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Analytical and BioanalyticalChemistry ISSN 1618-2642Volume 400Number 2 Anal Bioanal Chem (2011)400:547-560DOI 10.1007/s00216-011-4757-1

Structural and biological evaluation of amultifunctional SWCNT-AgNPs-DNA/PVAbio-nanofilm

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ORIGINAL PAPER

Structural and biological evaluation of a multifunctionalSWCNT-AgNPs-DNA/PVA bio-nanofilm

Ramesh P. Subbiah & Haisung Lee &

Murugan Veerapandian & Sathya Sadhasivam &

Soo-won Seo & Kyusik Yun

Received: 2 December 2010 /Revised: 1 February 2011 /Accepted: 1 February 2011 /Published online: 19 February 2011# Springer-Verlag 2011

Abstract A bio-nanofilm consisting of a tetrad nano-material (nanotubes, nanoparticles, DNA, polymer) wasfabricated utilizing in situ reduction and noncovalentinteractions and it displayed effective antibacterial activityand biocompatibility. This bio-nanofilm was composed ofhomogenous silver nanoparticles (AgNPs) coated on single-walled carbon nanotubes (SWCNTs), which were laterhybridized with DNA and stabilized in poly(vinyl alcohol)(PVA) in the presence of a surfactant with the aid ofultrasonication. Electron microscopy and bio-AFM (atomicforce microscopy) images were used to assess the mor-phology of the nanocomposite (NC) structure. Functional-ization and fabrication were examined using FT–Ramanspectroscopy by analyzing the functional changes in thebio-nanofilm before and after fabrication. UV–visiblespectroscopy and X-ray powder diffraction (XRD) con-firmed that AgNPs were present in the final NC on the basisof its surface plasmon resonance (370 nm) and crystalplanes. Thermal gravimetric analysis was used to measurethe percentage weight loss of SWCNT (17.5%) and finalSWCNT-AgNPs-DNA/PVA (47.7%). The antimicrobialefficiency of the bio-nanofilm was evaluated against majorpathogenic organisms. Bactericidal ratios, zone of inhibi-

tion, and minimum inhibitory concentration were examinedagainst gram positive and gram negative bacteria. Apreliminary cytotoxicity analysis was conducted usingA549 lung cancer cells and IMR-90 fibroblast cells.Confocal laser microscopy, bio-AFM, and field emissionscanning electron microscopy (FE-SEM) images demon-strated that the NCs were successfully taken up by the cells.These combined results indicate that this bio-nanofilm wasbiocompatible and displayed antimicrobial activity. Thus,this novel bio-nanofilm holds great promise for use as amultifunctional tool in burn therapy, tissue engineering, andother biomedical applications.

Keywords Hybrid . Characterization . Antibacterial .

Cytotoxicity . Cellular uptake . Skin film

Introduction

Burns are a serious issue because incidences occur widelyas a result of the use of fire, electricity, and chemicals.Burns can cause severe damage to different tissues and thelevel of damage depends on the degree of classification.Morbidity and mortality rate are directly proportional to theincrease in the degree of the burn, and improper treatmentcan result in amputation [1]. All burn classifications can becomplicated by shock, infection, multiple organ dysfunc-tion, and respiratory distress, and these factors can result indestruction of tissues and increase the risk of infection,which is exacerbated by immunosuppression [2, 3]. Onestudy reported that 90% of all burns occur in developingcountries where survival is rare when the burns cover morethan 40% of the total body surface area [4]. Studies in the1990s examined the relationship between the hospital stayand infection risk in burn patients. In these studies,

Electronic supplementary material The online version of this article(doi:10.1007/s00216-011-4757-1) contains supplementary material,which is available to authorized users.

R. P. Subbiah :M. Veerapandian : S. Sadhasivam :K. Yun (*)College of Bionanotechnology, Kyungwon University,Gyeonggi-do 461-701, South Koreae-mail: ykyusik@kyungwon.ac.kr

H. Lee : S.-w. SeoInterdisciplinary Graduate Program of Biomedical Engineering,School of Medicine, Sungkyunkwan University,50 Irwon-Dong( Gangnam-Gu( Seoul 135-710, Korea

Anal Bioanal Chem (2011) 400:547–560DOI 10.1007/s00216-011-4757-1

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Staphylococcus epidermidis was shown to be the mostcommon organism isolated within the first 24 h in burns. S.aureus was shown to be the most predominant organism inthe first week, and gram negative pathogens such asPseudomonas, which is the most dangerous cause of deathin burn patients, dominated after 8 days [2, 5, 6]. Theseinfections are dominant in burns and can also causesepticemia, bronchopneumonia, and pyelonephritis.

Because of the risk of pathogen infection, damaged skinneeds to be covered using dressings to prevent complica-tions. The preferred covering for burns is an autograft;however, patients may not have enough available naturalskin to transplant and it may get immediately rejected bythe body. The alternative is to use artificial skin bandages or

films according to the severity of the burn (Scheme 1). Thisfilm should have effective antimicrobial activity; should beinert and biocompatible; should reduce the rate of resorp-tion by the body; should have foam-like architecture tomimic the layers of the skin; should be wet and draped overthe wound; and has to be hydrophilic in nature so that it canbe easily washed from the skin after treatment. In addition,the film should be able to deliver therapeutics or proteinsand have the ability to uphold the autograft [7]. Hence, inthis study, we developed a novel film to fulfill theaforementioned criteria.

Though effective antibiotics are available to treatinfections, prophylaxis in burns has been widely studiedto prevent post-nosocomial infection in burns. A number of

Scheme 1 Criteria for the arti-ficial skin film to be effective.The model of the leaching andtransportation mechanism ofnanomaterials from the film tothe cells. Illustration of theantimicrobial mechanisms ofAgNPs, factors affecting itsactivity (above the skin), andDNA delivery for therapy(below the skin). Possibletoxicity of film is also shown.(Mechanism derived from theliterature). RES reticuloendothe-lial system

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special precautions may be stressed and attention shouldcenter on the three major pathogens described above.Recently, research has focused on finding new and effectiveantibacterial agents because the number of infectiousdiseases and antibiotic resistance pathogens has beenincreasing [8]. Silver nanoparticles (AgNPs) and hybridmaterials have unique physicochemical and effectiveantibacterial properties; thus, they can be used to developcost-effective antimicrobial agents against bacteria, viruses,and fungi [8–11]. In addition, because of the antiangiogenicproperties of AgNPs, they have also been used as anti-inflammatory agents and in cancer treatment [12]. Impor-tantly, control over size, shape, and stability of AgNPs isvery important for successful application, and stabilizationcan be achieved by using a carrier [13]. Single-walledcarbon nanotubes (SWCNTs) are the best choice as a carrierbecause of their excellent chemical and physical properties.Several studies have reported on methods to synthesisSWCNT-AgNPs with dramatic improvement in physico-chemical properties particularly in terms of their surfaceand surface area [14]. These AgNPs-functionalizedSWCNT can readily undergo chemical reactions, andtherapeutics can be conjugated to the hybrid.

Binding biomolecules such as DNA and protein to thehybrid will allow them to act as a carrier for gene therapyapplications and for producing artificial muscle [15].Biofunctionalization is a pertinent technique to increasethe biocompatibility of SWCNT-AgNPs for biomedicalapplications [16]. In this regard, we considered conjugatingDNA to the SWCNT-AgNPs via noncovalent interactions.Stabilization of this SWCNT-AgNPs-DNA (hybrid) can be

achieved by introducing them into the appropriate polymermatrix. Poly(vinyl alcohol) (PVA) is the best choice as apolymer matrix because of its biocompatibility and excel-lent properties such as high water solubility, low cytotox-ocity, easy processabiity, and high transmittance [17]. Theincorporation of a networked hybrid into the PVA matrixcan significantly improve mechanical strength and waterresistance [18]. Moreover, PVA has been used in severalbiomedical applications such as developing artificial pan-creas, hemodialysis, and medical materials [19–24].

Several studies on burn dressings containing AgNPshave been reported [10, 25]; other related studies concernedsilver-doped thin silica films formed via the sol-gel process[26], AgNPs-coated PVA thin films [27], a nanofibrousscaffold containing silver for tissue engineering applica-tions [28], and production of a silver nanocoated fabric fora biomedical antibacterial dressing [11]. However, theproduction of these silver-doped thin films requiredmultiple steps and complex reagents and was expensive;moreover, these films also can cause new epithelial tissuedamage, severe pain, and could only be used in a fewapplications. Hence, there is a need to develop novelmethods to fabricate silver-coated films with multifunction-al applications.

In this study, a strategy was developed to incorporateAgNPs into SWCNT by in situ reduction under ultra-sonication and to conjugate DNA via noncovalent bonding,which was further stabilized in a PVA film to be used as amultifunctional bio-nanofilm for biomedical applications.The method used to fabricate the film and its appearanceare shown in Scheme 2. The antimicrobial activity and

Scheme 2 Schematic representation of the stepwise process for the fabrication of the bio-nanofilm, and photographic images of the bio-nanofilm

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biological properties of the films were also examined.Interest in the preparation of such films is driven by theirpossible application in the biomedical field particularly inburn therapy as a prophylactic, a scaffold for tissueengineering, and therapeutic delivery. Thus, this multifunc-tional bio-nanofilm shown in Scheme 2 holds promise foruse as a covering/antimicrobial protection film and in bio/therapeutics delivery to regenerate the damaged tissues inburn patients.

Experimental

Fabrication of bio-nanofilm

SWCNT, AgNO3, NaBH4, sodium dodecyl sulfate (SDS),DNA (D6898), and Mueller–Hinton agar were purchasedfrom Sigma. Acetone and PVA were obtained from JunseiChemicals. A 20-mg portion of SWCNT was placed in areflux condenser containing 15 mL of 2.6 M HNO3 andheated at reflux for 48 h. The carboxyl-functionalizedSWCNT was centrifuged and the supernatant was decanted.The sediment was washed several times in deionized (DI)water to completely remove the by-products. At eachcleaning step, the sediment was suspended in a sonicationbath and recovered again by centrifugation. The formedSWCNT-COOH was dispersed in acetone, mixed with the1 mM AgNO3 precursor, and magnetically stirred for 2 h.The reducing agent, 6 mM NaBH4, was slowly added to themixture using a syringe pump at a rate of 10 mL/h duringstirring and this was continued for 6 h. The synthesizedSWCNT-AgNPs were purified several times as mentionedpreviously and placed in a beaker to which 2 mg of SDSwas added and ultrasonicated for 2 h. A 2-mg portion ofDNA in DI water was added to the above sample andultrasonicated for 1 h. A 500-mg aliquot of PVA in waterwas added to the hybrid and ultrasonicated for 1 h to obtaina uniform solution. The SWCNT-AgNPs-DNA/PVA nano-composite (NC/bio-nanofilm) was formed by evaporation atroom temperature.

Morphological characterization

Images of the samples were obtained by transmissionelectron microscopy (TEM; JEOL-1010), bio-AFM (bio-atomic force microscopy; Nanowizard II, JPK), and fieldemission scanning electron microscopy (FE-SEM; JEOL7500F). The sample was air dried and sputter coated withplatinum before examination by FE-SEM. The cross-sectional view of the film was observed using a focusedion beam FIB-SEM (FEI Nova Nanolab dual-beam sys-tem). Cell transfection images were obtained using aconfocal microscope (Nikon C1), bio-AFM, and FE-SEM.

Analytical characterization

The functional groups and composition of the fabricatedbio-nanofilm were characterized by FT–Raman spectrosco-py (Vertex 70 with FT–Raman; Bruker). UV–Vis spectra ofthe sample were measured in an Optizen 3220 (Mecasys)using a 100-μL cell at room temperature. The X-raypowder diffraction (XRD) patterns were recorded on a StoeSTADI-P2. The purity was determined by the energydispersive X-ray analysis (EDXA) combined with the FE-SEM (S-4700 Hitachi). The thermal stability of the samplewas characterized using a thermogravimetric analyzer (SDTQ600) at a scan rate of 10 °C/10 min under N2 atmospherein the temperature range 25–600 °C. Contact angle wasdetermined using a Kruss DSA 10-MK2 and analyzed withdrop shape analysis software.

Antimicrobial study

Bacterial strains

The antimicrobial activity of the fabricated films was testedagainst seven pathogenic bacterial strains: Staphylococcusaureus KCTC 1916, Enterococcus faecalis KCCM 13807,Bacillus subtilis KCTC 1021, Staphylococcus epidermidisKCTC 1917, Escherichia coli KCTC 1682, Salmonellatyphimurium KCCM 40253, and Salmonella entericaKACC 10763.

Film applicator coating assay (FAC)

A FAC assay was used to measure the bactericidal ratio ofthe prepared film. In this assay, bacterial cultures weredirectly applied or coated on the film and incubated, thelog-phase bacterial cells were washed with phosphate-buffered solution (PBS), and adjusted to 1×107 CFU/mL.The prepared films were placed on sterile plates. A 100-μLaliquots of each bacterial suspension were added to thecontrol group (bio-nanofilm) and blank group (CNT-DNA/PVA). The plates were incubated at 37 °C for 24 h, thebacterial suspensions on the plate were then washed with10 mL sterilized PBS, and vortexed for 5 min. The viablecells were enumerated after serial dilution, which wasfollowed by plating on Mueller–Hinton agar. The plateswere incubated for 48 h; the viable cells were counted andexpressed as colony forming units (CFU/sample). Theantibacterial effect in each group was represented by thebactericidal ratio, which was calculated as follows:

Bactericidal ratio %ð Þ ¼ CFUblank group � CFUcontrol group

CFUblank group

� 100:

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Disc diffusion assay

The antimicrobial activity of the fabricated films was alsoexamined using the disc diffusion assay method. Eachbacterial strain grown in LB broth was swabbed on thesurface of Mueller–Hinton agar plates. Filter paper discs(Fisher Scientific, MO) saturated with 30 μg of theSWCNT-DNA/PVA (blank), bio-nanofilm (control), andkanamycin (standard) samples were placed on the plate.They were incubated at 37 °C overnight and the diametersof the clear zones around the discs, called zones ofinhibition (ZOI), were recorded.

Micro titer broth dilution method

This method was used to determine the minimum inhibitoryconcentration (MIC) of the film [29]. Inocula were preparedby suspending growth from overnight cultures in sterile LBmedia. The samples and standards were serial dilutedtwofold in the 96-well plates. Approximately 107 CFU/mL cells were inoculated, where the final volume in eachmicrowell plate was 0.2 mL, and incubated at 35 °Covernight. After incubation, the microwell plates were readat 590 nm using an ELISA reader prior to and afterincubation to determine the MIC values. The MIC isdefined as the lowest concentration of the compound thatinhibited 90% of the bacterial growth.

Cytotoxicity evaluation

Cell culture

Human alveolar basal epithelial cells (A549) and fibroblastcells (IMR-90) were provided from the Korea Cell LineBank and were used for the in vitro study to examine thetoxicology of the NC. At day 1, 1.0×106 cells were placedin each well of a 96-well plate in 500 μL of Dulbecco’smodified Eagle’s medium containing 10% fetal bovineserum (FBS) and cultured for 24 h at 37 °C.

XTT assay

A 50-μL aliquot of the colloidal solution of NC in PBS (pH7.0) was added to the cell culture plate and incubated. Thefinal concentration of the samples was ranged from 10×10−3 to 1.0 mg/mL. SWCNT-DNA/PVA and the bio-nanofilm contained no fluorescent dye molecules so as tonot interfere with the interactions between the NCs and theXTT tetrazolium salt, which was used as the detectionreagent. XTT solution was added to the plate wells, and thecells were then incubated at 37 °C for 4 h. During thisincubation period, the living cells converted the tetrazoliumcomponent of the dye solution into a formazan product,

which absorbed UV light at wavelengths of 470 nm and560 nm.

Confocal laser scanning microscopy

Rhodamine B isothiocyanate (RITC) was conjugated to thebio-nanofilm using the following procedure. A 100 μg/mLcolloidal solution of bio-nanofilm was placed in V-shapedsmall volume vial and then a 5 mM RITC solution in dimethylsulfoxide (DMSO) was added to the solution, which had a pHbetween ca. 7.4 and 8.0. The reaction mixture was stirred atroom temperature for 12 h in the dark. The mixture was thenwashed with dichloromethane twice, and the aqueous colloidalsolution was separated by dialysis (MWCO: 50K) for 2 days.A549 cells and IMR-90 cells were cultured in RPMI 1640medium (Invitrogen) supplemented with 10% heat-inactivatedFBS and 1% antibiotics. Cells were routinely cultivated at37 °C in a humidified atmosphere of 5%CO2. A549 cells wereseeded in a 12-well culture plate at a density of 2×105 cells/well and allowed to attach overnight. The nucleus was stainedblue with 4′,6-diamidino-2-phenylindole (DAPI) and thecomplex formed with RITC-labeled bio-nanofilm appearedred. Transfection was carried out by adding the RITC-labeledNC to the wells in a dropwise manner. After 4-h incubation,the medium was exchanged again with fresh medium andtransfected cells were incubated for 24 h.

Statistical analysis

Each group was compared using the independent Student’st test. For all analyses, the probability of type I error lessthan or equal to 0.05 was considered statistically signifi-cant. Each test was repeated five separate times.

Results and discussion

In this study, we developed a novel method to fabricate a bio-nanofilm (Scheme 2) that displayed antimicrobial activityand could be utilized as a multifunctional tool in biomedicalapplications based on tetrad materials (nanotubes, nano-particles, DNA, polymer). The bio-nanofilm was systemat-ically synthesized and the DNA was successfully preservedby evaporation at room temperature. The biocompatibility,antibacterial activity, and cellular uptake of the bio-nanofilmwere examined and, on the basis of the results, this novelbio-nanofilm holds promise for use in burn therapy, as ascaffold in tissue engineering, and delivery system.

Structural characterization

The functional groups in the film were confirmed by FT–Raman analysis. The single plotted curve clearly demon-

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strated the presence of the COOH group in the SWCNT.The band at 1,591 cm−1 was assigned to the G band of theSWCNTs, which was due to the graphite Raman signaturein the plane E2g vibration mode (Electronic SupplementaryMaterial Fig. S1a). The band at 1,277 cm−1 confirmed thepresence of the D band, and the second harmonic D bandwas observed at 2,595 cm−1. The D band is generallyattributed to defects in the curved graphite and tube ends[30, 31]. Additionally, the disappearance of the peak at2,705 cm−1 demonstrated that the AgNPs were incorporated,which was also confirmed by the decrease in the G band andincreased intensity of the D band. The band at 1,508 cm−1

was attributed to the base electronic structure in the DNAfragments due to stacking, ligation, etc. The band at1,158 cm−1 was due to the PO2− interaction with the DNAbackbone [32]. The guanine nucleoside marker between 620and 685 cm−1 was due to the sugar pucker and glycosyltorsion, and the band near 727 cm−1 represented adenine.Bands in the region between 800 and 1,100 cm−1 aregenerally sensitive to the backbone geometry and secondarystructure [33, 34]. The presence of SWCNTs and DNAchemical fragments was verified from these results. Thecharacteristic vibration modes of the PVA polymer werealso observed. The C–C stretching modes at 855 cm−1

(C–O stretch), 1,092 cm−1 (O–H bends), 1,147 cm−1

(C–O, C–C stretch), 1,447 cm−1 (C–H, O–H bend), andat 1,747 cm−1 (CO stretch) were observed [35]. Overall,the Raman spectrum demonstrates that the AgNPs wereimmobilized on the SWCNT-COOH and DNA wasconjugated to the particles; thus, the bio-nanofilm wassuccessfully fabricated.

UV spectra of the NC sample exhibited spectral peaksknown as Van Hove singularities (Electronic SupplementaryMaterial Fig. S1b), which result from the electronic structureand are responsible for electronic conduction. The decreasein the intensity of the Van Hove singularities resulted fromfunctionalization of the SWCNT. The first pair of Van Hovetransitions, which was observed in the peaks centeredbetween 500 and 660 nm, was due to the singularitytransition of the semiconducting SWCNTs [36, 37]. Thepresence of AgNPs in the SWCNTs can be confirmed by themaximum absorption peak observed at 370 nm, whichcorresponds to the characteristic surface plasmon resonanceof covalently incorporated AgNPs. It is worth noting that themaximum absorption peak was observed at 275 nm, whichconfirms that DNA formed a layer around the SWCNT-AgNPs. This occurred because of the variation in the bondsbetween the bases of guanine and adenine under the partialunfolding of the DNA double spiral [38]. The surfactant SDShelped the DNA wrap around the SWCNT-AgNPs andprevented re-aggregation.

The strong signals in the EDX spectrum obtained fromFE-SEM further verified that the AgNPs were immobilized

on the SWCNTs surface and DNA was present on theSWCNT-AgNPs (Electronic Supplementary MaterialFig. S2). Peaks for carbon, silver (AgNPs), nitrogen, andphosphorus (DNA bases) were observed in the EDXspectrum. Whereas the SWCNT-COOH had peakscorresponding to carbon, oxygen, and silica, platinumpeaks were due to the glass cover slip and sputter coatingused for the analysis. The missing peaks corresponding toAg, N, and P in the SWCNT-COOH clearly demonstratedthat the AgNPs were successfully incorporated. Moreover,from the EDX pattern, the percentage loading of therespective metals in the SWCNT surface was determinedto be 29.5 wt% by using the carbon peaks as a reference.

The XRD profile of the crystal plane of the nano-materials in the film is shown in Electronic SupplementaryMaterial Fig. S1c. The major diffraction peaks at ∼38.10°,∼44.25°, ∼64.45°, ∼77.40°, and ∼81.50° corresponded tothe (111), (200), (220), (311), and (222) planes of the Agphase, and no silver oxide peaks were observed. A humpwas observed around 34°, which corresponded to thesignature for reflections from (101), (102), and (301)planes, and the small peak observed around 2θ ∼ 89°represented the reflections from the (200) planes of carbon[39]. The diffraction pattern showed peaks for PVAmolecules at 19.3° and 22.7° that were assigned as (101)and (200), respectively, which resulted from the crystallin-ity in the PVA polymer [40].

The surface properties of the film were characterized bywater contact angle. The average water contact angle wasfound to be 65±5° (Electronic Supplementary MaterialFig. S3), indicating that the polymer-conjugated hybrid wasmoderately hydrophilic. The thermal stability of thefabricated film was examined using thermal gravimetricanalysis (Electronic Supplementary Material Fig. S4). Amoderate weight loss was observed for the fabricated bio-nanofilm. The weight loss observed for the SWCNT-AgNPs and bio-nanofilm was 17.5% and 47.7% for thisregion (0–600°). The first-stage thermal reduction temper-ature was observed in the low temperature region around64.23 °C. This was attributed to the dehydration reaction,which leads to the evaporation of weakly adsorbed watermolecules 1.0623% (0.006 mg). The second weight lossobserved at around ∼136.3 °C was ascribed to thedecomposition of the carboxyl groups. The weight loss atthis stage was nearly 6.05% (0.0325 mg). In the third stage,a weight loss of 6.05% (0.325 mg) was observed at∼254.3 °C, which may be from the oxidation of AgNPsto form silver oxides. The fourth weight loss (4.321%,0.0232 mg) around 366.8 °C was attributed to the lessstable amorphous carbon. The SWCNT-AgNPs displayedgood thermal stability, indicating that their molecularstructure was maintained well. In the case of the film, thefirst thermal reduction was observed at 349.33 °C,

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Fig. 1 Bio-AFM, FE-SEM, and TEM images of SWCNT (a, e, i), SWCNT-AgNPs (b, f, j), hybrid (c, g, k), and bio-nanofilm (d, h, i),respectively

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indicating that the sample was anhydrous and highly stable.The weight loss of 2.11 mg (30%) in the second stage wasdue to a loss of amorphous carbon in the SWCNT,oxidation of AgNPs, and deformation of PVA molecules[41]. Furthermore, a maximum total weight percentage losswas observed between 350–550 °C. This was due to theoxidation of DNA bases and destruction of PVA moleculesin the film, which ensures the successful functionalizationof the fabricated bio-nanofilm. However, the final bio-nanofilm was thermally stable up to 350 °C, which makes itsuitable for biomedical applications under high thermalconditions.

Film morphology

Figure 1 shows the bio-AFM, FE-SEM, and TEM imagesof SWCNT (a, e, i), SWCNT-AgNPs (b, f, j), hybrid (c, g,k), and bio-nanofilm (d, h, l) respectively. These imagesclearly show the smooth surface of SWCNT, sphericalshaped NPs on the SWCNT after functionalization, suc-cessful conjugation of DNA to the SWCNT-AgNPs andtheir systemic distribution in the film. Furthermore, darkhybrids in the PVA film were observed in these images,which proves that the AgNPs were strongly conjugated tothe SWCNT and the hybrid was tightly adhered to the PVA.Also, the FIB-SEM images indicate that the dispersed metalNPs bound to the SWCNT were incorporated into thepolymer matrix. The SWCNT-AgNPs were clearlyobserved in the film and were shown to be homogenouslydistributed (Electronic Supplementary Material Fig. S5).Bio-nanofilm had a distinguished morphology compared toprevious unreacted SWCNT and SWCNT-AgNPs. This isin good agreement with bio-AFM (3D images shown inElectronic Supplementary Material Fig. S6a–d), FE-SEM,

and TEM analysis. Also, the AgNPs on the SWCNT werefound to be 6±3 nm in diameter (Fig. 1b, f, j), and theappearance of cloudy mass (Fig. 1c, g, k) around theSWCNT-AgNPs was indicative of successful DNA conju-gation. Thus, the hybrids were shown to be well dispersedand uniformly distributed in the bio-nanofilm, which provesthat the height and length distribution was consistent.

Antibacterial activity

Formation of a biofilm occurs when bacteria attach to thefilm, which eventually leads to the movement of bacteriainto the interior region of the film. Because of this, thebacteria are protected against phagocytosis and antibiotics;thus, they are far more resistant to first-generation antimi-crobial therapy. Therefore, it is very important to evaluatethe antimicrobial properties of the film. In this study, FACwas used to measure the antibacterial activity of the film. Inthe FAC assay, the fabricated film’s surface exhibitedmoderate to strong antibacterial activities. Figure 2a showsthe CFU of the microorganisms in the presence of thecontrol (bio-nanofilm) and blank (SWCNT-DNA/PVA).Figure 2b shows the bactericidal ratio of the film againstthe major pathogenic organisms examined in this study: (1)S. aureus, 72.89%; (2) E. faecalis, 69.31%; (3) B. subtilis,57.78%; (4) S. epidermidis, 71.76%; (5) E. coli, 70.91%;(6) S. typhimurium, 62.85%; (7) S. enterica, 70.53% ofbacteria in media were inhibited on the film surfacecompared to the blank.

We evaluated the antibacterial activity of the blank,control, and standard (kanamycin) on the basis of the ZOIand MIC of dispersed NPs in batch cultures. The discdiffusion assay method was used to examine the growthinhibition of a variety of bacterial species. Figure 3 shows

Fig. 2 Inhibition of bacterial growth on the bio-nanofilm surface. aColony forming units of SWCNT-DNA/PVA (blank) and bio-nanofilm(control) film surfaces after 24-h incubation. b Bactericidal ratio of the

bacteria in relation to bio-nanofilm. 1, S. aureus; 2, E. faecalis; 3, B.subtilis; 4, S. epidermidis; 5, E. coli; 6, S. typhimurium; 7, S. enterica

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the mean diameters of growth inhibition; the ZOI for thecontrol was (1) 17.33 mm, (2) 13.26 mm, (3) 12.4 mm, (4)14.5 mm, (5) 18.5 mm, (6) 16.5 mm, and (7) 17.4 mm.Growth inhibition was clearly observed for the controlgroup, which was compared with the standard, whereas nogrowth inhibition was observed for the blank not containingAgNPs. This result is consistent with other reports and wefound that the NC inhibited both gram positive and gramnegative bacteria [42].

The microtiter plate method was used to study the MICprofile against various pathogens. Blank, undiluted control,and kanamycin were used for this antimicrobial screening.Table 1 shows the list of bacteria and their MIC values. Theantibacterial mechanism of the film was attributed to theAgNPs attached to the SWCNT and release of silver ionsfrom the film. The AgNPs provided an extremely largesurface area for interaction with bacteria in all directions

due to the 3D exposure of the NPs on the SWCNT of thefilm, which provided better contact with microorganismsand enhanced antimicrobial activity (Scheme 1). Thepositively charged silver ions that were released from thesurface of the hybrid of the bio-nanofilm attached to the cellmembrane and penetrated into the bacteria. The silver ionsalso strongly bind to electron donor groups in biologicalmolecules containing sulfur, oxygen, or nitrogen. Pharma-cological investigations into the mechanisms of silveragainst pathogens demonstrate that bacterial inactivationoccurs and bacterial replication in vitro is prevented leadingto bacterial death. This process involves the interaction ofsilver ions with microbial DNA and disulfide or sulfhydrylgroups on metabolic enzymes in the bacterial electrontransport chain, causing physiological changes that lead tothe disruption of bacterial metabolic processes, which resultin death [43, 44]. The inhibitory action of AgNPs is alsobased on the release of silver ions [45]. The increasedpotential for the release of silver ions results in accumula-tion in the bacterial cytoplasmic membrane, and thegeneration of free radicals from the surface of NPs causesdamage to the membrane which results in bacterial death[46–48]. Scheme 1 illustrates the process of AgNPsleaching from the film and the interaction of silver ionswith the bacterial cell wall, toxicity, and factors influencingantibacterial activity of the film. As the concentration ofNC was increased to the MIC of the respective strains, noabsorbance was measured in the 96-well plates. Among theseven strains selected for the study, maximum growthinhibition was observed for S. aureus, S. epidermidis, E.coli, and S. enterica. The MICs for these strains rangedfrom 32 to 64 μg/mL, which were equal to the MICsobtained using standard antibiotics. The MIC of the filmagainst S. aureus was 32 μg/mL; thus, this bio-nanofilmcan be used as a prophylactic in burn infection because S.aureus is the most predominant pathogen in the first weekof the burn. The MIC value against gram negativepathogens was very low, which indicates that this film willprevent septicemia because gram negative pathogens are

Table 1 MIC of the blank, control, and standard against pathogenic bacterial strains after overnight incubation

Bacterial strains Blank MIC (μg/mL) Control MIC (μg/mL) Standard MIC (μg/mL)

Staphylococcus aureus KCTC 1916 >512 32 8

Enterococcus faecalis KCCM 13807 >512 128 16

Bacillus subtilis KCTC 1021 >512 256 8

Staphylococcus epidermidis KCTC 1917 >512 32 8

Escherichia coli KCTC 1682 >512 32 8

Salmonella typhimurium KCCM 40253 >512 256 8

Salmonella enterica KACC 10763 >512 64 16

Blank SWCNT-DNA/PVA; Control bio-nanofilm; Standard kanamycin

Fig. 3 Growth inhibition of SWCNT-DNA/PVA (blank), bio-nanofilm (control), and kanamycin (standard) against 1, S. aureus; 2,E. faecalis; 3, B. subtilis; 4, S. epidermidis; 5, E. coli; 6, S.typhimurium; 7, S. enterica

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predominant in the second week of the burn. In addition,nosocomial infection can also be prevented by using thisfilm because hospital instruments and appliances are themajor cause of infection in burn patients. Collectively,dissolution and release of silver ions from the film(Scheme 1) inhibit respiratory enzymes, reactive oxygenspecies (ROS) production, and disruption of membraneintegrity and transport processes [49, 50].

Cellular uptake profiles

The cells were exposed to NCs as described in the“Experimental” section. Figure 4 shows the bio-AFM andFE-SEM images of A549 and IMR-90 cells before and aftertransfection. Figure 4a, e, c, g shows images of the A549and IMR-90 cells before transfection and Fig. 4b, f, d,h shows images of the A549 and IMR-90 cells after

Fig. 4 Bio-AFM and FE-SEM images of A549 cells before (a, e) and after transfection (b, f) and IMR-90 cells before (c, g) and after transfection(d, h). Highly magnified images are shown on the right side of the corresponding images

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transfection. Most importantly, significant morphologicalchanges were observed (3D images of NC transfected cellsare shown in Electronic Supplementary Material Fig. S6e, f,g, h). Both before and after transfection, NCs wereobserved in the cytosol area of the cells. The hybridinteracts with medium proteins and cells surface receptorsto form a complex that can help NC uptake into the cells.The uptake of NC in A549 cells was higher when comparedto IMR-90 cells due to the increased charge on the surfaceof the hybrid [51]. NC entering into the cells was alsoevaluated using confocal laser microscopy 4 h aftertransfection. Figure 5 shows confocal images of A549 cellsand IMR-90 cells that were transfected with RITC (redfluorescence dye)-conjugated NC. The nucleus was stainedwith DAPI (blue fluorescence dye). The differentialinterference contrast (DIC) image, RITC excitation image,DAPI excitation image, and merged image of A549 cellsare shown in Fig. 5a–d, respectively, and the DIC image,RITC excitation image, DAPI excitation image, and mergedimage of IMR-90 cells are shown in Fig. 5e–h, respectively.RITC-conjugated NC were located throughout the entirecell, indicating efficient cellular uptake of NC. Based on thebio-AFM, SEM and confocal microscopic analysis, weconfirmed that NC was successfully transfected intomammalian cells and we think that these results will alsobe applicable to malignant cells for cancer treatment andnormal fibroblast cell for diagnostics (Scheme 1).

Cytocompatibility

In this study A549 cells and IMR-90 cells were used to testthe cytotoxicity of the hybrid in the bio-nanofilm.Figure 6b, d shows the viability measured using the XTTassay of A549 and IMR-90 cells cultured for 1 day with theNC solution. In these experiments, a low toxicity of NCwas observed against A549 and IMR-90 cells. The toxicitywas evaluated by comparing the control (bio-nanofilm)with the blank (SWCNT-DNA/PVA) using the same cellsdescribed above, and the results clearly show that the blankwas more toxic than the control, whereas only a low levelof toxicity was observed in the control group for both celltypes (Fig. 6b, d). For both cells, an increase in viabilitywas observed at decreased NC concentrations. Severalstudies reported that SWCNT or MWCNT were cytotoxicto mammalian cells. SWCNTs or MWCNTs have ahydrophobic surface due to the sp2 carbon (π-electron-richstructure), which is highly toxic. However, carboxylation ofSWCNT produced a hydrophilic surface, reduced thetoxicity of SWCNT, and made it easy for conjugation ofAgNPs. Thus, conjugation of AgNPs to the carboxylSWCNT further diminished the toxicity of SWCNT. Also,the SWCNT size increased after incorporation of AgNPsand its surface was modified with DNA, which was further

stabilized with PVA. Furthermore, bio-functionalization andPVA stabilization reduced the toxicity of the hybrid. Thefinal novel structure of the bio-nanofilm displayedimproved properties with diminished toxicity, which is inagreement with previous studies [51, 52]. The lowertoxicity may have originated from several species such asreactive oxygen species (ROS), DNA or RNA breakage ofprotein or cytokine in the cells, which is described inScheme 1.

Fig. 5 Confocal laser scanning microscopy images of A549 andIMR-90 cells 4 h after transfection with the nanocomposite. DICimages (a, e), RITC (red) excitation images (b, f), DAPI (blue)excitation images (c, g), merged images (d, h)

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The toxicity of AgNPs was also evaluated by comparingthe control with the blank, and the results clearly show thatthe blank exhibited a higher toxicity (Fig. 6a, c). Thisfurther proves that the AgNPs were not toxic. The decreasein toxicity of the AgNPs may be due to the larger size andhigher structural and superfluous functions of eukaryoticcells relative to prokaryotic cells. Eukaryotic cells requirehigher silver ion concentrations to produce comparabletoxic effects observed in bacterial cells [53]. The bacterialcells are successfully attacked by the silver ions, whereasharmful effects on eukaryotic cells require higher silver ionconcentrations. As reported in this study, no cell cytotox-icity with a high antibacterial activity against S. aureus, S.epidermidis, E. coli, and S. enterica was observed for the

bio-nanofilm containing AgNPs. Also, macrophage engulf-ment of SWCNT could be exploited for targeted delivery oftherapeutics [52]. The combined results of this studydemonstrate that this novel bio-nanofilm could be used incell repair and in the development of transdermal andtargeted delivery system as well as tissue engineeringapplications (Scheme 1).

Conclusions

Covalent incorporation of AgNPs into SWCNT via in situreduction reaction using ultrasonication, noncovalentattachment of DNA to the surface of the SWCNT-AgNPs

Fig. 6 Cell viability estimated from the XTT assay using A549 cells with a SWCNT-DNA/PVA (blank), b bio-nanofilm (control), and IMR-90cells with c SWCNT-DNA/PVA (blank), d bio-nanofilm (control)

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using surfactants, and fabrication of a bio-nanofilm weredemonstrated in this study. We validated the antibacterialactivity of this novel bio-nanofilm against major pathogenicorganisms. In addition, we demonstrated that this filmdisplayed improved properties with preserved biogroups,had a high biocompatibility, and cellular uptake capability.The interactions of the film with A549 and IMR-90 cellswere also investigated. The cellular uptake and cytotoxicityof the film were almost at the same level in both cells. Theuse of a surfactant and PVA significantly enhanced thecellular uptake and diminished toxicity. Furthermore, aversatile method to fabricate a bio-nanofilm with improvedproperties was developed in this study and this bio-nanofilm may serve as a new tool in the development ofnovel burn therapies, wound healing, artificial skin prepa-ration, scaffolds for tissue engineering, and targeteddelivery of therapeutics to macrophages.

Acknowledgements This work was supported by KyungwonUniversity research fund in 2010 and Grant No. 10032112 from theRegional Technology Innovation Program of the Ministry of Knowl-edge Economy. This study was supported by a grant of the Ministry ofHealth & Welfare (A040041) and Samsung Biomedical ResearchInstitute, Republic of Korea (PB00021).

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