journal of biomaterials applications ‘green’ biocompatible ... biomater appl-20… · pva/...

Nanotechnology in Biomaterials

‘Green’ biocompatible organic–inorganichybrid electrospun nanofibers forpotential biomedical applications

R Manjumeena1, T Elakkiya2, D Duraibabu2, A Feroze Ahamed3,PT Kalaichelvan1 and R Venkatesan4

Abstract

Gold nanoparticles were prepared by green route using Couroupita guianensis leaves extract. The green synthesized gold

nanoparticles exhibited maximum absorbance at 526 nm in the ultraviolet spectrum. By incorporating the green synthe-

sized gold nanoparticles in poly(vinyl alcohol) matrix, unique green organic–inorganic hybrid nanofibers (poly (vinyl

alcohol)–gold nanoparticles) were developed by electrospinning. Contact angle measurements showed that the prepared

poly (vinyl alcohol)–gold nanoparticles were found to be highly hydrophilic. The crystallinity of gold nanoparticles was

analyzed using XRD. The synthesized gold nanoparticles and poly (vinyl alcohol)–gold nanoparticles were characterized

using high-resolution transmission electron microscope, Fourier transform-infrared spectroscopy and energy-dispersive

analysis of X-ray. The ultimate aim of the present work is to achieve optimum antibacterial, antifungal, biocompatibility

and antiproliferative activities at a very low loading of gold nanoparticles. Vero cell lines showed a maximum of 90% cell

viability on incubation with the prepared poly (vinyl alcohol)–gold nanoparticles. MCF 7 and HeLa cell lines proliferated

only to 8% and 9%, respectively, on incubation with the poly (vinyl alcohol)–gold nanoparticles, and also exhibited good

antibacterial and antifungal activities against test pathogenic bacterial and fungal strains. Thus, the poly (vinyl alcohol)–

gold nanoparticles could be used for dual applications such as antimicrobial, anticancer treatment besides being highly

biocompatible.

Keywords

Green gold nanoparticles, organic- inorganic hybrid, nanofibers, biocompatibility, antiproliferative, antimicrobial

Introduction

Nanobiotechnology is a branch of applied sciences thatdeals with materials at the nanoscale (10�9m) focusingon biology, biochemical processes and their applica-tions. Nanobiotechnology offers potential develop-ments in pharmaceuticals, medical imaging, diagnosis,implantable materials, tissue regeneration, cancer treat-ment etc.1 Polymer nanofiber mats have unique proper-ties, such as a high surface area-to-volume ratio andhigh porosity. In addition, the polymer nanofiber scaf-fold composition can be controlled to achieve desiredproperties and functionality. Due to these advantages,nanofibrous scaffolds have been widely investigated inthe past several years with materials of different com-positions for applications of varying end uses, such asbiological scaffolds, wound dressings, optical and biosensors.2–18 There are many well-established techniques

namely centrifugal spinning, solution blowing, electro-spinning, pressurized gyration, etc.19,20 to generate awide variety of polymeric fibers across the micro- tonanometer-scale range. Electrospinning is a conven-tional process by which a polymer solution is chargedto a high voltage to produce fibers with a diameterranging from 10 to 500 nm. Over the years, a numberof electrospun nanofibers have been developed for

Journal of Biomaterials Applications

2015, Vol. 29(7) 1039–1055

! The Author(s) 2014

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DOI: 10.1177/0885328214550011

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1CAS in Botany, University of Madras, Guindy Campus, Chennai, Tamil

Nadu, India2Department of Chemistry, Anna University, Chennai, Tamil Nadu, India3Department of Microbial Technology, School of Biological Sciences,

Madurai Kamaraj University, Madurai, Tamil Nadu, India4National Institute of Ocean Technology, Chennai, Tamil Nadu, India

Corresponding author:

R Manjumeena, CAS in Botany, University of Madras, Guindy Campus,

Chennai 600 025, Tamil Nadu, India.

Email: [email protected]

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biomedical and pharmaceutical applications, many ofwhich are summarized in synoptic Table 1.

Aiming at the combination of the beneficial proper-ties of nanomaterials and electrospun nanofibers, inrecent years, more attention has been paid to the prep-aration of metal nanoparticles dispersed polymer nano-fiber film.21,22 Among the metal nanoparticles that areembedded in polymer film, gold nanoparticles (AuNPs)have been drawing much interest because of theirremarkable biocompatibility, antimicrobial and antic-ancer activities. These properties serve to make thesemetal nanoparticles a novel platform for biomedicine,pharmacology, labeling, drug-delivery, photo thermaltherapy, tissue, tumor imaging and sensing.23–27

Owing to its excellent water-solubility, high biocom-patibility, hydrophilicity, sound mechanical and ther-mal properties, poly (vinyl alcohol) (PVA) is apromising carrier of metal nanoparticles for biomedicaland pharmaceutical applications.28,29

The ease of synthesizing AuNPs and their affinityfor binding many biological molecules makes themattractive candidates for study. The green methodof nanoparticle synthesis employing plant extracts isa simple and viable alternative to chemical proced-ures and physical methods.30 Chemical and physicalmethods are harmful because the chemicals used aretoxic, flammable and are not disposed of easily in theenvironment.31 In recent years, biosynthesis of nano-particles has received considerable attention due tothe growing need to develop clean and nontoxic

chemicals, eco-friendly solvents and renewablematerials.32

With the increase in resistance of bacteria and fungito multiple antibiotics, there is a growing need todevelop antibacterial and antifungal agents withbroad spectrum and multitude mode of action. Thereare several reports on the antibacterial and antifungalactivities of nanosilver.33,34 So, we were inclined toextend the same application to green synthesizedAuNPs as AuNPs possess well-developed surface chem-istry, chemical stability and appropriate smaller size,which make them easier to interact with the micro-organisms causing structural changes, degradationand finally cell death as already reported.35

Breast and cervical cancer are the most commonforms of malignancy prevalent among middle-agedwomen that cause major mortality worldwide.Moreover, the incidence and mortality of breast andcervical cancer keep on rising every year.36 Over thepast decade, treatments to these life-threatening formsof cancer has become more challenging owing to theprevalence of multiple drug resistance, detrimentalside effects and the lack of innovative approaches.Chemotherapy is one of the most effective methodsfor the treatment of metastatic cancers, it is nonspecificand causes significant toxic damage. The developmentof drug resistance to chemotherapeutic agents throughvarious mechanisms also limits their therapeutic poten-tial. The success of cancer therapy depends on theability of a therapeutic agent to destroy the tumor

Table 1. Synoptic table of electrospun nanofibers developed for biomedical and pharmaceutical applications.

Electrospun nanofiber formulation Applications References

Chitosan–organic rectorite (OREC) /polyvinyl alcohol (PVA) Antibacterial [2]

PVA-AgNPs/carboxymethyl-chitosan Antibacterial [3]

AgNPs-PVA/ hydroxypropyl-beta-cyclodextrin Antibacterial [4]

Triclosan/cyclodextrin complexes Antibacterial [5]

PVA/sodium alginate (ALG)/OREC composite Antibacterial [6]

Chitosan-AgNPs blended with PVA Antibacterial [7]

Pullulan/PVA/silver hybrid Antibacterial [8]

Chitosan-blended polyamide Cytotoxicity [9]

Multiwalled carbon nanotube incorporated PVA/chitosan Cytotoxicity [10]

Gold nanoparticles and lysozyme deposited cellulose Antibacterial [11]

PVA–AgNPs Cytotoxicity [12]

Poly(L-lactide) ultrafine fibers containing nanosilver Antibacterial [13]

N-carboxyethylchitosan and poly(ethylene oxide) nanofibres containing AgNPs Antibacterial [14]

Gelatin fiber mats containing silver nanoparticles Antibacterial [15]

PVA/ chitosan/nano-ZnO composite nanofibrous membranes Antibacterial

and antifungal

[16]

Paclitaxel incorporated pHEMA-bamboo cellulose nanocomposite fibers Cytotoxicity and

anticancer

[17]

Hydrolyzed poly[2-(3-thienyl) ethanol butoxy carbonyl-methyl urethane]/cellulose acetate Biosensors [18]

1040 Journal of Biomaterials Applications 29(7)



cells while minimally affecting normal nonmalignantcells.37 The nonspecific nature of many current antic-ancer agents severely limits their effectiveness if thedosage is too high and the systemic toxic effects out-weigh the beneficial anticancer effect.38

This demonstrates the need to use AuNPs as a pro-mising alternative for the treatment of various diseasesin general and cancer in particular.39 The rationale forusing AuNPs for cancer treatments can be ascribed totheir large surface area for volume, porosity, solubility,increased bioavailability, ease of passing through thecellular barriers, strongly interacting with functionalbiomolecules and different structural properties.40 Theuse of green synthesized AuNPs to prepare organic–inorganic hybrid nanofibers has marked the start of‘green’ practices to take their place in the electrospin-ning process by integrating with recent developments inscience and industry in attempts to reduce the gener-ation of hazardous waste in the environment. Thisattempt aims at minimizing the use of unsafe productsand maximizing process efficiency while using environ-mentally safe nontoxic materials.4

In pursuit of overcoming the above-said shortcom-ings of chemical synthesis of nanoparticles, microbialresistance towards conventional antibiotics, anticancerdrugs, treatments, we have developed a unique greenorganic–inorganic hybrid nanofibers using electrospin-ning. The prepared organic–inorganic hybrid nanofi-bers scaffold was assessed for biocompatibility withVero cell lines, antiproliferative activity on breastcancer cell lines (MCF7) and cervical cancer cell lines(HeLa), antibacterial and antifungal activities. Theultimate aim of the present work is to achieve optimumantibacterial, antifungal, biocompatibility and antipro-liferative activities at a very low loading of AuNPs.

Experimental

Materials

Chloroauric acid (SRL, Mumbai, India), Muller-Hinton agar (Himedia Mumbai, India), Poly (vinylalcohol) (PVA)-Mw 89,000–98,000 (Sigma-Aldrich,Bangalore, India) were used as received without furthertreatment or purifications.

Cell lines and maintenance

Minimal Essential Media (HiMedia Laboratories,Mumbai, India) were used as received without furthertreatment or purifications. Vero cell lines, MCF 7,HeLa cancer cell lines were obtained from Nationalcentre for cell sciences (NCCS), Pune, India. The cellswere maintained in Minimal Essential Media whichwere supplemented with 10% fetal bovine serum

(Cistron Laboratories, Chennai, India), penicillin(100U/mL) and streptomycin (100mg/mL) in a humidi-fied atmosphere of 50 mg/mL CO2 at 37�C. Trypsin,methylthiazolyldiphenyl-tetrazoliumbromide (MTT)and dimethyl sulfoxide (DMSO) from Sisco ResearchLaboratory Chemicals, Mumbai, India, were used asreceived.

Source of microorganisms

The strains Candida albicans and C. krusei wereobtained from VHS hospital, Chennai, India. Pure cul-tures of Bacteria Escherichia coli (ATCC 8739)Staphylococcus aureus (ATCC 6538), Micrococcusluteus (ATCC 4698), Klebsiella pneumoniae (ATCC13883), Bacillus subtilis (ATCC 6633) andPseudomonas aeruginosa (ATCC 15442) were obtainedfrom American Type Culture Collection.

Green synthesis of AuNPs

Aqueous extract of Couroupita guianensis was preparedfollowing the procedure as reported in our previouswork.41 The steps in green synthesis of AuNPs aregiven in Figure 1.

Characterization of the green synthesized AuNPs

After the synthesis process was completed by reducingmetal ion solution with leaves extract, surface plasmonresonance of AuNPs was easily confirmed by Diffusereflectance ultraviolet–visible (UV-Vis) spectroscopy.The reaction mixture was sampled at regular intervalsand the absorption maxima was scanned at the wave-length of 400–800 nm using Shimadzu UV-Vis spectro-photometer (model 2450; Tokyo, Japan). Thebiosynthesized AuNPs gave sharp peak in the visibleregion of the electromagnetic spectrum. The X-raypowder diffraction data was acquired by PAN analyticalX’Pert PRO diffractometer in Bragg–Brentano geometryusing step scan technique and Johanssonmonochromatorto produce pure Cu Ka1 radiation (1.5406 A; 45kV,30mA) in the range of 30�–80�. The peaks were matchedwith (JCPDSNo. 01-1174). The obtained pattern was forfcc cubic crystal structure. The peak plane matched withthe card. The crystalline size was calculated from the full-width at half-maximum (FWHM)of the diffraction peaksusing the Debye–Sherrer formula. The Fourier trans-form-infrared spectroscopy (FTIR) spectra for biosynthe-sized AuNPs were recorded on an IR Affinity-1SHIMADZU spectrophotometer in transmittance modein the range of 400–4000 cm�1 at a resolution of 4 cm�1.For high-resolution transmission electron microscope(HRTEM) measurements, a drop of solution containingsynthesized AuNPs was placed on the carbon-coated

Manjumeena et al. 1041



grids andkept under vacuumdesiccation overnight beforeloading them onto a specimen holder. HRTEM micro-graphs were taken by analyzing the prepared grids on300 kV field emission TEM-STEM (FEI F30) with cap-ability of HAADF, EELS and EDX.

Electrospinning process to fabricateorganic–inorganic hybrid nanofiber (PVA-AuNPs)

The electrospinning set-up used in the present work wasdesigned and developed in Anna University,

Department of Chemistry, Chennai. Optimized weightpercentage of 10% (w/v) PVA and 0.1% (w/v) of greensynthesized AuNPs were dissolved in double-distilledwater by continuous stirring for 2–4 h to get a homo-geneous solution. The polymer solution mixed withAuNPs was taken in a 2-mL syringe to which aneedle tip of 0.56mm inner diameter was attached.The positive electrode of the high-voltage powersupply was connected to the needle and the negativeterminal to the collector. The polymer solutions wereelectrospun at a distance of 12 cm from the needle tipwith a flow rate of 0.35mL/h and an optimized appliedelectric voltage of 15 kV to produce beadles organic–inorganic hybrid nanofibers.

Characterization of electrospunorganic–inorganic hybrid nanofibers(PVA-AuNPS)

Spectral analysis

The functional groups in the electrospun organic–inor-ganic hybrid nanofibers were identified by Fouriertransform-infrared spectroscopy (FTIR) IR Affinity-1SHIMADZU spectrophotometer in transmittancemode in the range of 400–4000 cm�1 at a resolutionof 4 cm�1. Diffuse reflectance UV-Vis spectroscopywas used to obtain the spectra for electrospunorganic–inorganic hybrid nanofibers. The spectra wererecorded between 400 and 800 nm on a Shimadzu UV-Vis spectrophotometer (model 2450; Tokyo, Japan).

Surface morphological analysis

The morphology of electrospun organic–inorganichybrid nanofibers was analyzed by Field emissionScanning electron microscope (FESEM) HITACHISu-6600 with an energy-dispersive analysis of X-ray(EDAX) attachment and HRTEM FEI, TECHNAIG2 30 S-twin D905. The mean diameter and distribu-tion of the hybrid nanofibers were measured from atleast 100 nanofibers from various FESEM images usingUTHSCSA image tool. Atomic force microscope(AFM, Seiko SPI3800N, series SPA-400 (Tokyo,Japan)) was used to study the surface topography andsurface morphology of the organic–inorganic hybridnanofibers.

Contact angle measurements

The hydrophilicity of the electrospun organic–inor-ganic hybrid nanofibers was evaluated using contactangle measurements by placing the sample on theholder of Euromex Optical Microscope equipped witha CCD camera. A drop of deionised water (10mL) was

Couroupita guianensis

i) Washing/Water

Filter nylon mesh

ii) Boiled withdistilled water (100ml)60°C/10min

Leaf extract (9ml)

Cl

ClCl

Cl

Au

Chloroauric acid 1mM (1ml)

Incubation at 37°C/10min(static condition)

AuNPs

H

Figure 1. Green synthesis of AuNPs.




deposited on the sample surface. The contact angle ofthe drop on the surface was measured at room tempera-ture (27�C). Five measurements were performed at dif-ferent locations and the contact angles were calculatedwith the help of ‘UTHSCSA Image tool’ software.

Assessment of biocompatibility and antiproliferativeactivities of organic–inorganic hybrid nanofibers(PVA-AuNPs)

The cytotoxic activity of organic–inorganic hybridnanofibers was assessed on Vero cell lines and the antic-ancer activity was assessed on MCF 7 (breast cancercell lines) and HeLa (cervical cancer cell lines) by theMTT assay method.42 The Vero cell lines, MCF 7 andHeLa cell lines were plated in 0.2mL of the MinimalEssential Medium in 96-well plates. These cells reachedconfluence after 72 h of incubation. Then the Verocelllines, MCF 7, HeLa cell lines were incubated with PVAnanofibers without AuNPs (control) and organic–inor-ganic hybrid nanofibers (disinfected by ultraviolet Cirradiation for 1minute) in 0.1% DMSO at variousdilutions for a period of 72 h at a temperature of37�C. After 72 h of incubation, 0.5% of 3 -(4,5-dimethyl–2-thiazolyl)�2,5-diphenyl-tetrazolium brom-ide cells (MTT) in phosphate-buffer saline solutionwas added. The absorbance was measured at 630 nmusing UV spectrophotometer.

The results are expressed as mean standard error ofthe absorbance. Data were analyzed by Student’s t-testand differences at the 95% confidence level were con-sidered to be significant. The cells after biocompatibil-ity and antiproliferative tests were visualized usingFLoid Cell Imaging Station, California, USA.

Assessment of antibacterial and antifungal activitiesof hybrid nanofibers (PVA-AuNPs)

The antibacterial and antifungal activities of organic–inorganic hybrid nanofibers were assessed by an inhib-ition zone method.4 A loop of the bacterial culture andfungal culture were inoculated from fresh colonies onagar plates into 100mL Muller Hinton culture mediumseparately. The culture was allowed to grow until theoptical density reached 0.2 at 600 nm (OD of 0.2 cor-responding to a concentration of 108 CFU mL�1 ofmedium). This indicates that the bacterial and fungalculture are in exponential or log phase of growth, whichis ideal for the experiment. Then it was swabbed uni-formly onto individual Mueller Hinton agar platesusing sterile cotton swabs. Organic–inorganic hybridnanofibers and control (PVA nanofibers withoutAuNPs) disinfected by ultraviolet C irradiation for1minute were cut into 5-mm discs and placed in theculture-swabbed Petri plate. The plates were examined

for possible clear zone formation after overnight incu-bation at 37�C. The diameter of the clear zone formedaround the organic–inorganic hybrid nanofibers on theplates was measured and recorded as an inhibitionagainst the test bacterial strains and fungal species.

Results

Characterization of the biosynthesized AuNPs

The UV-vis spectra show a well-defined surface plas-mon band centered at around 526 nm (Figure 2), whichis the characteristic of AuNPs and clearly indicates theformation of AuNPs in solution. It may be due to theexcitation of surface plasmon resonance (SPR) effectand reduction of AuCl4– ions. The stability resultsfrom a potential barrier that develops as a result ofthe competition between weak Vander Waals forcesof attraction and electrostatic repulsion.43 The solutionwas stable even after 120 days of reaction, with no evi-dence of aggregation of particles. Figure 3(a) shows theXRD pattern of the synthesized AuNPs. The five dif-fraction peaks observed at 38�, 45�, 67�, 78� and 81� inthe 2y range can be indexed to the (111), (200), (220),(311), (222) reflection planes of face-centred cubicstructure of metallic gold nanopowders. The averagesize of the AuNPs was found to be about 15 nm.HRTEM micrographs and the particles size distribu-tion histogram determined from micrograph of theAuNPs are shown in Figure 3(b and c), respectively.Monodispersed, discrete spherical-shaped gold particlewas observed in HRTEM micrograph. HRTEM micro-graph showed that the size of the synthesized AuNPswere in the range of 4–13 nm. EDX spectrum of AuNPsshows different X-ray emission peaks with strong

Abs

orba

nce

526

500 520 540 560 580

Wavelength (nm)

Figure 2. Diffuse reflectance spectrum of AuNPs.




signals from the atoms in the AuNPs (Figure 4). Thisindicates the reduction of gold ions in the chloroauricacid to elemental gold. The crystalline nature of AuNPswas confirmed from X-ray diffraction (XRD) analysis.The FTIR spectrum of AuNPs (Figure 5b) shows thepresence of peaks at 1637, 1471, 1372 cm�1. The strongabsorption peak at 1637 corresponds to Alkenyl C¼Cstretch which is the characteristic of gold atoms.44 Theband at 1471 and 1372 cm�1 correspond to aromaticring stretch and methyl C-H symmetrical bend,respectively.

Spectral analysis

FTIR spectrum of organic–inorganic hybrid nanofibersis shown in Figure 5(c). The spectra for PVA andAuNPs are depicted in Figure 5(a and b), respectively.In the spectrum shown in Figure 5(a), the polymerhydroxy band at 3419 cm�1 appears due to the presenceof CH-OH stretching vibrations of PVA. The bandsappearing at 2938 cm�1 and 1423 cm�1 correspond tothe aliphatic stretching and bending vibrations of PVA.The spectrum obtained for AuNPs (Figure 5(b)) showsseveral absorption peaks at 1637 cm�1, 1471 cm�1 and1372 cm�1. The spectrum for organic–inorganic hybridnanofibers shows appearance of band at 1728 cm�1

formed due to a slight shift of band at 1637 cm�1 inthe AuNPs spectrum. The remaining bands at1447 cm�1 and 1383 cm�1 appear due to the bendingvibrations of AuNPs. This confirms that the AuNPsdistributed in the PVA matrix are stable after electro-spinning. The observed drastic decrease in the absorb-ance of the peak at 3419 cm-1 could be attributed to theinteractions between the AuNPs and some functional-ities of the PVA matrix molecules particularly the O-H

(a)500

400

300

200

100

0

40 50 60 70 802θ (degrees)

Size of nanoparticles (nm)

(c)16

14

12

10

8

6

4

No

of p

artic

le in

%

2 4 6 8 10 12 14

(222)

(311)

(220)

(200)

(111) (b)

Inte

nsity

(a,

u)

Figure 3. (a) XRD pattern of the synthesized AuNPs. (b) HRTEM micrographs of AuNPs. (c) Particles size distribution histogram.

Cou

nts

180

160

140

120

100

80

60

40

20

1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00Energy (kev)

o

Au

AuAuAu Au

c

Figure 4. EDX spectrum of AuNPs.




group.45 Figure 6 shows the diffused reflectance spec-trum of PVA-AuNPs hybrid nanofibers.

The spectrum shows a peak at 568 nm formed due toa slight shift in the original absorption peak (526 nm) ofAuNPs. However, the slight shift in the peak maximumthat corresponds to AuNPs could be attributed to theinteraction between the polymer hydroxy group ofPVA and AuNPs in the hybrid nanofiber leading tosome degree of local particle aggregation.46 Theseobservations throw light on the probability of existenceof hydrogen bonding between the organic and inor-ganic components, which led to better compatibilitybesides the absence of an organic surfactant or a com-patibilizer which are normally added to improve the

dispersion of inorganic nanoparticles in the polymermatrix.47

Surface morphological analysis

PVA was miscible with AuNPs and had good electro-spinnability when blended. Figure 7(a and b) show theFESEM and HRTEM micrographs. They were used tovisualize the morphology of the hybrid nanofiberand distribution of AuNPs in the hybrid nanofiber.Elemental composition of hybrid nanofibers wasanalyzed by energy-dispersive X-ray spectroscopy.Figure 7(c) shows the optical absorption peak for Auat 2 keV which is typical for metallic AuNPs. Atomicforce micrographs (Figure 7d) showed that AuNPs cansignificantly influence the surface topography of thenanofiber. Uniform nanoparticles dispersion can beseen on the surface of the hybrid nanofiber. Increasein surface roughness of the hybrid nanofiber may beattributed to the presence of AuNPs on the surface.

From the FESEM and HRTEM micrographs, simi-lar morphologies and homogeneous appearance ofAuNPs were observed in the beadless fibers.Mahalingam and Edirisinghe reported that bead-freecontinuous fibers could be formed when the polymerconcentration is above the critical concentration. As inthe present study, the concentration of PVA was higherand the loading of AuNPs was minimum, the overlap-ping of polymer chains formed sufficient entanglementnetworks of polymer chains and yielded smooth, bead-less fibers.20

Figure 8 show the diameter distribution of thehybrid nanofibers. The diameter of the hybrid nanofi-bers was in the range of 50–450 nm. The increase in thediameter of the hybrid nanofibers when compared tothe diameter of neat PVA nanofibers which was in therange of 50–300 nm as reported by one of the authors48

could be attributed to the increase in charge density andshear viscosity upon addition of AuNPs to PVA,thereby increasing the electrical force which consecu-tively can also cause the actual mass throughput toincrease. Thus, the increase in the hybrid fiber diam-eters from that of the neat PVA fibers should be due tothe addition of AuNPs.49 The Quasi-spherical-shapedAuNPs were slightly larger and were distributed in anencircling manner on the surface of the individual PVAfiber (Figure 7b), yet the same smooth and beadlessmorphology of hybrid nanofibers were retained.50

It can be seen from the FESEM micrographs thatthe asymmetry of the nanofiber was apparent and thedispersion of AuNPs did not visibly alter the nanofiberstructure. It can be also noticed when HRTEM micro-graphs are compared with FESEM ones, the FESEMmicrographs depict AuNPs sparsely dispersed in themesh-like hybrid nanofiber which is informative only

(a)

(b)

(c)

Tran

smitt

ance

(%

)

3419

2938

1728

1447

1383

1637 13

7214

7114

23

Wavenumber (cm−1)

4000 3500 3000 2500 2000 1500 1000 500

Figure 5. (a) FTIR spectrum of PVA. (b) FTIR spectrum of

AuNPs. (c) FTIR spectrum of organic–inorganic hybrid

nanofibers.

568

500 520 540 560 580 600 620

Wavelength (nm)

Ab

sorb

ance

Figure 6. Diffuse reflectance spectrum of organic–inorganic

hybrid nanofibers.




about the surface morphology of the hybrid fiber, theelectron beam in HRTEM analysis passes through thenanofibers, which could perhaps detect the exact denseencircling orientation of the AuNPs on the surface ofthe nanofibers.51 Though there might be hydrogenbonding between PVA and AuNPs which apparentlyleads to van der Walls gaps, the organic and inorganic

components retained their respective characteristics,and surface modification of the AuNps might beneeded to promote interfacial adhesion between PVAand AuNps which would have led to the distribution ofthe AuNps into the nanofiber.52 This would probablyexplain the reason for orientation of AuNPs on thesurface of the hybrid nanofiber. The nature of distribu-tion and the bonding interaction of AuNps in thehybrid nanofiber is illustrated graphically in Figure 9.This observation might add a novel feature to thehybrid nanofiber, since there are not many reports toour knowledge on such a distribution of AuNPs on thesurface of the nanofiber and such a distribution did nothinder the biocompatibility and antiproliferative prop-erties of the hybrid nanofiber. Moreover, this type ofdistribution of AuNPs encircling individual fibersmight aid in the rapid release of gold ions whichwould play a major role in enhanced antimicrobialproperty of the hybrid nanofiber.53

Contact angle measurements

Figure 10(a and b) show the contact angle measure-ments for PVA nanofiber (control) the hybrid nanofi-ber. The results show that the hydrophilicity increases

Cou

nts

Energy (keV)0 5 10

49.4μmμm

μmμm

0

050

Y’

X’

(a) (b)

(c) (d)

Figure 7. (a) FESEM micrograph of organic–inorganic hybrid nanofibers. (b) HRTEM micrograph of organic–inorganic hybrid

nanofibers. (c) EDX spectrum of organic–inorganic hybrid nanofibers. (d) AFM micrograph of organic–inorganic hybrid nanofibers.

Fiber diameter (nm)

Freq

uenc

y (%

)

20

16

12

8

4

0

0 50 100 150 200 250 300 350 400 450 500

Figure 8. Organic–inorganic hybrid nanofiber diameter

distribution.




after AuNPs incorporation. The contact angle for PVAnanofiber and hybrid nanofiber was 41� 3� and33� 4�, respectively, for 0min, which decreased withrespect to time. The contact angle for PVA nanofiberand hybrid nanofiber was 36� 4� and 20�3�, respect-ively, for 5min. Thus, the AuNPs which are localizedon the surface of the individual fibers control thehydrophilicity of the hybrid nanofiber. The increase inhydrophilicity would precisely yield a positive feedbackon cell adhesion studies.54

Assessment of biocompatibility and antiproliferativeactivities of organic–inorganic hybrid nanofiber(PVA-AuNPs)

For the application of organic–inorganic hybrid nano-fibers therapeutically it is essential to evaluate its bio-compatibility particularly its nontoxicity to normalcells. The bar chart in Figure 11(a) shows the percent-age of biocompatibility of organic–inorganic hybridnanofibers with Vero cell lines. There was a maximumpercentage of Vero cell viability (90%) at the end of72-h incubation. Figure11(b) shows that the cell prolif-eration increased in direct proportion to the time ofincubation. This indicates that the nutrition to the celllines was not hindered by the organic–inorganic hybridnanofibers, leading to increased viability of the cells. Itcan be seen from the FESEM micrograph (Figure 11c)that the cell lines proliferated in the direction of the

fiber orientation according to the architecture of thenanofibers densely covering the voids by cytoplasmicextensions, and some cells migrated underneath thefibers maintaining their morphology. The existence ofan optimally dispersed mesh-like morphology in theorganic–inorganic hybrid nanofibers which has beendemonstrated from FESEM micrographs could bebelieved to have yielded a suitable environment forcell adhesion and proliferation on the surface.55

Figure 12 shows the increasing cell density in theVero cells treated with hybrid nanofibers. Cell adhesionbehavior of Vero cell lines onto organic–inorganichybrid nanofibers could have also been mediated viathe electrostatic interaction between the positivelycharged PVA and the negatively charged cell mem-branes and the AuNPs did not hinder this electrostaticinteraction.56 The cell adhesion and proliferation couldhave been activated due to good hydrophilicity oforganic–inorganic hybrid nanofibers and the presenceof recognition sites. Thus, it can be concluded that theorganic–inorganic hybrid nanofibers can obviouslyimprove the cell growth behaviors.54 The presence ofAuNPs in the electrospun hybrid nanofiber shows anti-proliferative effects in MCF-7 and HeLa cell lines. Thepercentage of antiproliferation activity of the organic–inorganic hybrid nanofibers on MCF 7 and HeLa celllines is given in Figure 13(a and b), respectively. Thepercentage of proliferation of MCF 7 and HeLa celllines were only 8% and 9%, respectively at the end of72-h incubation with hybrid nanofibers. It can be seenfrom the optical density values (Figure 14a and b) thatthe MCF 7 and HeLa cell proliferation decreased indirect proportion to the time of incubation of the celllines treated with hybrid nanofibers, whereas in the celllines treated with only PVA nanofibers (withoutAuNPs) the cell proliferation was high. It can be seenfrom the FESEM images (Figure 15a and b) at the endof 72-h incubation, the organic–inorganic hybrid nano-fibers architecture was disrupted which was evidentfrom the broken fibers. The dead MCF 7 and HeLacell lines could have made the fibers brittle leading tobreakage. Figures 16 and 17 show the reducing celldensities of MCF 7 and HeLa cell lines, respectively,

Organic-inorganic hybrid nanofibrous mat

Bonding interactionFiber

AuNPs

Figure 9. Graphical illustration of the distribution and interaction of gold nanoparticles (AuNps) on the surface of the PVA

nanofiber.

(a)

(b)

0

0

min

min min

min41

+ +

++ _

_ _

_

3°

3°

2°

4°

5

5

33

36

20

Figure 10. (a) Contact angle measurements of PVA nanofibers

at 0 min and 5 min. (b) Contact angle measurements of organic–

inorganic hybrid nanofibers at 0 min and 5 min.




(a) 100

80

60

40

20

024h 48h 72h

% o

f cel

l vla

bllit

y

Incubation period in hours Incubation period in hours

(c)

Control ControlPVA-AuNPs PVA-AuNPs

(b)

0.8

0.6

0.4

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0 4 8 12 16 20 24 28 3236 40 44 48 5256 60 64 68 72

Figure 11. (a) Bar chart showing percentage of cell viability of Vero cells on treating with organic–inorganic hybrid nanofibers.

(b) OD values of Vero cells proliferation with respect to time. (c) FESEM micrograph showing high cell density of Vero cells on treating

with organic–inorganic hybrid nanofibers at the end of 72 h.

Figure 12. (a) Control-Vero cells treated with PVA nanofibers at the end of 72 h. (b) Vero cells treated with organic–inorganic

hybrid nanofibers at the end of 24 h incubation. (c) Vero cells treated with organic–inorganic hybrid nanofibers at the end of 48-h

incubation (D) Vero cells treated with organic–inorganic hybrid nanofibers at the end of 72-h incubation.




at 24, 48 and 72 h of incubation with hybrid nanofiber.The AuNPs accumulate inside the cancer cells andsequesters in large clusters in vacuoles in the peri-nuclear areas of the cytoplasm.57 The size of AuNPsis an important factor that influences the rate of endo-cytosis and exocytosis, and thus the level of cellularaccumulation.58

The mechanism of antiproliferative activity ofAuNPs may be linked to the ATP consumption inthe DNA repair process. ATP is generally known tocontrol apoptotic signals since apoptosis is inhibited atphysiological ATP concentrations. A decrease in theintracellular ATP concentration induces apoptosis, byregulating the activity of bax, which is also crucial forcaspase-3 activation which is a common downstreameffector of both extrinsic and intrinsic apoptosis path-ways, thus contributing to cell death.59,60 AuNPs alsoinduced an increase in the mRNA expression of bax

and bak, which are pro-apoptotic members of the Bcl-2family and responsible for the induction of intrinsicmitochondria apoptosis. AuNPs loaded in the PVAnanofibers target the signaling molecules that arehighly expressed in cancer cells and the normal cellsremain unaffected.61 Higher cytotoxicity of smaller par-ticles compared to larger ones is related to the amountof reactive oxygen species (ROS) generated at the rela-tively larger surface area of small nanoparticles.Smaller AuNPs release more gold ions from its surfacethan larger nanoparticles. Oxidative stress is inducedwhen the generation of ROS exceeds the cell’s antioxi-dant capacity. Besides the damaging effects to cellularproteins, lipids and DNA, an increasing level of ROStriggers the cell to respond by activating pro-inflamma-tory signaling cascades, and ultimately induces pro-grammed cell death.62 As we observed aggregation ofAuNps on the organic–inorganic hybrid nanofibers

Control ControlPVA-AuNPs PVA-AuNPs

Opt

ical

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at 6

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(b)(a)

0.0 0.0

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Incubation period in hours Incubation period in hours00 4 4 88 1212 161620 20 2424 2828 323236 3640 4044 44484852 525656 606064 6468 6872 72

Figure 14. (a) OD values of MCF 7 cells proliferation with respect to time. (b) OD values of HeLa cells proliferation with respect to

time.

% o

f cel

l pro

lifer

atio

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Incubation period in hours Incubation period in hours

Figure 13. (a) Bar chart showing percentage of cell proliferation of MCF 7 cells on treating with organic–inorganic hybrid nanofibers.

(b) Bar chart showing percentage of cell proliferation of HeLa cells on treating with organic–inorganic hybrid nanofibers.




after incubation with the cancer cell lines from theFESEM images (Figure 12c), the increase in antiproli-ferative activity can be attributed to this aggregation ofAuNPs which leads to long-term retention.Consequently, the membrane potential of the mito-chondria is decreased and the levels of reactiveoxygen species are increased, leading to cell death asreported by Cui et al.63 Thus, it is obvious that theorganic–inorganic hybrid nanofibers that are biocom-patible as well as toxic to cancer cells can combinediagnosis and therapy and can contribute broadly tobiomedicine.

Antibacterial and antifungal activities oforganic–inorganic hybrid nanofibers

The results showed significant inhibitory activity oforganic–inorganic hybrid nanofibers against all thetested microorganisms, as shown in Figure 18. Whilethe PVA nanofibers without AuNPs (control) did notshow any antibacterial and antifungal activity. Thediameter of the zone of inhibition of organic–inorganichybrid nanofibers against the test pathogenic bacterialand fungal strains is given in Figure 19. The resultsmanifested that the antibacterial and antifungal ability

Figure 16. (a) Control-MCF 7 cell lines treated with PVA nanofibers at the end of 72 h. (B) MCF 7 cell lines treated with organic–

inorganic hybrid nanofibers at the end of 24-h incubation. (c) MCF 7 cell lines treated with organic–inorganic hybrid nanofibers at the

end of 48-h incubation. (D) MCF 7 cell lines treated with organic–inorganic hybrid nanofibers at the end of 72-h incubation.

Figure 15. (a) FESEM micrograph showing broken organic–inorganic hybrid nanofibers due to MCF 7 cell death at the end of 72 h.

(b) FESEM micrograph showing broken organic–inorganic hybrid nanofibers due to HeLa cell death at the end of 72 h.




of the PVA nanofibers depend on the presence ofAuNPs on the surface of the fibers. AuNPs on the sur-face of organic–inorganic hybrid nanofibers manifesteffective antibacterial and antifungal property due totheir smaller dimension and higher specific area. Goldions are released when the organic–inorganic hybridnanofibers were brought in contact with the test

bacterial and fungal cultures in the Petri plate, whichresulted in the formation of zone of inhibition.4 Thismay be due to the fact that the release of AuNPsbecomes easier as the particle size decreases, so thatAuNPs can more effectively reach the microbialregion subsequently increasing their contact with themicroorganism. In addition, smaller dimensions and

Figure 17. (a) Control-HeLa cell lines treated with PVA nanofibers at the end of 72 h. (b) HeLa cell lines treated with organic–

inorganic hybrid nanofibers at the end of 24-h incubation. (c) HeLa cell lines treated with organic–inorganic hybrid nanofibers at the

end of 48-h incubation. (d) HeLa cell lines treated with organic–inorganic hybrid nanofibers at the end of 72-h incubation.

Figure 18. Zone of inhibition of organic–inorganic hybrid nanofibers on test pathogenic bacterial and fungal strains.




higher surface-to-volume ratios of AuNPs also enhancetheir contact with the microorganism.64

The inhibitory activity of the organic–inorganichybrid nanofibers against the Gram-positive bacteriais better than that against Gram-negative bacteria.These results are in close agreement with those reportedby Thiel et al.65 The antibacterial properties of AuNPscould be believed to be the same as silver nanoparticlesthat are associated with its slow oxidation and liber-ation of silver ions (in the case of AgNPs) and goldions (in the case of AuNPs) to the microbial environ-ment, making it an ideal biocidal agent. Moreover, thesmall size of these particles facilitates the penetration ofthese particles through cell membranes to affect intra-cellular processes from inside.66 It was reported thatAgNPs exhibited excellent antifungal activity onCandida albicans by disrupting the cell membrane andinhibiting the normal budding process.67 Similar mech-anism could be attributed to the antifungal activity ofAuNPs in the present work. Exposure of AuNPs tobacterial and fungal cells resulted in alterations in theexpression of a panel of envelope and heat sock protein.Consequently, these particles can penetrate and can dis-rupt the membranes of microorganisms. A massive lossof intracellular potassium was induced by AuNPs.Furthermore, the AuNPs decreased the ATP levels.The possible molecular targets for the AuNPs couldbe protein thiol groups present in enzymes such asNADH dehydrogenases and disrupt the respiratorychain, facilitating the release of reactive oxygen species,leading to oxidative stress, and resulting in significantdamage to the cell structures and ultimate cell death.The phospholipid portion of the bacterial membranemay also be the site of action for the AuNPs.68 Asthe antimicrobial effect of AuNPs was believed to beclosely related to that of AgNPs, which was brought

about by the formation of pits in the cell wall leading tochange in morphology and significant increase in per-meability, leaving bacterial cells incapable of properlyregulating transport through the plasma membrane,resulting in cell death. The increase in permeability ofthe cell membranes would allow the AuNPs to pene-trate the cell and cause cell death by breaking thedouble-stranded DNA.69

Conclusions

AuNPs were synthesized by a green route usingCouroupita guianensis leaves extract and characterized.The green synthesizedAuNPswere loaded into PVAandelectrospun to develop organic–inorganic hybrid (PVA-AuNPs) nanofibers. Surfacemorphological analyses likeSEM, HRTEM revealed the presence of AuNPs on thesurface of the electrospun hybrid nanofibers. PVA beinga hydrophilic polymer matrix by itself, became morehydrophilic upon very low loading of AuNPs. Thisincrease in hydrophilicity led to good cell adhesion andproliferation when tested for biocompatibility on Verocell lines. Organic–inorganic hybrid nanofibers impartedgood antiproliferative activity against MCF 7 (breastcancer cell lines), HeLa (cervical cancer cell lines) andalso exhibited promising antibacterial and antifungalactivities against test pathogenic bacterial strains,E. coli, S. aureus, M. luteus, K. pneumoniae, B. subtilis,P. aeruginosa and pathogenic fungal strains Candidaalbicans, and C. krusei. The organic–inorganic hybridnanofibers thus developed showed great potential forbiomedical applications namely antimicrobial wounddressing and cancer treatment. Organic–inorganichybrid nanofibers may also be beneficial in overcomingsome of the challenges prevailing in current cancer treat-ment procedures and antibiotic resistance in micro-organisms. Another direction for future research of thisorganic–inorganic hybrid nanofiber is AuNPs may beconjugatedwith the prevailing anticancer drugs, antibio-tics and electrospun with PVA matrix to assess theirsynergistic anticancer and antimicrobial activities. Thisaims at reducing the dosage and side effects caused by theprolonged use of conventional drugs and antibioticswhich is currently under investigation in our laboratory.

Acknowledgements

We thank NCNSNT, University of Madras, Guindy campus,for providing characterization studies. We also thank Prof. N.Raaman, Director, CAS in Botany, University of Madras,

Guindy campus, for providing lab facilities.

Funding

This research received no specific grant from anyfunding agency in the public, commercial, or not-for-profit

sectors.

a- B.subtilisb- E.colic- K.pneumoniaed- M.luteuse- P.aeruginosaf- S.aureusg- C.albicansh- C.krusei

Dia

met

er z

one

of In

hibt

ion

(mm

)

1.6

1.2

0.8

0.4

0.0

a b c d e f gOrganisms

h

Figure 19. Bar diagram showing diameter of zone of inhibition

of organic–inorganic hybrid nanofibers on test pathogenic

bacterial and fungal strains.




Declaration of conflicting interests

The authors declared no potential conflicts of interest with

respect to the research, authorship, and/or publication of thisarticle.

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