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Biomaterials 32 (2011) 4704e4713

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Biomaterials

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The targeted antibacterial and antifungal properties of magnetic nanocompositeof iron oxide and silver nanoparticles

Robert Prucek a,*, Ji�rí Tu�cek b, Martina Kilianová a, Ale�s Paná�cek a, Libor Kvítek a, Jan Filip b, Milan Kolá�r c,Kate�rina Tománková d, Radek Zbo�ril a,**aRegional Centre of Advanced Technologies and Materials, Department of Physical Chemistry, Faculty of Science, Palacky University, Slechtitelu 11, 78371 Olomouc, Czech RepublicbRegional Centre of Advanced Technologies and Materials, Department of Experimental Physics, Faculty of Science, Palacky University, Slechtitelu 11, 78371 Olomouc, Czech RepubliccDepartment of Microbiology, Faculty of Medicine and Dentistry, Palacky University, Hn�evotínská 3, Olomouc 77520, Czech RepublicdDepartment of Medical Biophysics, Faculty of Medicine and Dentistry, Palacky University, Hn�evotínská 3, Olomouc 77146, Czech Republic

a r t i c l e i n f o

Article history:Received 5 March 2011Accepted 12 March 2011Available online 19 April 2011

Keywords:NanocompositeNanoparticlesSilverMagnetismIron oxidesAntimicrobial agentCytotoxicity

* Corresponding author. Tel.: þ420 585 634 765; fa** Corresponding author. Tel.: þ420 585 634 947; fa

E-mail addresses: robert.prucek@upol.cz (R. P(R. Zbo�ril).

0142-9612/$ e see front matter � 2011 Elsevier Ltd.doi:10.1016/j.biomaterials.2011.03.039

a b s t r a c t

Two types of magnetic binary nanocomposites, Ag@Fe3O4 and g-Fe2O3@Ag, were synthesized andcharacterized and their antibacterial activities were tested. As a magnetic component, Fe3O4

(magnetite) nanoparticles with an average size of about 70 nm and monodisperse g-Fe2O3 (maghe-mite) nanoparticles with an average size of 5 nm were used. Nanocomposites were prepared via in situchemical reduction of silver ions by maltose in the presence of particular magnetic phase and mole-cules of polyacrylate serving as a spacer among iron oxide and silver nanoparticles. In the case of theAg@Fe3O4 nanocomposite, silver nanoparticles, caught at the surfaces of Fe3O4 nanocrystals, werearound 5 nm in a size. On the contrary, in the case of the g-Fe2O3@Ag nanocomposite, ultrafine g-Fe2O3

nanoparticles surrounded silver nanoparticles ranging in a size between 20 and 40 nm. In addition, themolecules of polyacrylate in this nanocomposite type suppress considerably interparticle magneticinteractions as proved by magnetization measurements. Both synthesized nanocomposites exhibitedvery significant antibacterial and antifungal activities against ten tested bacterial strains (minimuminhibition concentrations (MIC) from 15.6 mg/L to 125 mg/L) and four candida species (MIC from1.9 mg/L to 31.3 mg/L). Moreover, acute nanocomposite cytotoxicity against mice embryonal fibroblastswas observed at concentrations of higher than 430 mg/L (Ag@Fe3O4) and 292 mg/L (g-Fe2O3@Ag). Withrespect to the non-cytotoxic nature of the polyacrylate linker, both kinds of silver nanocomposites arewell applicable for a targeted magnetic delivery of silver nanoparticles in medicinal and disinfectionapplications.

� 2011 Elsevier Ltd. All rights reserved.

1. Introduction

Magnetic nanocomposites constitute one of major contributionsof current nanotechnology approaches. When exposed to anexternal magnetic field of various inductions and gradients, thesenanocomposites can be transported purposely to a certain location(e.g., in the human body) and may thus act as effective drugcarriers. As an example, such nanocomposites are composed of ironoxide nanoparticles (Fe3O4 and/or g-Fe2O3 in most cases) serving asmagnetic cores that are covered by a layer of porous silica oxides in

x: þ420 585 634 761.x: þ420 585 634 761.rucek), zboril@prfnw.upol.cz

All rights reserved.

pores of which molecules of particular anticancer drug areemplaced. The surface of this nanocomposite is modified by bothfluorescent polymethacrylate and folic acid as a cancer targetingmoiety [1].

Magnetic nanoparticles of iron oxides (Fe3O4 and/or g-Fe2O3)represent one family of the most suitable candidates for prepara-tion of magnetic nanocomposites owing to their application-convenient magnetic (e.g., superparamagnetism) and biochemical(e.g., non-toxicity, biocompatibility, biodegradability) propertiesand low price. Iron oxide nanoparticles are currently used ascontrast agents in magnetic resonance imagining (MRI) investiga-tions [2]. Another possible application of magnetic iron oxidenanoparticles fastens in the field of hyperthermia cancer treat-ments where hysteresis looses of magnetic nanoparticles generatedduring their cyclic remagnetizations under applied alternatingmagnetic fields are exploited to destroy cancer cells and tissues [3].

R. Prucek et al. / Biomaterials 32 (2011) 4704e4713 4705

Silver nanoparticles represent another important branch ofnanotechnology interest. It is well-known that they exhibitremarkable optical, catalytic and antimicrobial properties [4e8].Nowadays, antimicrobial effects are intensively studied due to anenormously increasing bacterial resistance against excessively andrepeatedly used classical antibiotics. Thus, day after day, thetreatment of bacterial infections utilizing classical antibiotics iscertainly becoming more serious global problem. As an evidence,let us mention the recent discovery of MDM-1 bacteria againstwhich almost all known antibiotics are inefficacious. Since most ofused efficacious antibiotics come from the 70th and 80th of the20th century, it is certainly essential to develop newmedical drugsfor an effective fight with bacteria. Silver nanoparticles may be ofpromising help in this struggle as they effectively eliminate bacteriaat relatively low concentrations of silver nanoparticles; concen-trations that are not toxic for human cells. In addition, bacterialresistance against silver nanoparticles has not been documented sofar. Besides antimicrobial activity [9e13], silver nanoparticles havebeen found effective in the field of surface-enhanced Ramanspectroscopy [14e19] and/or photothermal cancer therapy [20,21].In these areas, silver nanoparticles are more advantageous thantheir relevant rivals as they exhibit a very intense absorption bandthe position of which is dependent on the size and shape of silvernanoparticles.

Combining magnetic iron oxide nanoparticles and silver nano-particles, we thus get nanocomposites possessing all above-mentioned unique properties. For example, such nanocompositeswere utilized as a new type of a broad temporal optical limiter. Due tothe presence of Ag nanoparticles (7 nm) in a nanocomposite withFe3O4 (13 nm), nonlinear scaterring gives rise to enhanced opticallimiting responses to532-nmnanosecond laserpulses,with a limitingthreshold comparable to carbon nanotubes [22]. Combination ofmagnetic and antibacterial features, exhibited by these iron oxi-deesilver nanocomposites, predestinates to exploit them inmedicinewhere they can be used for a targeted transport of antimicrobial agentand its subsequent removal by an external magnetic field.

However, the aggregation instability of nanocomposite nano-particles caused by magnetic and electrostatic interactions isregarded as one of serious problems when synthesizing them andespecially applying them in practice. To prevent their coalescence,one can place them into suitable polymer matrix which can becomposed of, for example, polysaccharides [23], carboxymethyl-cellulose [24,25], sulfonated polyanilines [26], polyethyleniminesor eventually carboxyl polyethylenimines [27], poly(styrene-block-isoprene) [28], amino-terminated poly(amidoamine) dendrimers[29] and polymethacrylic acid [1]. However, in certain cases, poly-mer matrices into which magnetic and/or “active” particles areincorporatedmay evoke a certain limitation of application potentialof synthesized nanocomposites unveiling especially from a restric-tion of their possible transport due to substantial sizes of compactpolymer matrix.

In literature, the synthesis methods of binary iron oxideesilvernanocomposites when polymer matrix is not exploited arerarely described. Ying et al. [30] reported a method for prepa-ration of AgeFe3O4 heterodimeric nanoparticles which weresynthesized by reducing Ag in the presence of magnetite seedsnanoparticles. The same type of nanocomposite was formed bya chemical bond linkage. Li and Liu [31] presented a method forfabrication of Ag/g-Fe2O3 composite particles with a diameter inthe range of 200e300 nm whereas Cho [32] reported a synthesisof magnetic SiO2/Fe3O4/Ag nanostructures with a size of about200 nm.

In the present work, we report on a synthetic procedure ofbinary nanocomposite of the Ag@Fe3O4 and g-Fe2O3@Ag typeemploying polyacrylate acid as an effective linker.

2. Materials and methods

2.1. Chemicals

Following chemicals have been used: Silver nitrate (99.9%, Tamda), sodium salt ofpolyacrylic acid (MW 8000, 45% aqueous solution, SigmaeAldrich), ammonia (p.a.,28% aqueous solution, SigmaeAldrich), sodium hydroxide (p.a., SigmaeAldrich),D(þ)-maltosemonohydrate (p.a., Riedel-deHaën), FeCl2$4H2O (99%, SigmaeAldrich),FeCl3 (99%, SigmaeAldrich) and a-FeOOH nanoparticles (SigmaeAldrich). All chem-icals were used as-received without further purification.

2.2. Synthesis of g-Fe2O3 nanoparticles by hydrolysis of ferrous and ferric salt with10 M NaOH

To prepare g-Fe2O3 nanoparticles, wemodified a procedure reported by Cui et al.[33] Firstly, 172 mg of FeCl2$4H2O and 280 mg of FeCl3 were dissolved in 190 mL ofdeionized water. The amount of respective iron salts was calculated with regard tothe resulting concentration of 1 g/L of g-Fe2O3. Under intensive stirring, 10 mL of10 M NaOHwas added to the solution of iron salts. The color of the solution changedimmediately from orange to dark brown after addition of NaOH. The reactionmixture was then stirred for 1 h. After that, the mixture was stirred for another 1 hwhile placed in water bath at 90 �C. The as-acquired nanoparticles were separatedfrom the solution by an external magnetic field and washed several times bydeionized water.

2.3. Synthesis of Fe3O4 nanoparticles by thermally induced solid-statetransformation of a-FeOOH

Fe3O4 nanoparticles were synthesized according to the well-known sequentialtemperature-induced solid-state reaction employing goethite as a precursor, whichcan be summarized as follows [34,35]:

a� FeOOH/300� C

aira� Fe2O3/

400� C

H2 ;1hFe3O4:

2.4. Synthesis of g-Fe2O3@Ag and Ag@Fe3O4 nanocomposites

20 mg of either g-Fe2O3 or Fe3O4 nanoparticles were added to 160 mL of deion-ized water together with 220 mg of 45% aqueous solution of polyacrylate acid sodiumsalt (molecular weight of 8000 e NaPA 8000). In order to well disperse the iron oxidenanoparticles, the solutionwas placed to an ultrasound bath for 1 min. Subsequently,20 mL of 10�3

M AgNO3 solution and 0.2 mL of 0.1 M aqueous solution of ammoniawere added to the reaction mixture under steady stirring. The pH of the reactionsystem was then adjusted to 11.5 by 0.1 M NaOH solution. Finally, 20 mL of 5$10�2

M

maltose solutionwas added to the reaction systemwhichwas then stirred for 20min.The final volume of the mixture was 200 mL and final concentrations of iron oxides,NaPA 8000, Ag, NH3 and maltose were 100 mg/L, 500 mg/L, 10�4

M, 10�4M and

5$10�3M, respectively. Finally, the as-prepared nanocomposite was separated by an

application of external magnetic field and washed several times by deionized water.

2.5. Characterization techniques

Prior to analyses, samples of iron oxides and nanocomposites were washedseveral times by deionized water and subsequently dried in oven at 90 �C.

Transmission electronmicroscopy (TEM) imageswere obtained using a JEM2010microscope operated at 200 kV. A drop of very dilute suspension was placed ona carbon-coated grid and allowed to dry by evaporation at ambient temperature.

The X-ray powder diffraction (XRD) patterns of all solid samples were recordedon PANalytical X’Pert PRO (The Netherlands) instrument in BraggeBrentanogeometry with Fe-filtered CoKa radiation (40 kV, 30 mA). Samples were placed ona zero-background and rotating single-crystal Si slides, gently pressed in order toobtain sample thickness of about 0.5 mm and scanned in the 2q range of 10e90� insteps of 0.017�. The XRD patterns were evaluated using the X’Pert HighScore Plussoftware (PANalytical), PDF-4þ and ICSD databases.

Zero-field Mössbauer spectra were recorded at 5 and 300 K in a constantacceleration mode with a 50 mCi 57Co(Rh) source. The samples were placed ina cryomagnetic system (Oxford Instruments) and the values of the isomer shift arereported with respect to a-Fe at room temperature.

A superconducting quantum interference device (SQUID, MPMS XL-7, QuantumDesign) has been used for the magnetic measurements. The hysteresis loops werecollected at a temperature of 5 and 300 K in external magnetic fields from �5toþ5 T. The zero-field-cooled (ZFC) and field-cooled (FC) magnetization curves wererecorded on warming in the temperature range from 5 to 300 K and in an externalmagnetic field of 0.1 T after cooling in a zero magnetic field and a field of 0.1 T,respectively.

The content of silver present in the nanocomposites was determined by a PerkinElmer 3300 (Perkin Elmer, USA) device using a method of atomic absorption spec-troscopy with flame AAS ionization.

Fig. 1. (A) TEM image and (B) schematic representation of the g-Fe2O3@Ag nano-composite with the high-resolution inset showing an imminent surrounding of an Agnanoparticle.

R. Prucek et al. / Biomaterials 32 (2011) 4704e47134706

2.6. Antimicrobial testing

Antibacterial and antifungal activities of silver nanocomposites and separatesilver nanoparticles were tested by using of standard microdilution method whichenables to determine the minimum inhibition concentration (MIC) of an antibac-terial substance. The testing was carried out on microtitration plates employinga method when we tested a dispersion of silver nanocomposites 2-to-128 times, inthe geometrical progression, diluted by addition of 100 mL of the Mueller-Hintoncultivation medium inoculated by tested bacteria and yeast strain at a concentrationof 105e106 CFU mL�1. The MIC value, expressing a minimum concentration ofa tested compound that inhibited the growth of tested bacteria and yeasts, wasdetermined after 24 h of incubation at 37 �C. Following bacterial strains, obtainedfrom the Czech Collection of Microorganisms, Czech Republic (Masaryk University inBrno, Czech Republic), were used as standards: Staphylococcus aureus CCM 3953,Enterococcus faecalis CCM 4224, Escherichia coli CCM 3954 and Pseudomonas aeru-ginosa CCM 3955. For testing purposes, we also used bacterial strains isolated fromthe clinical material of the University Hospital in Olomouc, Czech Republic. Thesestrains included P. aeruginosa, Staphylococcus epidermidis, methicilline-resistantS. epidermidis, methicilline-resistant S. aureus (MRSA), vancomycine-resistantEnterococcus faecium (VRE) and ESBL-positive Klebsiella pneumoniae. Antimycoticactivity was tested using Candida albicans (I and II), Candida tropicalis and Candidaparapsilosis strains isolated from the blood of patients of the University Hospital inOlomouc, Czech Republic, who had confirmed candida sepsis. The yeasts wereidentified using conventional mycological procedures: (i) appearance on CHRO-Magar Candida (CHROMagar Microbiology); (ii) micromorphology on the rice agar;and (iii) by assimilation and fermentation tests including the ID 32C kit(bioMérieux).

2.7. Cytotoxicity assay

The cytotoxic effect of silver nanocomposites on NIH3T3 cells was determinedusing the MTT assay. The chemicals used included Dulbecco’s Modified EagleMedium (DMEM), phosphate buffered saline (PBS, pH 7.4 own preparation), 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT, Sigma Aldrich),dimethyl sulfoxide (DMSO, Sigma Aldrich). Measurements were carried out ona multi-detection microplate Synergy HT reader (BioTek, USA). We used 96 wellplates (P-Lab, Czech Republic) for cell lines cultivation, a centrifugal machine(Biotech, Czech Republic) and a glass cover slip (P-Lab, Czech Republic). Duringimpact of silver nanocomposites in concentrations of 100, 50, 25, 12.5, 6.25, 3, 1.5,0.7, 0.3, and 0%, cells were incubated at 37 �C and under 5% CO2 for 6 h. Beforestarting the MTT experiments, we replaced DMEM by PBS containing 5 mM glucose,subsequently added 20 ml 20 mM MTT (dissolved in PBS) and incubated the cells for3 h at 37 �C and under 5% of CO2. The MTT solutionwas carefully removed and 100 mlDMSO was added in order to solubilize theviolet formazan crystals. The absorbanceof the resulting solutionwas measured in a 96-well microplate Synergy HT reader at570 nm and 690 nm. The cell viability of the samples was determined asa percentage of control cell viability (100 times the average of a test group/theaverage of a control group).

3. Results and discussions

3.1. Synthesis and characterization of the g-Fe2O3@Agnanocomposite

The nanocomposite of the g-Fe2O3@Ag type was synthesized byreduction of silver ions in the presence of polyacrylatewith a relativemolecule weight of 8000 and addition of g-Fe2O3 nanoparticlessynthesized by a method mentioned in Section 2.2. RepresentativeTEM image of the g-Fe2O3@Ag nanocomposite (see Fig. 1A) revealedapresenceof silvernanoparticleswith sizes ranging from20 to40nmthat were surrounded and separated from each other by aggregatesof g-Fe2O3 nanoparticles with an average particle size of about 5 nm(see the schematic picture of the g-Fe2O3@Ag nanocomposite inFig.1B). The presence of both crystalline components was confirmedby an analysis of the corresponding XRD pattern (data not shown).

After washing and subsequent magnetic separation of theg-Fe2O3@Ag nanocomposite, the content of silver in the nano-composite was found to be equal to 10.5% (w/w) employing theASS method. Comparison of this value with the amount of silverincorporated into the nanocomposite during synthesis indicatesthat 70% of silver was bounded with g-Fe2O3 nanoparticles.

Zero-field 57Fe Mössbauer spectroscopy was exploited both tocheck the phase purity of iron oxide in the nanocomposite and

determine its magnetic properties. At 300 K (not shown), theMössbauer spectrum of the g-Fe2O3@Ag nanocomposite consists ofonly a doublet with the isomer shift d ¼ (0.34 � 0.01) mm/s andquadrupole splittingDEQ¼ (0.63� 0.01)mm/s. Since there is no signof any sextet component, all the iron oxide nanoparticles are ina superparamagnetic state with respect to the measuring time ofMössbauer spectroscopy. At 5 K, an asymmetric sextet is registeredas the only one spectral component (see Fig. 2A) with theaverage hyperfine parameters d ¼ (0.43 � 0.01) mm/s,DEQ ¼ (0.00 � 0.01) mm/s and hyperfine magnetic fieldBhf ¼ (51.1 � 0.3) T being close to the respective values reported forg-Fe2O3 [35]. At this temperature, the magnetic moments of all ironoxide nanoparticles in the nanocomposite are thus magneticallyblocked. The analysis of the Mössbauer spectra of Ag-contaningnanocomposite demonstrates that adding of sodium polyacrylateand Ag during the synthesis does not affect the iron oxide origin andevoke formation of any other iron oxide admixture. Thus, within the

Fig. 2. (A) Zero-field low-temperature Mössbauer spectrum, (B) hysteresis loops and(C) ZFC/FC magnetization curves of the g-Fe2O3@Ag nanocomposite.

R. Prucek et al. / Biomaterials 32 (2011) 4704e4713 4707

experimental error of the Mössbauer technique, g-Fe2O3 constitutesthe only one Fe-bearing phase in this Ag-containing nanocomposite.

The macroscopic magnetic properties of the preparedg-Fe2O3@Ag nanocomposite were monitored by measuring thehysteresis loops at a temperature of 5 and 300 K and ZFC/FCmagnetization curves under an external magnetic field of 0.1 T. Theresults are graphically depicted in Fig. 2B and c and correspondinghysteresis parameters are summarized in Table 1. Both hysteresisloops and ZFC/FC magnetization measurements demonstrate thatthe g-Fe2O3@Ag nanocomposite exhibits a transition from a super-paramagnetic state to a blocked regime on lowering the tempera-ture. Due to the small size of magnetic nanoparticles, the overallmagnetic features of the g-Fe2O3@Ag nanocomposite are domi-nantly driven by finite-size and surface effects as manifested bya reduced value of the maximum magnetization under 5 T andcoercive field in comparison with the corresponding values ofhysteresis parameters reported for bulk g-Fe2O3 at low tempera-tures. In addition, at 5 K, the hysteresis loop of the g-Fe2O3@Agnanocomposite is perfectly symmetric (see Fig. 2B) implying anabsence of the so-called exchange bias phenomenon frequentlyevolving in the case of a significant difference in the magnetichardness of the core and surface layers of the nanoparticle. As theexchange bias phenomenon is, among other factors, triggered bya presence of interparticle magnetic interactions, this thus suggeststhat the strength of interparticle magnetic interactions of anexchange origin seem to be reduced.

The fact that interparticle magnetic interactions are significantlysuppressed in the g-Fe2O3@Ag nanocomposite is further docu-mented by the profile of the FC magnetization curve that still keepsincreasing as the temperature lowers below the maximum in theZFC magnetization curve recognized as the average blockingtemperature (TB) of the magnetic fraction (the temperature belowwhich g-Fe2O3 nanoparticles with the most probable size in theirassembly enter the magnetically blocked regime) [36]. Further-more, the closeness of TB and Tirr (irreversibility temperature atwhich the ZFC and FC magnetization curves diverge from eachother, corresponding to the blocking of the largest g-Fe2O3 nano-particles in the nanocomposite) values (see Fig. 2C) predicatesa narrow particle size distribution of g-Fe2O3 nanoparticles in thenanocomposite which indicates that the synthesized g-Fe2O3@Agnanocomposite predominantly consists of well separated iron(III)oxide nanoparticles rather than their agglomerates.

Thus, it seems that the sodium-salt-based polyacrylate acts asa spacer (like a matrix with pores of definite size) which holds themagnetic nanoparticles apart. This significantly reduces the possi-bility of formation of agglomerates and consequently, the evolutionof interparticle magnetic interaction of exchange type. However,the interparticle magnetic interactions still exist in the nano-composite as documented by a slight bending of the FC magneti-zation curve below TB. These are of dipolar type, taking place viamagnetic moments of magnetic nanoparticles not necessarily ina close contact, which are considered as being much weaker thatinterparticle magnetic interactions of exchange nature [36].Nevertheless, the effect of interparticle magnetic interactions onmagnetic properties of g-Fe2O3@Ag nanocomposite is significantlysuppressed and the system behaves similarly as that composed ofalmost non-interacting magnetic nanoparticles. In addition, asdocumented by the room-temperature hysteresis loop, theg-Fe2O3@Ag nanocomposite exhibits a strong magnetic responseeven at a field of 1 T, being thus possibly controlled by a simplemagnet.

Since the sizes of g-Fe2O3 nanoparticles are in the range ofseveral units of nanoparticles, it is highly improbable that underused reaction conditions, silver nanoparticles with sizes of 1e2 nmwould form on the surface of g-Fe2O3 nanoparticles. In this case, as

Table 1Hysteresis parameters derived from the hysteresis loops measured for the g-Fe2O3@Ag and Fe3O4@Ag nanocomposite, where T is the temperature of measurement, Mmaxþdenotes the maximum magnetization under þ5 T, Mmax� stands for the maximum magnetization under �5 T, BCþ represents the positive coercive field, BCe is the negativecoercive field, MRþ denotes the positive remanent magnetization and MRe represents the negative remanent magnetization. The values of Mmaxþ, Mmax�, MRþ and MRe arenormalized to the overall weight of the corresponding sample.

Nanocomposite T (K) Mmaxþ � 0.01 (Am2/g) Mmax� � 0.01 (Am2/kg) BCþ � 0.0001 (T) BCe � 0.0001 (T) MRþ � 0.01 (Am2/kg) MRe � 0.01 (Am2/kg)

g-Fe2O3@Ag 5 35.85 �35.85 0.0211 �0.0211 2.36 �2.36300 21.92 �21.92 e e e

Fe3O4@Ag 5 66.80 �66.80 0.0438 �0.0586 31.58 �27.87300 60.90 �60.90 0.0122 �0.0122 10.68 �10.68

R. Prucek et al. / Biomaterials 32 (2011) 4704e47134708

proved by TEM and XRD analyses, ultrafine g-Fe2O3 nanoparticlesacted as a “particle matrix” surrounding silver nanoparticles (seeFig. 3). In order to prevent an evolution of magnetic interactionsamong g-Fe2O3 nanoparticles, polyacrylate acid sodium salt wasadded to the reaction system. As discussed above, magnetizationmeasurements (especially profiles of ZFC/FC magnetization curves)indicate the suppression of magnetic interactions among g-Fe2O3nanoparticles, confirming thus anti-agglomeration role of sodiumsalt of polyacrylic acid. A considerable number of dissociatedcarboxyl groups of this organic compound that are adsorbed on thesurfaces of g-Fe2O3 nanoparticles avoid their aggregation due toelectrostatic interactions. Free carboxyl groups, non-adsorbed onthe surfaces of g-Fe2O3 nanoparticles, attract free silver ions to theproximity of g-Fe2O3 nanoparticle surface and silver nanoparticlesthen form by reduction of these silver ions. In order to maximallysuppress the possibility of formation of silver nanoparticles inplaces not close to surfaces of g-Fe2O3 nanoparticles, ammonia wasadded to the reaction system. Ammonia forms a relatively stable

Fig. 3. Schematic representation of the reaction steps leadin

complex with silver ions (b1 ¼3.31 and b2 ¼ 7.22). At this place, oneshould stress that ammonia was added only in equimolar amountwith respect to silver ions. Thus, only part of silver ions were boundto [Ag(NH3)]þ and [Ag(NH3)2]þ complex and rest of silver ions wereleft to carboxyl groups of polyacrylate acid sodium salt. Thebonding of certain part of silver ions prevents their extensivereduction except places close to surfaces of g-Fe2O3 nanoparticles.After formation of silver nuclei, this ammonia complex serves asa “reservoir” of silver ions for their subsequent growth. The reasonwhy larger silver nanoparticles form can be explained adoptinghypothesis that g-Fe2O3 nanoparticles with a significantly highsurface area (due to increased surface-to-volume ratio for 3e6 nmlarge nanoparticles) bind to carboxyl groups of polyacrylate leavingless free carboxyl groups for binding of free silver ions in theproximity of surfaces of g-Fe2O3 nanoparticles. Since silver ionsbound to free carboxyl groups are reduced to silver nuclei, smallernumber of these silver nuclei results in formation of silver nano-particles with sizes of several tens of nanometers.

g to the preparation of the g-Fe2O3@Ag nanocomposite.

Fig. 4. (A) TEM image and (B) schematic representation of the Ag@Fe3O4 nano-composite with the high-resolution inset showing an imminent surrounding of anFe3O4 nanoparticle.

R. Prucek et al. / Biomaterials 32 (2011) 4704e4713 4709

3.2. Synthesis and characterization of Ag@Fe3O4 nanocomposite

To prepare the Ag@Fe3O4 nanocomposite, we used Fe3O4nanoparticles synthesized from a goethite precursor as mentionedin the Section 2.3. The synthesis procedure of this nanocompositewas carried out in the same manner as in the case of theg-Fe2O3@Ag nanocomposite. TEM image of Ag@Fe3O4 nano-composite (see Fig. 4A) showed an existence of silver nanoparticleswith an average size of z5 nm residing on the surfaces of Fe3O4nanoparticles with an average size of z70 nm (see the schematicpicture of the Ag@Fe3O4 nanocomposite in Fig. 4B). XRD analysis(data not shown) then confirmed the presence of both metallicsilver and Fe3O4. In this case, only 6% (w/w) of metallic Ag wascalculated from the quantitative phase analysis.

Similarly as in the case of the g-Fe2O3@Ag nanocomposite, 57FeMössbauer spectroscopy was used to identify the type of iron oxidein the nanocomposite and state its magnetic properties. At 300 K(see Fig. 5A), the Mössbauer spectrum of this nanocomposite israther complicated in contrast to the previously studied sample.With regard to the measuring time of Mössbauer technique, theappearance of sextets and absence of any doublet component implythat all nanoparticles of iron oxides are in a magnetically blockedstate at room temperature. The mathematical deconvolution of theroom-temperature Mössbauer spectrum leads to the two sextetcomponents with the hyperfine parameter values close to thosereported for bulk Fe3O4. In the case of Fe3O4 above the Verweytransition temperature, the two sextets are expected fulfilling theassumption of a spinel structure with the two nonequivalent crys-tallographic ironpositions. The Sextet 1with d¼ (0.32� 0.01)mm/s,DEQ ¼ (0.01 � 0.01) mm/s and Bhf ¼ (48.9 � 0.3) T thus correspondsto Fe3þ ions occupying the tetrahedral sites in the crystal structure ofFe3O4 whereas the Sextet 2 with d ¼ (0.60 � 0.01) mm/s,DEQ ¼ (0.01 � 0.01) mm/s and Bhf ¼ (45.1 � 0.5) T is ascribed to theFe ions with a valence state between 2þ and 3þ situated at theoctahedral sites of the Fe3O4 crystal structure. In the case of stoi-chiometric Fe3O4, an effective valence state of 2.5þ is observed forFe2þ and Fe3þ ions occupying neighboring octahedral sites [34]. Ifwe assume that the room-temperature ratio of the recoil-free frac-tions for the octahedral (O) and tetrahedral (T) sites is 0.97, theintensity ratio a¼ I(O)/I(T) for a perfect stoichiometric Fe3O4 shouldbe 1.94. In our case, a ¼ (1.14 � 0.01) which indicates that we havea non-stoichiometric Fe3O4 with a certain degree of cation vacanciesat the octahedral sites of the Fe3O4 crystal structure. Since any otheriron oxide admixtures were not detected in the room-temperatureMössbauer spectrum of this nanocomposite, bearing in mind theexperimental error ofMössbauer technique, one can conclude that itconsists solely of non-stoichiometric Fe3O4 nanoparticles with sizesmuch larger than in the case of g-Fe2O3 nanoparticles present in theg-Fe2O3@Ag nanocomposite.

The macroscopic magnetic properties of the prepared Ag@Fe3O4nanocomposite were again monitored by measuring the hysteresisloops at a temperature of 5 and 300 K and ZFC/FC magnetizationcurves under an external magnetic field of 0.1 T. The results aregraphically depicted in Fig. 5B and C and corresponding hysteresisparameters are summarized in Table 1. Analysis the hysteresis loopsof the Ag@Fe3O4 nanocomposite, at 300 K, a significant portion ofFe3O4 nanoparticles stays still in the magnetically blocked state asevidenced by non-zero values of coercivity and remanent magne-tization. This is in contrast to what we have observed in the case ofthe g-Fe2O3@Ag nanocomposite where all magnetic nanoparticleshave already entered the superparamagnetic regime below 300 K.This confirms bigger average size and different particle size distri-bution of Fe3O4 nanoparticles compared to size characteristics ofg-Fe2O3 nanoparticles as already derived from TEM and XRDanalyses. Similarly as for the g-Fe2O3@Ag nanocomposite, finite-

size effects most probably accompanied by interparticle magneticinteractions cause a reduction in the value of the maximummagnetization under 5 T compared to that expected for bulk Fe3O4(z95 Am2/kg). However, note that the magnetization values arenormalized to the weight of the whole sample including Fe3O4nanoparticles, sodium-salt-based polyacrylate and Ag nano-particles. Thus, the reduced value of the maximum magnetizationof the Ag@Fe3O4 nanocomposite partly reflects the presence ofsodium-salt-based polyacrylate and Ag nanoparticles which bothbehave as diamagnetic compounds. Nevertheless, from the appli-cation point of view, the Ag@Fe3O4 nanocomposite exhibits a strongmagnetic response achievable at relatively low applied magneticfields. The profile of the ZFC/FCmagnetization curves (see Fig. 5C) istypical of magnetic nanoparticles with wide particle size distribu-tion. Contrary to the g-Fe2O3@Ag nanocomposite, Tirr is found tooccur above 300 K which well corresponds with the hysteresisobserved for the room-temperature loop. A bigger average size ofFe3O4 nanoparticles is documented by a blocking temperature ofz166 K being much higher than TB derived for the g-Fe2O3@Agnanocomposite. The presence of interparticle magnetic interactionsacting here as a driving force responsible for a nanoparticle

Fig. 5. (A) Zero-field room-temperature Mössbauer spectrum, (B) hysteresis loops and(C) ZFC/FC magnetization curves of the Ag@Fe3O4 nanocomposite.

R. Prucek et al. / Biomaterials 32 (2011) 4704e47134710

aggregation is reflected in the FCmagnetization curvewhich slowlyapproaches a constant character as the temperature lowers [36].This implies that the sodium-salt-based polyacrylate does notproperly cover the surfaces of magnetic nanoparticles most prob-ably due to their small surface-to-volume ratio. Then, in order toreduce their total energy, magnetic nanoparticles come to eachother and form aggregates. In addition, the FC magnetization curvedoes not exhibit a decrease in magnetization suggesting an absenceof Verwey transition down to 5 K. Along with the results of analysisof room-temperature Mössbauer spectrum of the Ag@Fe3O4nanocomposite, this confirms a non-stoichiometry of Fe3O4 nano-particles which manifest itself by a shifting the Verwey transitiontemperature from z120 K (typical of bulk Fe3O4) to much lowertemperatures.

After washing and subsequent magnetic separation of theAg@Fe3O4 nanocomposite, the content of silver was determined tobe 5.8 (w/w) as documented by the ASS method. Comparing thisvalue with the amount of silver incorporated in the beginning ofnanocomposite synthesis, it follows that 36% of amount of silverwas bounded to Fe3O4 nanoparticles. In contrast to the g-Fe2O3@Agnanocomposite, the synthesis yield is lower. For the Ag@Fe3O4

nanocomposite, we observed a smaller size of bounded silvernanoparticles. This can be ascribed to a larger size of Fe3O4 nano-particles having a smaller surface area providing less bonds forcarboxyl groups of polyacrylate. Thus, a larger number of thesecarboxyl groups are left for free silver ions resulting in a largernumber of silver nuclei which turn into silver nanoparticles withmuch smaller sizes (see Fig. 6). Nevertheless, due to the smallersurface area of Fe3O4 nanoparticles, a significant number ofunbounded polyacrylate molecules are present in the reactionsystem. As a consequence, the silver nanoparticles also form inplaces off surfaces of Fe3O4 nanoparticles, which then leads toa smaller yield mentioned already above.

3.3. Antibacterial and antifungal assay

Antibacterial and antifungal activities of the synthesizedAg@Fe3O4 and g-Fe2O3@Ag nanocomposites were determined bymeans of a standard dilution method which enables to state theminimum concentration of the tested compound needed fora growth inhibition of tested bacteria and yeasts. For testing ofantimicrobial activity, we prepared concentrated aqueous disper-sion of the Ag@Fe3O4 and g-Fe2O3@Ag nanocomposites ata concentration of 1 g/L. The final mass concentrations of silverpresent in such concentrated dispersions of the studied nano-composites corresponded to the values of 58 mg/L (5.8% w/w of Agfrom AAS) and 105 mg/L (10.5% w/w of Ag from AAS) for theAg@Fe3O4 and g-Fe2O3@Ag nanocomposite, respectively. Final MICvalues of the investigated nanocomposites and MIC values relatedto the concentration of silver contained in the respective nano-composite (i.e., Ag-related MIC values) are listed in Table 2. The Ag-related MIC values were calculated in order to compare them withthose of 26 nm silver nanoparticles synthesized bymodified Tollensprocess based on reduction of [Ag(NH3)2]þ bymaltose in an alkalinemedium [37]. From the values of MIC, it follows that both nano-composites exhibit a high antibacterial and antifungal activities.The MIC values acquired for both nanocomposites fall into rangefrom 15.6 mg/L to 125 mg/L in the case of bacteria and from 1.9 mg/L to 31.3 mg/L in the case of yeasts. If related to the concentration ofsilver in the respective nanocomposite, Ag-related MIC values ofboth nanocomposites were foundwithin the range from0.9mg/L to13.2 mg/L of silver for bacteria and from 0.1 mg/L to 3.3 mg/L ofsilver for yeasts, depending on a type of the tested microbe. In thecase of the Ag@Fe3O4 nanocomposites, the Ag-related MIC valuesare even lower than those reported for 26 nm silver nanoparticles.

Fig. 6. Schematic representation of the reaction steps leading to the preparation of the Ag@Fe3O4 nanocomposite.

Table 2MIC values of the Ag@Fe3O4 and g-Fe2O3@Ag nanocomposites against testedbacteria and yeasts. For comparison purposes with the MIC values of 26 nm silvernanoparticles, the Ag-related MIC values of nanocomposites are also listed.

Tested bacteria and yeasts MIC of nanocomposite/MIC of silver (mg/L)

MIC of silvernanoparticles (mg/L)

Ag@Fe3O4/Ag g-Fe2O3@Ag/Ag Ag NPs (26 nm)

Enterococcus faecalisCCM 4224

125/7.2 125/13.2 6.8

Staphylococcus aureusCCM 3953

31.3/1.8 62.5/6.6 3.4

Escherichia coliCCM 3954

15.6/0.9 15.6/1.6 1.7

PseudomonasaeruginosaCCM 3955

15.6/0.9 62.5/6.6 3.4

Pseudomonas aeruginosa532

15.6/0.9 31.3/3.3 1.7

Staphylococcus epidermidis1879

31.3/1.8 31.3/3.3 1.7

Staphylococcus epidermidis2901

31.3/1.8 31.3/3.3 3.4

Staphylococcus aureus(4591MRSA)

62.5/3.6 125/13.2 6.8

Enterococcus faecium(1324 VRE)

62.5/3.6 125/13.2 3.4

Klebsiella pneumoniae(2486 ESBL)

31.3/1.8 31.3/3.3 3.4

Candida albicans I 1.9/0.1 1.9/0.2 0.2Candida albicans II 1.9/0.1 1.9/0.2 0.2Candida tropicalis 5 3.9/0.2 31.3/3.3 0.4Candida parapsilosis 6 7.8/0.5 31.3/3.3 0.8

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On the contrary, for the g-Fe2O3@Ag nanocomposite, the Ag-relatedMIC values are slightly higher than those observed for 26 nm silvernanoparticles. The different Ag-related MIC values reflectingdifferent antibacterial and antifungal activity of these nano-composites can be explained by different sizes of silver nano-particles present in the respective nanocomposite. Thenanocomposite of the Ag@Fe3O4 type consists of silver nano-particles with an average size of z5 nm exhibiting thus a highantimicrobial activity compared to aqueous dispersion of 26 nmsilver nanoparticles, whereas g-Fe2O3@Ag nanocomposite iscomposed of silver nanoparticles with an average size of z30 nmshowing thus lower antimicrobial activity than that of aqueousdispersion of 26 nm silver nanoparticles. Thus, it turns out thatantibacterial activity of silver nanoparticles depends on their size aspublished earlier [37e39].

Taking into account the acquired results, it is evident that silvernanoparticles incorporated into a nanocomposite practically do notloose their antimicrobial properties as they are comparable withthat of silver nanoparticles themselves [37]. Similarly, synthesizediron oxide nanoparticles preserve their magnetic properties whenforming a nanocomposite. This is important from the viewpoint ofapplication potential of these nanocomposites in, for example,medicine. Combination of magnetic properties of iron oxidenanoparticles and antimicrobial features of silver predestinatesthese nanocomposites for exploitation in human medicine wherethey can be used for targeted transport of antimicrobial drug and/orfor its removal by an external magnetic field. With regard to theiruniquemagnetic and antibacterial properties, they can be exploited

Table 3Comparison of antimicrobial activity and cytotoxicity of synthesized Ag@Fe3O4 andg-Fe2O3@Ag nanocomposites against tested microbes (MIC) and mouse fibroblasts(24 h LC50).

MIC (mg/L) 24 h LC50 (mg/L)

Ag@Fe3O4 1.9e125 430g-Fe2O3@Ag 1.9e125 292

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at their low concentrations so that their toxic effect on human cellsis minimized. In literature, no nanocomposite with such a highantibacterial activity has been reported so far. Dallas et al. [40]described a synthetic procedure of magnetic nanocompositebased on g-Fe2O3 and silver nanoparticles incorporated intomultifunctional phosphotriazine matrix, however, the lowest MICvalue of this nanocompositewere found to bez80mg/L dependingon the bacteria type. In our case, both investigated nanocompositesexhibit MIC values as low as 15 mg/L for tested bacteria, concludingthat the Ag@Fe3O4 and g-Fe2O3@Ag nanocomposites possess anantimicrobial activity several times higher. Moreover, both kinds ofnanocomposites work with the biocompatible polyacrylate linker,which extends their applicability for a targeted delivery inbiomedical and disinfection areas.

3.4. Cytotoxicity assay

Cytotoxic activity of the synthesized nanocomposites was testedby means of an MTT test against mice embryonal fibroblasts.Cytotoxicity was monitored for the Ag@Fe3O4 and g-Fe2O3@Agnanocomposites and for the Fe3O4 and g-Fe2O3 phases as

Fig. 7. Dependence of the cell viability on the concentration of the (A) g-Fe2O3@Ag and(B) Ag@Fe3O4 nanocomposite.

a verification. Cytotoxity was determined on the basis of viability ofcells in dependence on the nanocomposite concentration. In thisway, we obtained dependence of toxic activity of tested compoundon its concentration. Once this dependence was known, we eval-uated 24 h LC50 toxic index. The dependence of viability of cells onnanocomposite concentration is depicted in Fig. 7. As one can see,the Ag@Fe3O4 and g-Fe2O3@Ag nanocomposites exhibit differentlimits of toxic activity against tested fibroblasts. For the Ag@Fe3O4nanocomposite, we observed a weak (pale) cytotoxicity activity ata concentration above 125 mg/L; a significant cytotoxicity activitywas observable for the highest tested concentration (i.e., 500mg/L).The determined 24 h LC50 toxic index was equal to 430 mg/L forAg@Fe3O4 nanocomposite. For the g-Fe2O3@Ag nanocomposite,cytotoxic activity already appears at a concentration of 31.3 mg/L,however, the fall in surviving cells decreases below 50% ata concentration of 250mg/L and higher. As far as the 24 h LC50 toxicindex of the g-Fe2O3@Ag nanocomposite is concerned, we found itto be equal to 292 mg/L, a somewhat lower than that for Ag@Fe3O4nanocomposite. The different values of the 24 h LC50 toxic indexare explainable taking into account different amount of silver in therespective nanocomposite; a higher cytotoxicity activity of theg-Fe2O3@Ag nanocomposite is governed by a higher amount ofpresent silver (10.5% w/w in comparison to 5.8% for the Ag@Fe3O4nanocomposite). This is supported by a fact that Fe3O4 and g-Fe2O3did not exhibit cytotoxicity activity against tested fibroblast;a slight cytotoxicity activity of iron oxide phases was observed atthe highest concentration of 500 mg/L of Fe3O4 and g-Fe2O3;however, 70% of cells have been surviving in this case. It thusfollows that the 24 h LC50 toxic indices of Fe3O4 and g-Fe2O3 areexpected to be equal to values much higher than 500 mg/L.

When comparing theMIC and 24hod LC50 values of synthesizednanocomposites, one can conclude that both nanocompositeseffectively destroy microorganisms at concentrations far below theLC50 values (see Table 3), i.e., concentrations that are not toxic formammal (eukaryotic) cells. In most cases, this difference is of anorder which is appealing from the viewpoint of exploitation ofthese nanocomposites in various biomedical applications whereboth high antimicrobial activity and low cytotoxicity ofa compound is highly required. From this aspect, our resultsdemonstrate that the Ag@Fe3O4 nanocomposite is a more effectiveantimicrobial agent since, regardless of its lower content of silverand thus its lower cytotoxicity, it effectively destroys pathogenicmicroorganisms due to a smaller size of its silver nanoparticlescompared to that found for the g-Fe2O3@Ag nanocomposite.

4. Conclusion

In this work, we synthesized and characterized in details twotypes of magnetic nanocomposites exhibiting high antimicrobialactivities e Ag@Fe3O4 and g-Fe2O3@Ag. Molecules of polyacrylatewith a relative molar mass of 8000 were exploited as a spaceramong iron oxide and silver nanoparticles, synthesized via in situchemical reduction by maltose. In the case of the Ag@Fe3O4nanocomposite, ultrasmall silver nanoparticles (z5 nm) werecaught at the surfaces of Fe3O4 magnetic cores (z70 nm) and theirweight content was determined to be equal to 5.8%. The

R. Prucek et al. / Biomaterials 32 (2011) 4704e4713 4713

g-Fe2O3@Ag nanocomposite revealed a higher content (10.5%) oflarger silver nanocores (20e40 nm), which are surrounded byultrasmall g-Fe2O3 nanomagnets (z5 nm). Both studied nano-composites possess eminent magnetic properties (e.g., high valueof magnetization achievable at relatively low applied fields,superparamagnetic and soft magnetic behavior at room tempera-ture from the viewpoint of SQUID measurements, suppression ofinterparticle magnetic interactions) since they are very easilycontrolled by a low external magnetic field. Both synthesizednanocomposites also exhibited the highest antibacterial and anti-fungal activities among all magnetic silver nanocomposites devel-oped so far. At the observed minimum inhibition concentrations,both investigated nanocomposites did not exhibit acute cytotox-icity against mice embryonal fibroblasts. The combination ofmagnetic properties of iron oxide nanoparticles and antimicrobialfeatures of nanosilver thus predestinates these nanocomposites tobe possibly used in disinfection and biomedical applications wherethey can be exploited for a targeted transport of an antimicrobialagent and its subsequent removal bymeans of an external magneticfield.

Acknowledgments

The authors gratefully acknowledge the support by the Opera-tional Program Research and Development for Innovations e

European Social Fund (Project No. CZ.1.05/2.1.00/03.0058 andCZ.1.05/2.1.00/01.0030of theMinistryof Education, Youth and Sportsof theCzechRepublic). Thiswork has been supported by theMinistryof Education, Youth and Sports of the Czech Republic (ProjectsNo. 1M6198959201, MSM6198959218 and MSM6198959223),the Academy of Sciences of the Czech Republic (ProjectNo. KAN115600801) and the Czech Science Foundation (ProjectNo. GAP304/10/1316).

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