Journal ofMaterials Chemistry B

PAPER

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Chitosan films co

aDipartimento di Scienze Farmaceutiche, U

Liceo 1, 06123 Perugia, Italy. E-mail

0755855125; Tel: +39 755855125bDipartimento di Chimica, Biologia e Biotecn

Via Elce di Sotto, 8, 06123 Perugia, ItalycDipartimento di Scienze Farmaceutiche, M

Universita degli Studi di Perugia, Via del GidIstituto per lo Studio dei Materiali Nanostru

Roma 1, Via Salaria Km 29.3, 00015, Mont

† Electronic supplementary informa10.1039/c4tb00927d

Cite this: J. Mater. Chem. B, 2014, 2,6054

Received 7th June 2014Accepted 2nd July 2014

DOI: 10.1039/c4tb00927d

www.rsc.org/MaterialsB

6054 | J. Mater. Chem. B, 2014, 2, 605

ntaining mesoporous SBA-15supported silver nanoparticles for wound dressing†

Valeria Ambrogi,*a Anna Donnadio,b Donatella Pietrella,c Loredana Latterini,b

Federica Alunni Proietti,a Fabio Marmottini,b Giuseppina Padeletti,d Saulius Kaciulis,d

Stefano Giovagnolia and Maurizio Riccia

Chitosan films containing mesoporous SBA-15 supported silver nanoparticles (AgNPs) were prepared to be

applied as a potential wound dressing material. First SBA-15–silver nanoparticle (SBA-15–Ag) composite

materials were prepared by a controlled annealing process without the use of organic solvents and

reagents. The SBA-15–AgNPs were characterized in detail by X-ray powder diffraction, field emission

scanning electron microscopy and transmission electron microscopy which evidenced the presence of

uniformly distributed silver nanostructures inside the silicate pores. UV-vis spectra of the sample showed

a band at 430 nm characteristic of the surface plasmon resonance of silver nanoparticles with a diameter

below 10 nm and X-ray photoemission spectra confirmed the formation of metal-nanoparticles on the

silicate template. Then SBA-15–Ag was used to prepare chitosan films which were characterized in

detail. In particular, they showed good hydration properties, water vapor transmission rate and

mechanical properties. After hydration films exhibited good antimicrobial activity against both Gram-

negative (Pseudomonas aeruginosa) and Gram-positive (Staphylococcus epidermidis and S. aureus)

bacteria.

1. Introduction

The skin is the largest human organ and is the essential inter-face between the body and its environment. Its major functionsare the protection of underlying tissues from microbial patho-gens present in the environment and the control of water lossfrom the body. Injured skin is not able to guarantee its barrierfunction and wound healing, which is the process of tissueregeneration, is necessary for the survival. Healing failure orprolonged wound healing time results in chronic lesions. Thepresence of exudates and infections are among the main factorsin the healing process.1 In fact, the excessive production ofexudates can cause maceration of healthy skin around thewound, while bacterial or fungal infections can lead to severesystemic complications as well. Inadequate management ofinfected wounds can provoke cellulitis (cell inammation) and

niversita degli Studi di Perugia, Via del

: valeria.ambrogi@unipg.it; Fax: +39

ologie, Universita degli Studi di Perugia,

icrobiology and Immunology Laboratory,

ochetto, 06122 Perugia, Italy

tturati (ISMN) – CNR, Area della Ricerca

erotondo Stazione, Rome, Italy

tion (ESI) available. See DOI:

4–6063

ultimately fatal septicemia.1 Infection is the leading cause ofmorbidity and mortality in extensive burn injuries, traumaticinjuries and surgical procedures. Thus the control of infectionremains a major challenge in wound management. Conse-quently, broad-spectrum antimicrobial agents are required tocontrol infection and to facilitate the healing process. The useof systemic antibiotics is not always recommended due to theincreasing occurrence of bacterial resistance and sensitizingside effects. Therefore, topical antimicrobial agents have beenpreferred for wound dressing. Silver (Ag), in particular, has beena preferred additive to prepare medicated wound dressingsbecause it has a broad spectrum of antimicrobial and anti-fungal activity.2 Ionic Ag is active against Gram-negative andGram-positive microorganisms and shows antibacterial effectson antibiotic resistant bacteria, such as methicillin-resistantStaphylococcus aureus and vancomycin-resistant enterococci.Recently, Ag nanoparticles3–5 (AgNPs) have attracted interest asantimicrobial agents for applications in medicine. The reasonof this success can be ascribed to the enhanced antibacterialproperties of AgNPs compared to the Ag bulk material. In fact,AgNPs possess a much higher surface area and a high fractionof surface atoms, which make them effective at nanomolarconcentration.6 The antimicrobial mechanism of AgNPs is stillunclear but is mediated by partial and gradual oxidation andrelease of Ag ions and include: (i) photocatalytic production ofreactive oxygen species (ROS), (ii) interaction with the pepti-doglycan cell wall and the plasma membrane, (iii) inhibition of

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bacterial enzyme activity, (iv) impairment of DNA synthesis7,8

and (v) increase of membrane permeability of Gram-negativebacteria with consequent improvement of the action of existingantiobiotics.9

Recent strategies focused on the acceleration of woundrepair by the use of biomaterials such as chitosan.10,11 Chitosanis a linear, semi-crystalline polysaccharide composed of (1:4)-2-acetamido-2-deoxy-b-D-glucan (N-acetyl D-glucosamine) and(1:4)-2-amino-2-deoxy-b-D-glucan (D-glucosamine) units. It isobtained from the partial deacetylation of the natural polymerchitin. Chitosan is biocompatible and biodegradable, showsantimicrobial and hydrating properties, stimulates cell prolif-eration and histoarchitectural tissue organization, showshemostatic action and gradually depolimerizes to releaseN-acetyl-b-D-glucosamine, which initiates broblast prolifera-tion, helps in ordered collagen deposition, and stimulates anincreased level of natural hyaluronic acid synthesis at thewound dressing site.11,12 Moreover chitosan–inorganic nano-composites have been recently proposed as scaffolds for tissueengineering.13

This paper describes the preparation of AgNPs supported onmesoporous silicate SBA-15. Then the AgNP–silicate compositewas introduced into chitosan lms. To the best of our knowl-edge composite chitosan lms containing silver AgNPs inmesoporous silicates have not been prepared to date.

2. Experimental section2.1 Materials

Tetraethyl orthosilicate (TEOS) was purchased from Sigma-Aldrich GmbH (Steinheim, Germany). The surfactant Pluronic(P123, EO20PO70EO20) (BASF Corporation – New Jersey, USA)was kindly provided by BASF Italia SPA. Deionized water wasobtained by a reverse osmosis process with a MilliQ system(Millipore, Roma, Italy). Acetate silver and chitosanmedium PM(200–800 cP, 1 wt% in 1% acetic acid, 25 �C, Brookeld) werepurchased from Sigma Aldrich (Milan). Other reagents andsolvents were of reagent grade and were used without furtherpurication.

2.2 Characterization

XRPD patterns were recorded using a Philips X'PERT PRO MPDdiffractometer operating at 40 kV and 40 mA, with a step size of0.0170 2q� and a step scan rate of 30 s, using Cu Ka radiationand an X'Celerator detector.

Nitrogen adsorption–desorption isotherms at 77 K weredetermined using a computer controlled Micromeritics (Nor-cross, GA, USA) ASAP 2010 apparatus. Prior to adsorptionmeasurements, free SBA-15 and loaded samples were outgassedat room temperature overnight. The specic surface area wasdetermined by the Brunauer, Emmett and Teller (B.E.T.) tech-nique,14 while mesopore size distribution andmesopore volumewere detected by the BJH method.15

The morphology of the samples was investigated with aPhilips 208 transmission electron microscope (TEM) and with aFEG LEO 1525 scanning electron microscope (SEM). TEM

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samples were prepared by depositing a drop of aqueousdispersion on a Formvar-coated copper grid, which was thenevaporated in air at room temperature. SEM micrographs werecollected aer depositing the samples on stubs successivelysputter-coated with chromium for 20 seconds.

Elemental mapping of metals on SBA-15 samples was con-ducted by using energy dispersive X-ray spectroscopy (EDS).

Quantitative Ag analyses were performed with Varian 700-ESseries inductively coupled plasma-optical emission spectrome-ters (ICP-OES). A weighed amount of SBA-15–Ag (about 20 mg)was dissolved in concentrated HF and HNO3 and the solutionwas brought to a nal volume of 100 mL. The solution, appro-priately diluted, was analyzed by ICP.

The optical behaviour of the solid samples was investigatedthrough spectrophotometric measurements; the spectra wererecorded using a Varian (Cary 4000) spectrophotometer,equipped with a 150 mm integration sphere and a bariumsulphate tablet was used as a reference. The recorded spectrawere analysed with the Kubelka–Munk equation in order tomake possible the comparison.

X-ray photoemission spectra (XPS) were collected by using anEscalab 250Xi spectrometer (Thermo Fisher Scientic, UK),equipped with a monochromatic Al Ka excitation source, elec-tron and ion ood guns for sample charge neutralization and a6-channeltron detection system. In order to increase theelemental sensitivity, the photoemission spectra were collectedat 50 eV pass energy, and the diameter of the analyzed area waskept at about 1 mm. Obtained spectroscopic data were pro-cessed by the Avantage v.5 soware.

2.3 SBA-15 synthesis

12 g of Pluronic P123 were added to 432 mL of an aqueoussolution of HCl (1.7 M) and stirred for 4 h at 40 �C. Successively26 mL of TEOS (mass ratio TEOS : P123 ¼ 2) were addeddropwise and then stirred for 2 hours Aerwards, the mixturewas transferred to a Teon-lined sealed container and kept at100 �C for 48 hours The nal product was ltered, washedalternatively with water and ethanol and nally calcined at540 �C for 24 hours.16

2.4 Preparation of AgNPs

500 mg of silver acetate were solubilized in 50 mL of deionizedH2O. Successively 1.0 g of SBA-15 was added. This dispersionwas sonicated (Ultrasonic Cell MicrosonTM Distruptor XL) for15 minutes, then stirred for 2 hours and nally centrifuged at4000 rpm for 15 minutes at 20 �C (Hettich Universal 32 R). Thesediment was dried in an oven at 60 �C. The product was trit-urated and nally calcined at 300 �C for 2.5 hours to give SBA-15loaded AgNPs (hereaer SBA-15–Ag).

2.5 Film preparation

A 1.5% (w/w) chitosan dispersion was prepared in 0.5% (v/v)acetic acid and 0.1% (v/v) glycerol aqueous solution with stir-ring at room temperature for ca. 12 hours. Then, aer air bubbleremoval with vacuum, 20 g of the dispersion were cast in a Petridish (12 cm diameter) and then dried at 60 �C for 6 hours. Films

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Table 1 Film compositions and thickness

FilmChitosan amount(g per 100 g of dispersion)

Glycerol amount(g per 100 g of dispersion)

SBA-15 amount(g per 100 g of chitosan)

SBA-15–Ag amount(g per 100 g of chitosan)

Film thickness(mm)

Film 1 1.5 0.1 — — 44Film 2 1.5 0.1 5 — 50Film 3 1.5 0.1 10 — 58Film 4 1.5 0.1 15 — 60Film 5 1.5 0.1 — 5 64Film 6 1.5 0.1 — 10 70Film 7 1.5 0.1 — 15 81

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containing SBA-15 and SBA-15–Ag with different concentrations(Table 1) were prepared following the same procedure.

2.6 Water uptake

2 � 2 cm samples cut from the lms were weighed (W1) andthen immersed at 37 �C into phosphate buffer pH 7.4 (sodiumphosphate dibasic 2.38 g, potassium phosphate monobasic 0.19g and sodium hydrochloride 8.0 g in 1000 mL deionized water,according to Italian Pharmacopoeia XII). At predetermined timethe lm was removed, carefully bolted with a lter paper andweighed (W2). The water uptake was determined by thefollowing equation:

% of hydration ¼ ðW2 �W1ÞW1

� 100

2.7 Water vapor transmission rate

The water vapor transmission rate (WVTR) across the lms wasdetermined according to the ASTM E96-90 method, ProcedureD, with a Payne permeability cup.17 Films were mounted on themouth (3 cm diameter) of a Payne cup containing 20 g ofdeionized water and this system was weighed, thermostated at37 �C at 40% relative humidity and nally reweighed aer 24hours. The WVTR was calculated by using the followingformula:

WVTR ¼ [(Wi � Wf)/A] � 106 g m�2 per day

where A is the area of the cup mouth (706.5 mm2),Wi andWf arethe weights of the cup before and aer being placed in the oven,respectively.

2.8 Mechanical properties test

Stress–strain mechanical tests were carried out using a ZwickRoell Z1.0 testing machine, with a 200 N static load cell. TheYoung's modulus (the slope of the stress–strain curve in theelastic deformation region), tensile strength (the tensile stressat the breaking point of the specimen), and elongation at break(the percentage increase in length that occurs before the samplebreaks) were measured on rectangle shaped lm stripes,obtained using a cutting machine, length and width of whichwere 100 and 5 mm, respectively. Before the tests at roomtemperature, samples were equilibrated for 7 days in vacuum

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desiccators at 53% RH and room temperature (20–23 �C). Thethickness of the lm stripe, determined with an uncertainty of 5mm, was in the range of 45–50 mm. An initial grip separation of10.000 � 0.002 mm and a crosshead speed of 30 mm min�1

were used. At least ve replicate lm stripes were analyzed. Thedata were elaborated by the TestXpert V11.0 Master soware.

2.9 In vitro silver release

Ag in vitro release studies were performed by immersing lm 7(2 � 2 cm) into 50 mL of the phosphate buffered saline solutionpH 7.4 under stirring (100 rpm) at 37 �C. At desired timeintervals, aliquots (3 mL) of supernatant were removed andreplaced by fresh dissolution medium. They were treated withHNO3 solution 69% and then the silver released was detected byICP. All experiments were done in triplicate (n¼ 3) and the errorwas expressed as standard deviation (SD).

2.10 Microorganisms

The microbial strains used in this study were two Gram positivebacteria Staphylococcus aureus ATCC 29213 and Staphylococcusepidermidis ATCC 35984 and the Gram negative Pseudomonasaeruginosa PAO-1. The cultures were maintained in tryptic soyagar (TSA). The day before the test, one colony was inoculated intryptic soy broth (TSB) and incubated for 24 h at 37 �C.

2.11 Measurement of the zone of inhibition (ZOI)

The zone of inhibition was determined as previously describedwith some suitable modications.18 Bacterial suspensions werewashed in sterile saline, cell concentration was determined byspectrophotometric measurement (600 nm). A suspension of1 � 108 cells per mL was uniformly spread on a Muller HintonAgar (MHA) plate. All test lms were cut into disks (diameter of6 mm), sterilized by UV and placed on the surface of the MHAplate previously inoculated with bacteria, hydrated with 20 mLdistilled sterile water and incubated for 24 h at 37 �C. The ZOIwas determined by measuring the diameter of the clearing zonearound the lms using a metric ruler.

2.12 Effect of lms on the growth of S. epidermidis and P.aeruginosa

S. epidermidis and P. aeruginosa were cultured in Muller HintonBroth in a 96 well culture plate in the presence of different lmsat 37 �C for 13 and 24 h respectively. The absorbance A600 of

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culture media was revealed every 60 minutes aer shaking by anInnite M200Pro (Tecan).

2.13 Cell viability assay

All lms were treated with 2 wt% sodium hydroxide aqueoussolution to remove excessive acetic acid, then rinsed with a gooddeal of distilled water and dried at 40 �C. Human skin kerati-nocytes NCTC2544 (2 � 105) were grown overnight to conu-ence. Monolayers were treated for 24 h at 37 �C with theindicated lm. Aer incubation the cell viability was evaluatedby the use of an ATP bioluminescence kit (Via Light kit; Cam-brex). Results are expressed as relative light units (RLU).

Fig. 1 XRDP patterns of SBA-15 and SBA-15–Ag.

3. Results and discussion3.1 AgNP preparation and characterization

The synthetic methods of AgNPs reported so far usually requirethe use of organic solvents and reducing agents such ashydrazine, sodium borohydride and N,N-dimethylformamidethat are highly reactive and can be the source of potentialenvironmental and biological hazards. In this paper, AgNPswere obtained without the use of organic solvents and reagents,suitably modifying the method reported for the synthesis of Agnanowires within the pores of halloysite.19 AgNPs were obtainedby thermal decomposition of Ag acetate previously loaded intothe pores of the silicate SBA-15. This hard tubular template waschosen for the presence of ordered and suitable mesopores (7–8nm) with narrow distribution and high surface area.20 Suchstructures offer support to metal nanostructures, protectingmetal nanoparticles from oxidation and photoinducedmorphology changes. Moreover, the connement of Ag into thesilicate mesopores avoids the direct contact between nano-particles and wound preventing Ag absorption across thedamaged skin. This aspect is of paramount importance astoxicological AgNP issues are still discussed and in vitro and invivo studies demonstrated that AgNPs can exhibit signicanttoxicity and that long-term exposure is associated withincreased argyremia.7 In addition, SBA-15 and other orderedmesoporous silicates, such as MCM-41, have been proposed asbiomaterials21 for biological and pharmaceutical use22–24 andmany biocompatibility studies have been performed as well.25,26

The obtained SBA-15–Ag was characterized and its propertieswere compared to those of mesopores of SBA-15, in order tohave information on the silver nanostructures. The SBA-15typical XRPD pattern (Fig. 1) revealed the 2-D hexagonallystructured pores (P6mm space group) at low angles (data notreported) and a broad peak in the range of 2 theta ¼ 12–32�

indicated the amorphous nature of SBA-15. The SBA-15–Agdiffractogram showed reections at 2 theta ¼ 38.0�, 44.3�, 64.4�

and 77.1� corresponding to (111), (200), (220), and (331)diffraction planes of cubic metallic Ag [JCPDS no. 4-0783]. Thisconrmed the presence of Ag domains in the porous amor-phous SBA-15.27

The Ag content determined by ICP was 4.1 � 0.2% (g of Agper 100 g of SBA-15–Ag). With the aim of evaluating the inu-ence of AgNP deposition on the specic surface area and

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porosity of solids, nitrogen adsorption and desorptionisotherms at 77 K were performed. Important informationabout the space occupied by AgNPs could be obtained from thecomparison between the specic surface area, pore size andpore volume of SBA-15 before and aer loading.

The nitrogen adsorption–desorption isotherms recorded forSBA-15 before and aer loading (Fig. 2A) showed that, in the p/p0 value range, the nitrogen adsorption–desorption isothermvolume of SBA-15–Ag was below that of the precursor SBA-15.Thus, a decrease in the measured adsorbed nitrogen aer AgNPdeposition was evidenced. At the same time, it could be notedthat the adsorption and desorption isotherms of the precursorSBA-15 and those of SBA-15–Ag had similar proles indicating asimilar mesoporous structure for the two samples. This was alsoconrmed by BJH analyses of the adsorption and desorptiondata. A little diminution, from 0.83 cm3 g�1 to 0.80 cm3 g�1, inthe pore volume of SBA-15 and SBA-15–Ag was calculated whilea similar pore size distribution between the two samples wasfound (Fig. 2B). In order to understand if the AgNPs were placedinside or outside the mesopores of the precursor, the Ag contentwas taken into account: 1.00 g of loaded SBA-15–Ag contains ca.0.041 g of Ag. Considering that the density value for metallic Agis 10.49 g cm�3, the approximate volume occupied by 0.041 g ofAgNPs was 0.0039 cm3. This value is lower than the sensibility ofthe pore volume calculation. Nevertheless, the small decrease ofmesopore volume aer Ag loading as well as the similarity of theprecursor and loaded sample pore size distributions seemed toindicate that at least a part of the AgNPs were placed deep insidethemesopores and thus it could be hypothesized that Ag did notmodify the pore size and only a part of some pores were occu-pied by the AgNPs which partly blocked the pore space of themesoporous channels.

SBA-15 and SBA-15–Ag were characterized also by SEM andSEM-EDS. Fig. 3 shows the SEM images. SBA-15 micrographsshowed the typical rod-like morphology with size in the range of200 nm–1 micron. No relevant differences could be observedaer AgNP loading and the annealing process.

Fig. S-1† displays the elemental mapping of Si, O and Ag inSBA-15–Ag. The local relative concentration of each elementpresent in the sample is indicated by the relative brightness andthe intensity of the color. The Ag metal was uniformly distrib-uted throughout the particles of the sample under examination.

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Fig. 2 (A) Nitrogen adsorption and desorption isotherms at 77 K obtained for SBA-15 and SBA-15–Ag; (B) pore size distribution calculated for theSBA-15 and SBA-15–Ag.

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TEM images of SBA-15–Ag (Fig. 4) showed the orderedunidirectional mesopores with a hexagonal array of the silicate.The presence of AgNPs uniformly distributed inside the meso-pores of the silicate was denitely conrmed by the directvisualization. The spheroidal AgNPs were dispersed all over theinorganic framework, with no evidence of agglomeration. Asignicant amount of AgNPs on the outer surface of meso-porous silica could not be observed. The pore size was about 7nm while the AgNP size was 6–8 nm. Only a few NPs showedlarger size, likely due to an enlargement of the silicate meso-pores during the nanoparticle growth.

In order to conrm the presence of AgNPs on the SBA-15–Agsample the optical properties of the samples were investigatedthrough UV-vis spectrophotometric measurements. In Fig. 5Athe extinction spectra of SBA-15–Ag and the pristine SBA-15 arepresented. The spectrum of the pristine inorganic matrix wasdominated by radiation scattering phenomena and no denedbands could be observed. On the other hand, the spectrum ofSBA-15–Ag showed a dened band centered at 430 nm, whichcould be assigned to the surface plasmon resonance (SPR) of theAg particles having dimensions below 10 nm.28–30 These datasupported the formation of nanostructures with metal inelemental form. The band presented an asymmetric shape(Fig. 5B) due to the particle shape, which deviated from thespherical one.

XPS analysis of SBA-15–Ag was carried out by placing thepowder on grated foil of pure Au (99.999%) in order to avoid any

Fig. 3 FE-SEM micrographs of SBA-15 and SBA-15–Ag. Scale barcorresponds to 200 nm.

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interference with photoemission peaks from the substrate andto minimize the sample charging under X-ray ux. However, aneffect of differential charging could be observed and somephotoemission peaks were shied by 4–5 eV, while other peaksremained unmoved. This effect, generated the Ag 3d with twoapparently different chemicals, is shown in Fig. 6.

Aer correction of differential charging, the Ag 3d doubletremained splitted in two species: metallic at BE ¼ 368.2 eV andthe second one at BE ¼ 368.6–369.1 eV (Fig. 6). As it is knownthat the chemical shi of Ag 3d is very small, the value of theAuger parameter31 is oen used for the identication of the Agchemical state. However, due to the low content of Ag in oursamples, the Ag MNN signals were too low and too noisy for theapplication of this method. In addition, when Ag NPs are smallenough (less than 10 nm), it is possible to observe a shi of Ag3d peaks caused by reduced dimensions of the particles.32,33

Therefore, it was possible to attribute the second spin–orbitdoublet of Ag 3d to this size-shi, whereas the rst doublet wasgenerated by bigger clusters of NPs corresponding to themetallic value of Ag 3d as shown in Fig. 6. The other peaks ofconstituent elements were attributed to SiO2 (Si 2p and O 1speaks).

3.2 Chitosan composite lm characterization

Firstly, chitosan lms (lms 2–4) with different SBA-15concentrations were prepared in order to dene the highest

Fig. 4 TEM images of SBA-15–Ag showing AgNPs within SBA-15tubular mesopores at different magnifications. Scale bar correspondsto 200 nm.

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Fig. 5 Diffuse reflectance spectra of SBA-15 (black line) and SBA-15–Ag (red line) (A) together with the absorption spectrum of SBA-15–Ag inKubelka–Munk units (B). Absorption spectra of film 5 (black line), 6 (red line) and 7 (blue line) (C).

Fig. 6 Ag 3d spectra of the sample SBA-15–Ag: dashed lines –synthetic components of both elements after correction of differentialcharging.

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ller percentage suitable to afford lms with macroscopicallygood properties. When the 20% SBA-15 lm was prepared,agglomeration of ller particles was obtained. Thus thispercentage was discarded and only lms with 5, 10 and 15%SBA-15–Ag (Table 1) were prepared. Diffractograms of lm 1 andlms containing AgNPs (lms 5–7) are shown in Fig. S2.† Thelm 1 diffractogram showed a very broad reection at 2 theta ca.19.9� due to the amorphous nature of chitosan.34 In lms 5–7diffractograms, among typical Ag reections, only that at 37.5�

was clearly visible, maybe because of the low silver concentra-tion. Only in the lm 7 diffractogram reection at 44.2�, alreadydescribed in the SBA-15–Ag sample, could be seen, conrmingthe presence of AgNPs.

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Fig. 7 reports the elemental mapping of C, Si, O and Ag of thelm 7 surface. The uniform distribution of the inorganic llerand consequently of the AgNPs could be observed on the lmsurface.

The absorption spectra of chitosan–SBA-15–Ag lms 5–7(Fig. 5C) presented two main bands, one centred at ca. 300 nmdue mainly to the inorganic matrix contributions and one withthe maximum at 420–430 nm, which was due to the SPR signalof AgNPs. In particular, the absorption intensity of the latterband changed with the SBA-15–Ag content in the lms. Thesimilarity of the Ag SPR band in SBA-15–Ag and in the lmsindicated that the procedure used to obtain the lms did notalter the location and the dispersion of AgNPs in the silicamatrix.

3.3 Water sorption

Water absorption capacity is an important property for a wounddressing material as it absorbs wound exudates, avoidingmaceration and enhancing wound healing.1

The water absorption capacities of the lms are shown inTable 2. Film 1 showed the highest hydration capacity reachinga maximum value of ca. 1840% aer 60 minutes. The presenceof SBA-15 slightly modied water absorption in lms 2–4. Theselms showed good hydration capacity, even if they absorbedwater more slowly and reached a maximum hydration aer 2hours. The percentage of hydration decreased with the increaseof SBA-15 percentage. The presence of SBA-15–Ag had only aslight effect on hydration. The decrease of hydration percentagewith the increase of SBA-15 concentration could be explained as

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Fig. 7 EDS images of elements (A) present on the film 7 surface and corresponding images of C, O, Si and Ag on the film 7 surface.

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some of the OH and NH2 groups interact with the silanols of theSBA-15 and thus are not available for hydrogen bonding withwater molecules.

The presence of SBA-15–Ag had only a slight effect onhydration and this is because the AgNPs are mostly inside themesopores and do not affect the interactions among watermolecules, silicate and the polymer.

3.4 Water vapor transmission rate

The WVTRs of all lms are reported in Table 2. As it can be seenWVTR values were between 1975 and 2151 g m�2 per day andthe presence of SBA-15 or SBA-15–Ag did not change the char-acteristics of the chitosan lm. The WVTR is another importantcharacteristic of wound dressings as the controlled rate of waterloss from the skin is important for wound healing. Wounddressing should avoid body liquid losses, which dramaticallyincrease in burned skin, and should maintain humidity in thewound area in order to accelerate the formation of granules andthe epithelization process. It was recommended that a rate of2000–2500 g m�2 per day would provide an adequate level ofmoisture without risk of wound dehydration.35 As shown inTable 2, the presence of SBA-15 or SBA-15–Ag did not change thecharacteristics of chitosan lms and the WVTR of all lms

Table 2 Hydration and WVTR of films 1–7

% hydration aer min

15 30 60 120 240

Film 1 1088 1452 1846 1720 116Film 2 732 762 1333 1434 102Film 3 685 765 943 1185 97Film 4 618 717 907 1016 95Film 5 711 745 1202 1298 92Film 6 636 725 904 1034 94Film 7 607 702 857 969 90

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covered the ideal range to maintain a proper uid balance onthe wound bed, which can facilitate cellular migration andenhance epithelization.

3.5 XPS analysis

As a starting point for the XPS investigation of chitosan lmswith SBA-15 and SBA-15–Ag, the reference sample of the chito-san lm was analyzed. It was characterized by the signals of C1s, N 1s and O 1s. The spectrum of C 1s was composed of fourpeaks at BE ¼ 284.8, 285.7, 286.6 and 288.3 eV, which arecharacteristic of chitosan.36,37 The same four components of theC 1s spectrum remained also in lm 7. The four components ofC 1s could be attributed to: C–H and C–C bonds at 284.8 eV, C–Nbonds at 285.6–286.0 eV, the C–O–C peak at 286.6–287.0 eV andthe N–C]O peak at 288.3–288.9 eV, respectively.36 Nevertheless,the content of Si and Ag on the surface of composite lms wasvery low: from z1 to 3% of Si and from z0.04 to 0.1% of Ag inthe SBA-15–Ag lms (lms 5–7). Namely for this reason, thecomposite samples were hydrated by immersion into deionizedwater for about 20 h, then dried in air at room temperature. Thecontent of Si on the surface increased to 5.6%, whereas theconcentration of Ag reached 0.3% (that is a 3-fold increase) inlm 7. Moreover, aer this treatment, the spectrum of Ag 3d

WVTR (g m�2 per day)aer 24 h360 480 1440

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contained only one spin–orbit doublet, with the Ag 3d5/2 peak atBE ¼ 368.6 eV, indicating the size-shi of about 0.4 eV, whichcorresponded to a AgNP size of about 6 nm.33 All the signals ofAg were increased aer the sample hydration, therefore itbecame possible to determine modied Auger parameter a0,31

which was equal to 726.3 eV and indicated the metallic state ofAg NPs. In conclusion, the presence of small NPs of Ag (z6 nm)in the composite material was conrmed by XPSmeasurements.

3.6 Mechanical properties

The mechanical properties of chitosan/SBA-15 and chitosan/SBA-15–Ag composite lms, containing from 5 to 15 wt% ller,were investigated at room conditions (T¼ 20 �C; RH 50%). In alllms, the presence of the ller did not modify the prole of thestress–strain curve of the neat polymer, as shown in Fig. S3†where the stress–strain curves of lm 3 with 10 wt% SBA-15 andlm 6 with 10% SBA-15–Ag are compared with that of the neatpolymer lm.

The Young's modulus (E) and the tensile strength derivedfrom the analysis of the stress–strain curves are shown in Fig. S4and S5† respectively for lms with different ller loadings. Theplot clearly shows that the Young's modulus of lms containingthe ller (both SBA-15 and SBA-15–Ag) increased as comparedwith that of the neat chitosan lm (from a minimum of �25%for 5 wt% to a maximum of �40% for 10 wt% higher than thecorresponding value of the neat chitosan lm), while the tensilestrength beneted from the presence of the ller mainly for 10wt% loading. The slight decrease in the strength and modulusof the composites with 15 wt% SBA-15 and SBA-15–Ag contentcould be related to a partial re-aggregation of the inorganicller. Nevertheless, the mechanical properties were still supe-rior than those of pure chitosan.

Fig. 8 The effect of films on bacterial growth was evaluated after 24 hourcontrol. Data represent the mean � SD of values obtained from three diStudent's t-test (two-tailed). *P-values of <0.05 were considered significthree independent experiments.

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Interestingly, even with the introduction of an inorganicphase, the composite lms could sustain a deformation similar,within the errors, to that of the neat polymer (Fig. S6†).

As the mechanical properties of nanocomposites depend notonly on the amount, size, shape, but also on the ability of thematrix to transfer the stress, the increase in the mechanicalproperties should be due to the intermolecular hydrogenbonding between the matrix molecules and the ller.13 Sohydrogen bonds between chitosan and SBA-15 silanols makethe loaded stress to be easily transferred to the ller. Finally itcould be observed that the presence of AgNPs did not modifythe mechanical properties of the lms and this can be explainedbecause they were mainly inside the silicate mesopores.

These ndings proved that the SBA-15 and SBA-15–Ag werewell dispersed in the polymer matrix as conrmed by EDSimages (Fig. 7) and effectively acted as a stiffener of the polymermatrix which, however, kept a good ductile behaviour up to aloading of 15 wt%. The reinforcing effects of nanoparticles onpolymers were related to the dispersion state of nanoparticles inthe matrix and their interfacial interactions favored by the highsurface area of SBA-15.

3.7 Antibacterial properties

The antibacterial properties of lms 5–7 were determined usinga disk-diffusion test (Kirby–Bauer Clinical Laboratory StandardsInstitute classication) and compared to those of lm 1 aschitosan is known to have antimicrobial activity.38 The chitosanlm (lm 1) showed antimicrobial activity against the Grampositive Staphylococcus aureus and S. epidermidis but no effectwas observed against Pseudomonas aeruginosa. Films 5–7 con-taining AgNPs showed antimicrobial activity against all testedmicroorganisms. It is worth highlighting that in these lms the

s. a A Paper disk soaked with 20 mg of gentamicin was used as a positivefferent experiments. The statistical significance was evaluated with theant. The results reported in the picture are representative of one of the

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zone of inhibition (ZOI) increased as a function of the AgNPconcentration Fig. 8.

To obtain more insight into the antimicrobial activity ofchitosan lms containing AgNPs on Staphylococcus epidermidisand Pseudomonas aeruginosa, a growth curve in the presence oflm 5 (containing the lower dose of Ag) and lms 1 and 2 as acontrol was performed. As reported in Fig. S7,† the resultsdemonstrated that both the Gram positive S. epidermidis andGram negative P. aeruginosa bacteria were highly susceptible tolm 5. In parallel we analyzed the growth curve of lm 1 andlm 2 containing only SBA-15 and both lms did not show anysignicant inhibitory activity on the growth of both bacteria.

3.8 Cell viability

The in vitro cell viability was evaluated by the use of an ATPbioluminescence kit (Via Light kit, Cambrex), in the humanskin keratinocyte cell line (NCTC 2544). All in vitro experimentswere repeated in triplicate. According to the data presented inFig. S8,† lm 1, containing chitosan, and lm 2, containingchitosan and SBA-15 5% were not cytotoxic. Considering lmscontaining AgNPs, lm 5 and 6 were not cytotoxic, while the lm7, with the higher amount of AgNPs, induced a signicantreduction of cell viability.

3.9 Silver release

In order to support the hypothesis that silver release causedimprovement of lms 5–7 antimicrobial activity, lm 7, whichwas loaded with the highest amount of AgNPs and providedwith the best antimicrobial activity, was submitted to in vitrosilver release for 21 days in phosphate buffer 7.4 saline solution.The cumulative released Ag amount (mg g�1 of specimen) isreported as a function of time in Fig. 9. The Ag release proleshowed an initial burst effect in the rst hour, maybe due to thesilver release from the few AgNPs present close to the lmsurface, then, aer a plateau until 24 h, silver release slowlyincreased. This was maybe due to lm hydration that allowedthe slow AgNP diffusion towards the lm surface and then thesilver release. As shown by XPS analysis, before hydration Agcontent on the lm 7 surface was very low (0.1%) and then, aer

Fig. 9 Silver release from film 7.

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lm swelling, the presence of metallic AgNPs on the lm surfaceincreased (0.3%) and this allowed a slow silver release.However, considering the low amount of silver released, itcannot be excluded that the lms exert antimicrobial activityboth by the release of silver ions and by direct contact betweenthe lm surface containing silver and the microorganisms.

4. Conclusions

In the current study chitosan lms containing AgNPs connedwithin the channels of the mesoporous silicate SBA-15 havebeen described. AgNPs were obtained by following a simpleapproach, which furnished homogeneous size nanoparticles.Films, obtained by dispersing SBA-15–Ag in chitosan, showedgood mechanical properties, water absorption, vapor trans-mission properties and antimicrobial activity and, with theexception of lm 7 with the highest amount of AgNPs, lowcytotoxicity.

It is remarkable to point out that the prepared lms con-tained a very low silver concentration (ca. 0.22% w/w, that is7.73 mg cm�2 for lm 5, 0.44% w/w, that is 17.39 mg cm�2 forlm 6 and 0.59% w/w, that is 26.14 mg cm�2 for lm 7), less thanthat of most commercial wound dressings.3,39

This has two important advantages, rst a reduced amountof silver dispersed in the environment aer dressing removal,secondly an exposure to low silver doses with improvement ofthe dressing safety. Moreover, the connement of silver into themesopores avoids the direct contact between nanoparticles andwound reducing the possible toxicity of nanoparticles. Thus,this research work suggests that the proposed lms could besuitable for biomedical applications.

Acknowledgements

This work was partially performed at the University of PerugiaLaboratory for nanomaterials (LUNA).

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