispeak intensity is even more than what was observed in the caseof just PEG alone. Therefore, this simply indicates that chitosanalso played a major role in the formation of silver nanoparticles.

K. Vimala et al. / Colloids and Surfa

ith increase of concentration. Though, the reduction potentialas further increased with 2% (w/v) of PEG-6000, its SPR peakas slightly red shifted which implies again for aggregative phe-omenon.

From the foregoing experiments, the optimum reaction con-itions to obtain uniform AgNPs in PEG solution were found as:emperature = 80 ◦C; Molecular weight of PEG = 6000; Concentra-ion of PEG = 1% (w/v); and concentration of AgNO3 (silver ion) = 1%w/v).

.2. Fabrication of chitosan–silver nanocomposite (CSSNC) andorous chitosan–silver nanocomposite (PCSSNC)

The CSB, CSSNC, and PCSSNC were prepared as explained incheme 1A, and the developed films were depicted Scheme 1B.owever, we have studied especially for chitosan–PEG (1:1)anocomposites because of its stability in water. Other two com-ositions were brittle in water and difficult in handling.

.2.1. Swelling capacityThe swelling capacity of the antibacterial film/gels and

anocomposites play an important role in the antibacterial activity,ound healing process, and other biomedical applications. Fig. 2,

llustrates the swelling capacity of the CSB, CSSNC, and PCSSNClms with time. The order of the swelling capacity of films followss, PCSSNC > CSB > CSSNC. The CSB blend films show the moderatewelling ratio (9 g/g) than the CSSNC films. Lower swelling capacityf CSSNC film may be attributed by binding of AgNPs with electronich O and N atoms of ether and amine groups of PEG/chitosan thatre present in the composite, which are responsible to produce thedditional cross-links within the chain networks. The higher cross-inks restrict the water penetration for swelling. Interestingly, theorous chitosan silver nanocomposite films show higher swellingapacity because of presence porosity in their network structureshat allow more water to enter inside the film. With increase of chi-osan content in the composites (1:1, 2:1, and 5:1, chitosan: PEG)esult a slightly improved swelling capacity Fig. 2A–C). But, furthernvestigations are limited to only 1:1 chitosan:PEG films becausef easy handling.

.2.2. FTIR spectra [26]The FTIR spectra of CSB, CSSNC and PCSSNC films are depicted in

ig. 3. The CSB film (Fig. 3A) shows absorption peaks at 1642 cm−1,568 cm−1, and 1341 cm−1 belonging to amide I, II, and III of C Otretching, N–H/C–N stretching, and CH2 wagging coupled with OHroups of chitosan. A band near 1093–1023 cm−1 is due to the COtretching and the peak near 1463 cm−1 is due to CH bending. Theeak observed at 3442 corroborates to hydrogen-bonding of OH/NHtretching.

In the case of chitosan–silver nanocomposite (Fig. 3B), whichlso contains PEG chains throughout the cross-linked polymer filmas exhibited intense peak at 2880 cm−1 due to increased C–Htretching vibrations of PEG. In addition, the stretching vibrationst 3432 cm−1 are highly shortened, and the absence of 1568 cm−1

eak that exists in plain chitosan films and the appearance ofdditional peak at 1724 cm−1, indicate that the silver is boundo the functional groups of chitosan. Peak shifting occurs due too-ordination bond between the heavy metal atom (silver in thisase) and electron rich groups (oxygen/nitrogen). This causes anncrease in the bond length, ultimately shifting the frequency.

ore importantly, an additional peak appeared at 953 cm−1 due

o C-O-C stretching peaks which arise due to the presence ofEG chains. Therefore, it can be concluded that chitosan–silveranocomposite consists of all three major components, i.e., chi-osan, PEG and silver nanoparticles basing on the above spectraltudies.

Biointerfaces 76 (2010) 248–258 253

In the FTIR spectrum of porous chitosan–silver nanocompos-ite (Fig. 3C), the disappearance of peak at 953 cm−1 (C–O–C), thereduced intensity of C–H stretching peak (2920 cm−1), increasedhydrogen-bonding peaks (3318 cm−1) clearly suggest that almostall PEG was leached out and the silver nanoparticles are efficientlybound by chitosan film through their –OH and –NH2 functionalgroups. This can be supported from the increased peak intensity at1718 cm−1.

3.2.3. UV–vis spectroscopy

Fig. 5. Optical micrograph images of CSB, CSSNC, and PCSSNC films.

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urther, the silver nanoparticles Plasmon peak of PCSSNC wasot diminished much even after extracting the PEG chains inhe porogenator process in which there is a possibility of loss ofanoparticles.

.2.4. XRD analysisThe X-ray diffraction (XRD) was used to evaluate the crystal

tructure of films that is widely used to confirm the formation ofilver nanoparticles (Fig. 4B). The XRD patterns of all the films (CSB,SSNC and PCSSNC) below 30 ◦C are related to the crystalline naturef major component i.e., chitosan (Fig. 4B(a)). Fig. 4B(b-c), exhibitstrong reflections around 33◦ and 40◦ characteristic to (1 1 1) and2 0 0) planes of the face centered cubic (fcc) of the silver nanopar-icles [35]. Therefore, this gives a clear evidence for the presencef silver nanoparticles in the chitosan nanocomposite and poroushitosan nanocomposite films.

.2.5. ThermogravimetryFig. 4C illustrates the thermogravimetric curves of CSB, CSSNC

nd PCSSNC films. Chitosan film show good thermal stability upo 358.53 ◦C, maximum decomposition occurs at 488.53 ◦C, and00% weight loss noticed at 700 ◦C. In the case of chitosan–silver

ig. 6. Scanning electron micrographs of (A and B) chitosan, (C and D) chitosan–silver nndicates 5 �m.

Biointerfaces 76 (2010) 248–258

nanocomposites films (CSSNC and PCSSNC), the degradation startsearlier than chitosan film because of loose networks or mois-ture present due to nanoparticles incorporation process leadingto porosity. However, major weight loss starts from 488 ◦C. Inter-estingly, silver nanocomposites have overall higher stability thanchitosan film because even at 700 ◦C, 8.23 and 11.27% of materialis not degraded. That means, chitosan film contains 8–11% of silvernanoparticles in their matrix.

3.2.6. Microscope analysisIt can be believed that chitosan polymer helps in nucleation

and stabilization of the silver nanoparticles formed with the helpof PEG chains and thus, promotes the uniform distribution of sil-ver nanoparticles throughout the chitosan film (CSSNC). Even afterremoving PEG chains from the chitosan silver nanoparticles film(PCSSNC) by a leaching process in hot water (80 ◦C), silver nanopar-ticles are anchored by chitosan functional groups. This can be seen

clearly in Fig. 5. The light optical micrograph of the chitosan–silvernanocomposite and porous chitosan–silver nanocomposite showsbrown color AgNPs, which are uniformly distributed in the films(Fig. 5B-C). Whereas, the chitosan film (Fig. 5A) shows completeabsence of these silver nanoparticles.

anocomposite, and (E and F) porous chitosan–silver nanocomposite films. Bar size

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osan–

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To get good acceptance of presence of silver nanoparticles inhe nanocomposites as well as to inspect the presence of poros-ty in chitosan–silver nanoparticles, SEM studies were performed.he SEM photographs of the CSB, CSSNC and PCSSNC films are pre-

ig. 8. Transmission electron images of silver nanoparticles of (A–C) chitosan–silver nanize calculated from these TEM images using ImageJ software for (G) chitosan–silver nan

silver nanocomposite films. Bar size indicates 1 �m.

sented in Fig. 6. The pure CSB film (Fig. 6A and B) exhibited a denseand uniform plain microstructure. Whereas CSSNC film (Fig. 6C andD) showed the presence of nanoparticles in the entire film (whitedots). In the case of PEG extracted film i.e., PCSSNC (Fig. 6E and F)

ocomposite, and (D–F) porous chitosan–silver nanocomposite. Silver nanoparticlesocomposite, and (H) porous chitosan–silver nanocomposite.

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Fig. 9. Uniaxial stress–strain curves of (a) CSB, (b) CSSNC and (c) PCSSNC films.

Fig. 10. (A) The antibacterial activity of (a1–a3) CSB, (b1–b3) CSSNC, and (c1–c3) PCSSNC films against E. coli, Bacillus, and K. pneumonia, by disc diffusion method. (B) Theantibacterial activity of CSB, CSSNC, and PCSSNC films against E. coli, determined by absorbance count method.

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Table 1Mechanical properties of CSB, CSSNC, and PCSSNC films.

Sample code Stress at maximumload (MPa)

Modulus (MPa) Elongation atbreak (%)

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CSB 24.35 1500.00 4.49CSSNC 25.96 583.68 18.86PCSSNC 28.71 795.05 22.10

learly indicate the presence of hundreds of pores throughout thelm. Porous formation in PCSSNC can be seen in an enlarged SEM

mages (Fig. 7A–D).To find out the exact size and morphology of silver nanopar-

icles those were impregnated in CSSNC (Fig. 8A–C) and PCSSNCFig. 8D–F), the TEM studies were performed. The TEM resultsllustrates that the particles formed have an average size of2.5 nm ± 4.5 nm (Fig. 8G) and 12.2 ± 3.4 nm (Fig. 8H), obtainedrom CSSNC and PCSSNC, respectively. A significant decrease in theize of silver nanoparticles after porous formation in the chitosanlms may be due to the involvement of further reduction as well asalancing the stabilization process by chitosan polymeric chains.n the other hand, the processing temperature used for extrac-

ion of PEG could also be involved in reducing the size of silveranoparticles. According to our previous and the available litera-ure suggests that the particles we have generated in these filmsre within the useful range for essential antibacterial applications.

.2.7. Mechanical propertiesLack of good mechanical strength and porous nature of poly-

eric or hydrogel silver nanocomposites, may not serve an efficientntibacterial materials in water filtration units and in wound dress-ng applications. A number of chitosan–silver nanocomposites wereeen investigated for many applications. To improve their mechan-

cal strength further we have developed porous silver impregnatedhitosan-nanocomposites and expected to have superior mechani-al properties. Mechanical properties CPB, CSSNC and PCSSNC filmsre shown in Fig. 9 and Table 1. The higher stress at maximum loadnd elongation at break of the PCSSNC films was obtained than thatf CSB and CSSNC films. Whereas the modulus was decreased thanSB film when induced the silver nanoparticles, but when poresere induced into their structure some improvement was observed

n the modulus. The mail goal is to produce smaller pore size struc-ured chitosan, which can be induced by this method, leading toigher strength.

.3. Antimicrobial activity

Still there is a great interest to generate antibacteriallms because of their superior biomedical relevance [36]. Although,any synthetic polymer based nanocomposites have been

mployed as surgical and wound dressings but often regenerateskin irritations due to leaching of harmful chemicals causing sideffects to human beings [37]. Therefore, generation of antimicro-ial films using renewable natural sources including biopolymersould be a better option. Recently, Rujitanaroj et al. [38] pre-ared silver nanoparticles on gelatin fiber mats and tested for theirntimicrobial activity against E. coli, Pseudomonas aeroginosa, S.ureus and methicillin-resistant S. aureus (MRSA). Edible antimi-robial films prepared by using yam starch and chitosan inhibitedffectively the growth of S. enteritidis [39].

In view of this, herein we have evaluated our novel composites

pplicability as antibacterial materials. The antibacterial activityf developed CSSNC and PCSSNCs was determined by disc dif-usion method for E. coli, Bacillus, and K. pneumoniae (Fig. 10A).t was found that the CSSNC [Fig. 10A (a2, b2, c2)] and PCSSNCFig. 10A (a3, b3, c3)] exhibited an inhibition zone while the chi-

Fig. 11. UV–vis spectra of (A) CSSNC and (B) PCSSNC films after releasing silvernanoparticles in water at different time intervals.

tosan film [Fig. 10A (a1, b1, c1)] does not involve in the inhibitionzone process. CSSNC showed the growth inhibition ring size of E.coli, Bacillus, and Klebsiella pneumoniae 18, 18 and 15 mm, respec-tively. Whereas, PCSSNC expressed greater inhibition ring size of E.coli, Bacillus, and Klebsiella pneumoniae 20, 22, and 17 mm, respec-tively. This behavior can be explained based on the smaller sizeof silver nanoparticles which are more efficient for antibacterialapplication. Our previous results also support the same [40]. Thesuperior antibacterial activity with PCSSNC could be due to thepresence of porous structure which absorbs large quantity of waterand releases the silver nanoparticles efficiently into the media.Further, the CSSNC and PCSSNC were tested with E. coli suggestthat a drastic bacterial growth inhibition was observed with time(0–350 min). The absorbance counts recorded on UV–vis spec-trophotometer for E. coli after different time treatment with thesenanocomposites (Fig. 10B). After 350 min of incubation, there wasa 70–75% reduction in viable E. coli. A slight improved antibac-terial action was noticed with PCSSNC at all points of treatmenttimes. From these experiments, we can clearly understand thatthe porous chitosan–silver nanocomposite film exhibits excellentantimicrobial activity.

To prove the enhanced silver nanoparticles release from theporous nanocomposites which has greater effect as antibacterialcompound, a simple experiment was performed. Fig. 11, clearly

support more release of AgNPs from PCSSNC than CSSNC. Accord-ing to SN (Swiss norm) 195920 - ASTM E 2149-01, any agent showzone inhibition of >1 mm, is considered as a good antibacterialagent [41]. Therefore, we can conclude that the developed porous

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hitosan–silver nanocomposite exhibits excellent activity in all thehree bacteria.

. Conclusion

This study demonstrates preparation of porous chitosan–silveranocomposite films in a facile three step process. The employedhitosan and poly(ethylene glycol)s are not toxic and biocompati-le in nature. These polymers not only help in reducing the metal

ons into nanoparticles but also provide excellent stability for austained release of nanoparticles for antibacterial applications.he developed porous nanocomposite film has exhibited superiorntibacterial properties and good mechanical properties than thehitosan and chitosan–silver nanocomposites, suggesting that itan be applied for wound dressings and water purification purpose.

cknowledgements

KMR thanks the Defence Research and Development Organiza-ion (DRDO) and Ministry of Defence, Government of India, Newelhi and KV thank U.G.C-SAP, New Delhi for the partial financial

upport.

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