Biomaterials 32 (2011) 4704e4713

Contents lists available at ScienceDirect

Biomaterials
journal homepage: www.elsevier.com/locate/biomaterials

The targeted antibacterial and antifungal properties of magnetic nanocomposite

of iron oxide and silver nanoparticles
Robert Prucek a, *, Jirí Tu
cek b, Martina Kilianová a, Ales Paná
cek a, Libor Kvítek a, Jan Filip b, Milan Kolár c,
Katerina Tománková d, Radek Zboril a, **
a
Regional Centre of Advanced Technologies and Materials, Department of Physical Chemistry, Faculty of Science, Palacky University, Slechtitelu 11, 78371 Olomouc, Czech Republic
b
Regional Centre of Advanced Technologies and Materials, Department of Experimental Physics, Faculty of Science, Palacky University, Slechtitelu 11, 78371 Olomouc, Czech Republic
c
Department of Microbiology, Faculty of Medicine and Dentistry, Palacky University, Hn
evotínská 3, Olomouc 77520, Czech Republic
d
Department 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 a b s t r a c t

Article history: Two types of magnetic binary nanocomposites, Ag@Fe3O4 and g-Fe2O3@Ag, were synthesized and
Received 5 March 2011 characterized and their antibacterial activities were tested. As a magnetic component, Fe3O4
Accepted 12 March 2011 (magnetite) nanoparticles with an average size of about 70 nm and monodisperse g-Fe2O3 (maghe-
Available online 19 April 2011
mite) nanoparticles with an average size of 5 nm were used. Nanocomposites were prepared via in situ
chemical reduction of silver ions by maltose in the presence of particular magnetic phase and mole-
Keywords:
cules of polyacrylate serving as a spacer among iron oxide and silver nanoparticles. In the case of the
Nanocomposite
Ag@Fe3O4 nanocomposite, silver nanoparticles, caught at the surfaces of Fe3O4 nanocrystals, were
Nanoparticles
Silver
around 5 nm in a size. On the contrary, in the case of the g-Fe2O3@Ag nanocomposite, ultrafine g-Fe2O3
Magnetism nanoparticles surrounded silver nanoparticles ranging in a size between 20 and 40 nm. In addition, the
Iron oxides molecules of polyacrylate in this nanocomposite type suppress considerably interparticle magnetic
Antimicrobial agent interactions as proved by magnetization measurements. Both synthesized nanocomposites exhibited
Cytotoxicity very significant antibacterial and antifungal activities against ten tested bacterial strains (minimum
inhibition concentrations (MIC) from 15.6 mg/L to 125 mg/L) and four candida species (MIC from
1.9 mg/L to 31.3 mg/L). Moreover, acute nanocomposite cytotoxicity against mice embryonal fibroblasts
was observed at concentrations of higher than 430 mg/L (Ag@Fe3O4) and 292 mg/L (g-Fe2O3@Ag). With
respect to the non-cytotoxic nature of the polyacrylate linker, both kinds of silver nanocomposites are
well applicable for a targeted magnetic delivery of silver nanoparticles in medicinal and disinfection
applications.
Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction pores of which molecules of particular anticancer drug are

Magnetic nanocomposites constitute one of major contributions
of current nanotechnology approaches. When exposed to an
external magnetic field of various inductions and gradients, these
nanocomposites can be transported purposely to a certain location
(e.g., in the human body) and may thus act as effective drug
carriers. As an example, such nanocomposites are composed of iron
oxide nanoparticles (Fe3O4 and/or g-Fe2O3 in most cases) serving as
magnetic cores that are covered by a layer of porous silica oxides in
* Corresponding author. Tel.: þ420 585 634 765; fax: þ420 585 634 761.
** Corresponding author. Tel.: þ420 585 634 947; fax: þ420 585 634 761.
E-mail addresses: robert.prucek@upol.cz (R. Prucek), zboril@prfnw.upol.cz
(R. Zboril).
emplaced. The surface of this nanocomposite is modified by both
fluorescent polymethacrylate and folic acid as a cancer targeting
moiety [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) properties
and low price. Iron oxide nanoparticles are currently used as
contrast agents in magnetic resonance imagining (MRI) investiga-
tions [2]. Another possible application of magnetic iron oxide
nanoparticles fastens in the field of hyperthermia cancer treat-
ments where hysteresis looses of magnetic nanoparticles generated
during their cyclic remagnetizations under applied alternating
magnetic fields are exploited to destroy cancer cells and tissues [3].

0142-9612/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biomaterials.2011.03.039
 R. Prucek et al. / Biomaterials 32 (2011) 4704e4713
Silver nanoparticles represent another important branch of
nanotechnology interest. It is well-known that they exhibit
remarkable optical, catalytic and antimicrobial properties [4e8].
Nowadays, antimicrobial effects are intensively studied due to an
enormously increasing bacterial resistance against excessively and
repeatedly used classical antibiotics. Thus, day after day, the
treatment of bacterial infections utilizing classical antibiotics is
certainly becoming more serious global problem. As an evidence,
let us mention the recent discovery of MDM-1 bacteria against
which almost all known antibiotics are inefficacious. Since most of
used efficacious antibiotics come from the 70th and 80th of the
20th century, it is certainly essential to develop new medical drugs
for an effective fight with bacteria. Silver nanoparticles may be of
promising help in this struggle as they effectively eliminate bacteria
at relatively low concentrations of silver nanoparticles; concen-
trations that are not toxic for human cells. In addition, bacterial
resistance against silver nanoparticles has not been documented so
far. Besides antimicrobial activity [9e13], silver nanoparticles have
been found effective in the field of surface-enhanced Raman
spectroscopy [14e19] and/or photothermal cancer therapy [20,21].
In these areas, silver nanoparticles are more advantageous than
their relevant rivals as they exhibit a very intense absorption band
the position of which is dependent on the size and shape of silver
nanoparticles.
Combining magnetic iron oxide nanoparticles and silver nano-
particles, we thus get nanocomposites possessing all above-
mentioned unique properties. For example, such nanocomposites
were utilized as a new type of a broad temporal optical limiter. Due to
the presence of Ag nanoparticles (7 nm) in a nanocomposite with
Fe3O4 (13 nm), nonlinear scaterring gives rise to enhanced optical
limiting responses to 532-nm nanosecond laser pulses, with a limiting
threshold comparable to carbon nanotubes [22]. Combination of
magnetic and antibacterial features, exhibited by these iron oxi-
deesilver nanocomposites, predestinates to exploit them in medicine
where they can be used for a targeted transport of antimicrobial agent
and its subsequent removal by an external magnetic field.
However, the aggregation instability of nanocomposite nano-
particles caused by magnetic and electrostatic interactions is
regarded as one of serious problems when synthesizing them and
especially applying them in practice. To prevent their coalescence,
one can place them into suitable polymer matrix which can be
composed of, for example, polysaccharides [23], carboxymethyl-
cellulose [24,25], sulfonated polyanilines [26], polyethylenimines
or 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 are
incorporated may evoke a certain limitation of application potential
of synthesized nanocomposites unveiling especially from a restric-
tion of their possible transport due to substantial sizes of compact
polymer matrix.
In literature, the synthesis methods of binary iron oxideesilver
nanocomposites when polymer matrix is not exploited are
rarely described. Ying et al. [30] reported a method for prepa-
ration of AgeFe3O4 heterodimeric nanoparticles which were
synthesized by reducing Ag in the presence of magnetite seeds
nanoparticles. The same type of nanocomposite was formed by
a chemical bond linkage. Li and Liu [31] presented a method for
fabrication of Ag/g-Fe2O3 composite particles with a diameter in
the range of 200e300 nm whereas Cho [32] reported a synthesis
of magnetic SiO2/Fe3O4/Ag nanostructures with a size of about
200 nm.
In the present work, we report on a synthetic procedure of
binary nanocomposite of the Ag@Fe3O4 and g-Fe2O3@Ag type
employing polyacrylate acid as an effective linker.
4705
2. Materials and methods
2.1. Chemicals
Following chemicals have been used: Silver nitrate (99.9%, Tamda), sodium salt of
polyacrylic acid (MW 8000, 45% aqueous solution, SigmaeAldrich), ammonia (p.a.,
28% aqueous solution, SigmaeAldrich), sodium hydroxide (p.a., SigmaeAldrich),
D(þ)-maltose monohydrate (p.a., Riedel-de Haën), 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 with
10 M NaOH
To prepare g-Fe2O3 nanoparticles, we modified a procedure reported by Cui et al.
[33] Firstly, 172 mg of FeCl2$4H2O and 280 mg of FeCl3 were dissolved in 190 mL of
deionized water. The amount of respective iron salts was calculated with regard to
the resulting concentration of 1 g/L of g-Fe2O3. Under intensive stirring, 10 mL of
10 M NaOH was added to the solution of iron salts. The color of the solution changed
immediately from orange to dark brown after addition of NaOH. The reaction
mixture was then stirred for 1 h. After that, the mixture was stirred for another 1 h
while placed in water bath at 90  C. The as-acquired nanoparticles were separated
from the solution by an external magnetic field and washed several times by
deionized water.
2.3. Synthesis of Fe3O4 nanoparticles by thermally induced solid-state
transformation of a-FeOOH
Fe3O4 nanoparticles were synthesized according to the well-known sequential
temperature-induced solid-state reaction employing goethite as a precursor, which
can be summarized as follows [34,35]:
 
a  FeOOH / a  Fe2 O3 / Fe3 O4 :
300 C 400 C
air H2 ;1h
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 sodium
salt (molecular weight of 8000 e NaPA 8000). In order to well disperse the iron oxide
nanoparticles, the solution was placed to an ultrasound bath for 1 min. Subsequently,
20 mL of 103 M AgNO3 solution and 0.2 mL of 0.1 M aqueous solution of ammonia
were added to the reaction mixture under steady stirring. The pH of the reaction
system was then adjusted to 11.5 by 0.1 M NaOH solution. Finally, 20 mL of 5$102 M
maltose solution was added to the reaction system which was then stirred for 20 min.
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, 104 M, 104 M and
5$103 M, 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 washed
several times by deionized water and subsequently dried in oven at 90  C.
Transmission electron microscopy (TEM) images were obtained using a JEM2010
microscope operated at 200 kV. A drop of very dilute suspension was placed on
a carbon-coated grid and allowed to dry by evaporation at ambient temperature.
The X-ray powder diffraction (XRD) patterns of all solid samples were recorded
on PANalytical X’Pert PRO (The Netherlands) instrument in BraggeBrentano
geometry with Fe-filtered CoKa radiation (40 kV, 30 mA). Samples were placed on
a zero-background and rotating single-crystal Si slides, gently pressed in order to
obtain sample thickness of about 0.5 mm and scanned in the 2q range of 10e90 in
steps of 0.017. The XRD patterns were evaluated using the X’Pert HighScore Plus
software (PANalytical), PDF-4þ and ICSD databases.
Zero-field Mössbauer spectra were recorded at 5 and 300 K in a constant
acceleration mode with a 50 mCi 57Co(Rh) source. The samples were placed in
a cryomagnetic system (Oxford Instruments) and the values of the isomer shift are
reported with respect to a-Fe at room temperature.
A superconducting quantum interference device (SQUID, MPMS XL-7, Quantum
Design) has been used for the magnetic measurements. The hysteresis loops were
collected at a temperature of 5 and 300 K in external magnetic fields from 5
to þ5 T. The zero-field-cooled (ZFC) and field-cooled (FC) magnetization curves were
recorded on warming in the temperature range from 5 to 300 K and in an external
magnetic 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 Perkin
Elmer 3300 (Perkin Elmer, USA) device using a method of atomic absorption spec-
troscopy with flame AAS ionization.
4706 R. Prucek et al. / Biomaterials 32 (2011) 4704e4713

2.6. Antimicrobial testing determine its magnetic properties. At 300 K (not shown), the
Mössbauer spectrum of the g-Fe2O3@Ag nanocomposite consists of
Antibacterial and antifungal activities of silver nanocomposites and separate
silver nanoparticles were tested by using of standard microdilution method which
only a doublet with the isomer shift d ¼ (0.34  0.01) mm/s and
enables to determine the minimum inhibition concentration (MIC) of an antibac- quadrupole splitting DEQ ¼ (0.63  0.01) mm/s. Since there is no sign
terial substance. The testing was carried out on microtitration plates employing of any sextet component, all the iron oxide nanoparticles are in
a method when we tested a dispersion of silver nanocomposites 2-to-128 times, in a superparamagnetic state with respect to the measuring time of
the geometrical progression, diluted by addition of 100 mL of the Mueller-Hinton
Mössbauer spectroscopy. At 5 K, an asymmetric sextet is registered
cultivation medium inoculated by tested bacteria and yeast strain at a concentration
of 105e106 CFU mL1. The MIC value, expressing a minimum concentration of as the only one spectral component (see Fig. 2A) with the
a tested compound that inhibited the growth of tested bacteria and yeasts, was average hyperfine parameters d ¼ (0.43  0.01) mm/s,
determined after 24 h of incubation at 37  C. Following bacterial strains, obtained DEQ ¼ (0.00  0.01) mm/s and hyperfine magnetic field
from the Czech Collection of Microorganisms, Czech Republic (Masaryk University in Bhf ¼ (51.1  0.3) T being close to the respective values reported for
Brno, Czech Republic), were used as standards: Staphylococcus aureus CCM 3953,
Enterococcus faecalis CCM 4224, Escherichia coli CCM 3954 and Pseudomonas aeru-
g-Fe2O3 [35]. At this temperature, the magnetic moments of all iron
ginosa CCM 3955. For testing purposes, we also used bacterial strains isolated from oxide nanoparticles in the nanocomposite are thus magnetically
the clinical material of the University Hospital in Olomouc, Czech Republic. These blocked. The analysis of the Mössbauer spectra of Ag-contaning
strains included P. aeruginosa, Staphylococcus epidermidis, methicilline-resistant nanocomposite demonstrates that adding of sodium polyacrylate
S. epidermidis, methicilline-resistant S. aureus (MRSA), vancomycine-resistant
and Ag during the synthesis does not affect the iron oxide origin and
Enterococcus faecium (VRE) and ESBL-positive Klebsiella pneumoniae. Antimycotic
activity was tested using Candida albicans (I and II), Candida tropicalis and Candida evoke formation of any other iron oxide admixture. Thus, within the
parapsilosis strains isolated from the blood of patients of the University Hospital in
Olomouc, Czech Republic, who had confirmed candida sepsis. The yeasts were
identified 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 determined

using the MTT assay. The chemicals used included Dulbecco’s Modified Eagle
Medium (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 on
a multi-detection microplate Synergy HT reader (BioTek, USA). We used 96 well
plates (P-Lab, Czech Republic) for cell lines cultivation, a centrifugal machine
(Biotech, Czech Republic) and a glass cover slip (P-Lab, Czech Republic). During
impact 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. Before
starting 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 for
3 h at 37  C and under 5% of CO2. The MTT solution was carefully removed and 100 ml
DMSO was added in order to solubilize theviolet formazan crystals. The absorbance
of the resulting solution was measured in a 96-well microplate Synergy HT reader at
570 nm and 690 nm. The cell viability of the samples was determined as
a percentage of control cell viability (100 times the average of a test group/the
average of a control group).

3. Results and discussions

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

nanocomposite

The nanocomposite of the g-Fe2O3@Ag type was synthesized by

reduction of silver ions in the presence of polyacrylate with a relative
molecule weight of 8000 and addition of g-Fe2O3 nanoparticles
synthesized by a method mentioned in Section 2.2. Representative
TEM image of the g-Fe2O3@Ag nanocomposite (see Fig. 1A) revealed
a presence of silver nanoparticles with sizes ranging from 20 to 40 nm
that were surrounded and separated from each other by aggregates
of g-Fe2O3 nanoparticles with an average particle size of about 5 nm
(see the schematic picture of the g-Fe2O3@Ag nanocomposite in
Fig. 1B). The presence of both crystalline components was confirmed
by an analysis of the corresponding XRD pattern (data not shown).
After washing and subsequent magnetic separation of the
g-Fe2O3@Ag nanocomposite, the content of silver in the nano-
composite was found to be equal to 10.5% (w/w) employing the
ASS method. Comparison of this value with the amount of silver
incorporated into the nanocomposite during synthesis indicates
that 70% of silver was bounded with g-Fe2O3 nanoparticles. Fig. 1. (A) TEM image and (B) schematic representation of the g-Fe2O3@Ag nano-
Zero-field 57Fe Mössbauer spectroscopy was exploited both to composite with the high-resolution inset showing an imminent surrounding of an Ag
check the phase purity of iron oxide in the nanocomposite and nanoparticle.
 R. Prucek et al. / Biomaterials 32 (2011) 4704e4713 4707

experimental error of the Mössbauer technique, g-Fe2O3 constitutes

the only one Fe-bearing phase in this Ag-containing nanocomposite.
The macroscopic magnetic properties of the prepared
g-Fe2O3@Ag nanocomposite were monitored by measuring the
hysteresis loops at a temperature of 5 and 300 K and ZFC/FC
magnetization curves under an external magnetic field of 0.1 T. The
results are graphically depicted in Fig. 2B and c and corresponding
hysteresis parameters are summarized in Table 1. Both hysteresis
loops and ZFC/FC magnetization measurements demonstrate that
the 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 overall
magnetic features of the g-Fe2O3@Ag nanocomposite are domi-
nantly driven by finite-size and surface effects as manifested by
a reduced value of the maximum magnetization under 5 T and
coercive field in comparison with the corresponding values of
hysteresis parameters reported for bulk g-Fe2O3 at low tempera-
tures. In addition, at 5 K, the hysteresis loop of the g-Fe2O3@Ag
nanocomposite is perfectly symmetric (see Fig. 2B) implying an
absence of the so-called exchange bias phenomenon frequently
evolving in the case of a significant difference in the magnetic
hardness of the core and surface layers of the nanoparticle. As the
exchange bias phenomenon is, among other factors, triggered by
a presence of interparticle magnetic interactions, this thus suggests
that the strength of interparticle magnetic interactions of an
exchange origin seem to be reduced.
The fact that interparticle magnetic interactions are significantly
suppressed in the g-Fe2O3@Ag nanocomposite is further docu-
mented by the profile of the FC magnetization curve that still keeps
increasing as the temperature lowers below the maximum in the
ZFC magnetization curve recognized as the average blocking
temperature (TB) of the magnetic fraction (the temperature below
which g-Fe2O3 nanoparticles with the most probable size in their
assembly enter the magnetically blocked regime) [36]. Further-
more, the closeness of TB and Tirr (irreversibility temperature at
which the ZFC and FC magnetization curves diverge from each
other, corresponding to the blocking of the largest g-Fe2O3 nano-
particles in the nanocomposite) values (see Fig. 2C) predicates
a narrow particle size distribution of g-Fe2O3 nanoparticles in the
nanocomposite which indicates that the synthesized g-Fe2O3@Ag
nanocomposite predominantly consists of well separated iron(III)
oxide nanoparticles rather than their agglomerates.
Thus, it seems that the sodium-salt-based polyacrylate acts as
a spacer (like a matrix with pores of definite size) which holds the
magnetic nanoparticles apart. This significantly reduces the possi-
bility of formation of agglomerates and consequently, the evolution
of 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 via
magnetic moments of magnetic nanoparticles not necessarily in
a close contact, which are considered as being much weaker that
interparticle magnetic interactions of exchange nature [36].
Nevertheless, the effect of interparticle magnetic interactions on
magnetic properties of g-Fe2O3@Ag nanocomposite is significantly
suppressed and the system behaves similarly as that composed of
almost non-interacting magnetic nanoparticles. In addition, as
documented by the room-temperature hysteresis loop, the
g-Fe2O3@Ag nanocomposite exhibits a strong magnetic response
even at a field of 1 T, being thus possibly controlled by a simple
magnet.
Since the sizes of g-Fe2O3 nanoparticles are in the range of
Fig. 2. (A) Zero-field low-temperature Mössbauer spectrum, (B) hysteresis loops and
several units of nanoparticles, it is highly improbable that under (C) ZFC/FC magnetization curves of the g-Fe2O3@Ag nanocomposite.
used reaction conditions, silver nanoparticles with sizes of 1e2 nm
would form on the surface of g-Fe2O3 nanoparticles. In this case, as
4708 R. Prucek et al. / Biomaterials 32 (2011) 4704e4713

Table 1
Hysteresis parameters derived from the hysteresis loops measured for the g-Fe2O3@Ag and Fe3O4@Ag nanocomposite, where T is the temperature of measurement, Mmaxþ
denotes the maximum magnetization under þ5 T, Mmax stands for the maximum magnetization under 5 T, BCþ represents the positive coercive field, BCe is the negative
coercive field, MRþ denotes the positive remanent magnetization and MRe represents the negative remanent magnetization. The values of Mmaxþ, Mmax, MRþ and MRe are
normalized 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 2.36
300 21.92 21.92 e e e
Fe3O4@Ag 5 66.80 66.80 0.0438 0.0586 31.58 27.87
300 60.90 60.90 0.0122 0.0122 10.68 10.68

proved by TEM and XRD analyses, ultrafine g-Fe2O3 nanoparticles
acted as a “particle matrix” surrounding silver nanoparticles (see
Fig. 3). In order to prevent an evolution of magnetic interactions
among g-Fe2O3 nanoparticles, polyacrylate acid sodium salt was
added to the reaction system. As discussed above, magnetization
measurements (especially profiles of ZFC/FC magnetization curves)
indicate the suppression of magnetic interactions among g-Fe2O3
nanoparticles, confirming thus anti-agglomeration role of sodium
salt of polyacrylic acid. A considerable number of dissociated
carboxyl groups of this organic compound that are adsorbed on the
surfaces of g-Fe2O3 nanoparticles avoid their aggregation due to
electrostatic interactions. Free carboxyl groups, non-adsorbed on
the surfaces of g-Fe2O3 nanoparticles, attract free silver ions to the
proximity of g-Fe2O3 nanoparticle surface and silver nanoparticles
then form by reduction of these silver ions. In order to maximally
suppress the possibility of formation of silver nanoparticles in
places not close to surfaces of g-Fe2O3 nanoparticles, ammonia was
added to the reaction system. Ammonia forms a relatively stable
complex with silver ions (b1 ¼ 3.31 and b2 ¼ 7.22). At this place, one
should stress that ammonia was added only in equimolar amount
with respect to silver ions. Thus, only part of silver ions were bound
to [Ag(NH3)]þ and [Ag(NH3)2]þ complex and rest of silver ions were
left to carboxyl groups of polyacrylate acid sodium salt. The
bonding of certain part of silver ions prevents their extensive
reduction except places close to surfaces of g-Fe2O3 nanoparticles.
After formation of silver nuclei, this ammonia complex serves as
a “reservoir” of silver ions for their subsequent growth. The reason
why larger silver nanoparticles form can be explained adopting
hypothesis that g-Fe2O3 nanoparticles with a significantly high
surface area (due to increased surface-to-volume ratio for 3e6 nm
large nanoparticles) bind to carboxyl groups of polyacrylate leaving
less free carboxyl groups for binding of free silver ions in the
proximity of surfaces of g-Fe2O3 nanoparticles. Since silver ions
bound to free carboxyl groups are reduced to silver nuclei, smaller
number of these silver nuclei results in formation of silver nano-
particles with sizes of several tens of nanometers.

Fig. 3. Schematic representation of the reaction steps leading to the preparation of the g-Fe2O3@Ag nanocomposite.
 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 Fe3O4

nanoparticles synthesized from a goethite precursor as mentioned
in the Section 2.3. The synthesis procedure of this nanocomposite
was carried out in the same manner as in the case of the
g-Fe2O3@Ag nanocomposite. TEM image of Ag@Fe3O4 nano-
composite (see Fig. 4A) showed an existence of silver nanoparticles
with an average size of z5 nm residing on the surfaces of Fe3O4
nanoparticles with an average size of z70 nm (see the schematic
picture of the Ag@Fe3O4 nanocomposite in Fig. 4B). XRD analysis
(data not shown) then confirmed the presence of both metallic
silver and Fe3O4. In this case, only 6% (w/w) of metallic Ag was
calculated from the quantitative phase analysis.
Similarly as in the case of the g-Fe2O3@Ag nanocomposite, 57Fe
Mössbauer spectroscopy was used to identify the type of iron oxide
in the nanocomposite and state its magnetic properties. At 300 K
(see Fig. 5A), the Mössbauer spectrum of this nanocomposite is
rather complicated in contrast to the previously studied sample.
With regard to the measuring time of Mössbauer technique, the
appearance of sextets and absence of any doublet component imply
that all nanoparticles of iron oxides are in a magnetically blocked
state at room temperature. The mathematical deconvolution of the
room-temperature Mössbauer spectrum leads to the two sextet
components with the hyperfine parameter values close to those
reported for bulk Fe3O4. In the case of Fe3O4 above the Verwey
transition temperature, the two sextets are expected fulfilling the
assumption of a spinel structure with the two nonequivalent crys-
tallographic iron positions. The Sextet 1 with d ¼ (0.32  0.01) mm/s,
DEQ ¼ (0.01  0.01) mm/s and Bhf ¼ (48.9  0.3) T thus corresponds
to Fe3þ ions occupying the tetrahedral sites in the crystal structure of
Fe3O4 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 the
Fe ions with a valence state between 2þ and 3þ situated at the
octahedral sites of the Fe3O4 crystal structure. In the case of stoi-
chiometric Fe3O4, an effective valence state of 2.5þ is observed for
Fe2þ and Fe3þ ions occupying neighboring octahedral sites [34]. If
we assume that the room-temperature ratio of the recoil-free frac-
tions for the octahedral (O) and tetrahedral (T) sites is 0.97, the
intensity ratio a ¼ I(O)/I(T) for a perfect stoichiometric Fe3O4 should
Fig. 4. (A) TEM image and (B) schematic representation of the Ag@Fe3O4 nano-
be 1.94. In our case, a ¼ (1.14  0.01) which indicates that we have composite with the high-resolution inset showing an imminent surrounding of an
a non-stoichiometric Fe3O4 with a certain degree of cation vacancies Fe3O4 nanoparticle.
at the octahedral sites of the Fe3O4 crystal structure. Since any other
iron oxide admixtures were not detected in the room-temperature
Mössbauer spectrum of this nanocomposite, bearing in mind the size effects most probably accompanied by interparticle magnetic
experimental error of Mössbauer technique, one can conclude that it interactions cause a reduction in the value of the maximum
consists solely of non-stoichiometric Fe3O4 nanoparticles with sizes magnetization under 5 T compared to that expected for bulk Fe3O4
much larger than in the case of g-Fe2O3 nanoparticles present in the (z95 Am2/kg). However, note that the magnetization values are
g-Fe2O3@Ag nanocomposite. normalized to the weight of the whole sample including Fe3O4
The macroscopic magnetic properties of the prepared Ag@Fe3O4 nanoparticles, sodium-salt-based polyacrylate and Ag nano-
nanocomposite were again monitored by measuring the hysteresis particles. Thus, the reduced value of the maximum magnetization
loops at a temperature of 5 and 300 K and ZFC/FC magnetization of the Ag@Fe3O4 nanocomposite partly reflects the presence of
curves under an external magnetic field of 0.1 T. The results are sodium-salt-based polyacrylate and Ag nanoparticles which both
graphically depicted in Fig. 5B and C and corresponding hysteresis behave as diamagnetic compounds. Nevertheless, from the appli-
parameters are summarized in Table 1. Analysis the hysteresis loops cation point of view, the Ag@Fe3O4 nanocomposite exhibits a strong
of the Ag@Fe3O4 nanocomposite, at 300 K, a significant portion of magnetic response achievable at relatively low applied magnetic
Fe3O4 nanoparticles stays still in the magnetically blocked state as fields. The profile of the ZFC/FC magnetization curves (see Fig. 5C) is
evidenced by non-zero values of coercivity and remanent magne- typical of magnetic nanoparticles with wide particle size distribu-
tization. This is in contrast to what we have observed in the case of tion. Contrary to the g-Fe2O3@Ag nanocomposite, Tirr is found to
the g-Fe2O3@Ag nanocomposite where all magnetic nanoparticles occur above 300 K which well corresponds with the hysteresis
have already entered the superparamagnetic regime below 300 K. observed for the room-temperature loop. A bigger average size of
This confirms bigger average size and different particle size distri- Fe3O4 nanoparticles is documented by a blocking temperature of
bution of Fe3O4 nanoparticles compared to size characteristics of z166 K being much higher than TB derived for the g-Fe2O3@Ag
g-Fe2O3 nanoparticles as already derived from TEM and XRD nanocomposite. The presence of interparticle magnetic interactions
analyses. Similarly as for the g-Fe2O3@Ag nanocomposite, finite- acting here as a driving force responsible for a nanoparticle
4710 R. Prucek et al. / Biomaterials 32 (2011) 4704e4713

aggregation is reflected in the FC magnetization curve which slowly

approaches a constant character as the temperature lowers [36].
This implies that the sodium-salt-based polyacrylate does not
properly cover the surfaces of magnetic nanoparticles most prob-
ably due to their small surface-to-volume ratio. Then, in order to
reduce their total energy, magnetic nanoparticles come to each
other and form aggregates. In addition, the FC magnetization curve
does not exhibit a decrease in magnetization suggesting an absence
of Verwey transition down to 5 K. Along with the results of analysis
of room-temperature Mössbauer spectrum of the Ag@Fe3O4
nanocomposite, this confirms a non-stoichiometry of Fe3O4 nano-
particles which manifest itself by a shifting the Verwey transition
temperature from z120 K (typical of bulk Fe3O4) to much lower
temperatures.
After washing and subsequent magnetic separation of the
Ag@Fe3O4 nanocomposite, the content of silver was determined to
be 5.8 (w/w) as documented by the ASS method. Comparing this
value with the amount of silver incorporated in the beginning of
nanocomposite synthesis, it follows that 36% of amount of silver
was bounded to Fe3O4 nanoparticles. In contrast to the g-Fe2O3@Ag
nanocomposite, the synthesis yield is lower. For the Ag@Fe3O4
nanocomposite, we observed a smaller size of bounded silver
nanoparticles. This can be ascribed to a larger size of Fe3O4 nano-
particles having a smaller surface area providing less bonds for
carboxyl groups of polyacrylate. Thus, a larger number of these
carboxyl groups are left for free silver ions resulting in a larger
number of silver nuclei which turn into silver nanoparticles with
much smaller sizes (see Fig. 6). Nevertheless, due to the smaller
surface area of Fe3O4 nanoparticles, a significant number of
unbounded polyacrylate molecules are present in the reaction
system. As a consequence, the silver nanoparticles also form in
places off surfaces of Fe3O4 nanoparticles, which then leads to
a smaller yield mentioned already above.

3.3. Antibacterial and antifungal assay

Antibacterial and antifungal activities of the synthesized

Ag@Fe3O4 and g-Fe2O3@Ag nanocomposites were determined by
means of a standard dilution method which enables to state the
minimum concentration of the tested compound needed for
a growth inhibition of tested bacteria and yeasts. For testing of
antimicrobial activity, we prepared concentrated aqueous disper-
sion of the Ag@Fe3O4 and g-Fe2O3@Ag nanocomposites at
a concentration of 1 g/L. The final mass concentrations of silver
present in such concentrated dispersions of the studied nano-
composites corresponded to the values of 58 mg/L (5.8% w/w of Ag
from AAS) and 105 mg/L (10.5% w/w of Ag from AAS) for the
Ag@Fe3O4 and g-Fe2O3@Ag nanocomposite, respectively. Final MIC
values of the investigated nanocomposites and MIC values related
to 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 with
those of 26 nm silver nanoparticles synthesized by modified Tollens
process based on reduction of [Ag(NH3)2]þ by maltose in an alkaline
medium [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 range
from 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 of
silver in the respective nanocomposite, Ag-related MIC values of
both nanocomposites were found within the range from 0.9 mg/L to
13.2 mg/L of silver for bacteria and from 0.1 mg/L to 3.3 mg/L of
Fig. 5. (A) Zero-field room-temperature Mössbauer spectrum, (B) hysteresis loops and
silver for yeasts, depending on a type of the tested microbe. In the
(C) ZFC/FC magnetization curves of the Ag@Fe3O4 nanocomposite.
case of the Ag@Fe3O4 nanocomposites, the Ag-related MIC values
are even lower than those reported for 26 nm silver nanoparticles.
 R. Prucek et al. / Biomaterials 32 (2011) 4704e4713 4711

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

On the contrary, for the g-Fe2O3@Ag nanocomposite, the Ag-related Table 2

MIC values are slightly higher than those observed for 26 nm silver MIC values of the Ag@Fe3O4 and g-Fe2O3@Ag nanocomposites against tested
bacteria and yeasts. For comparison purposes with the MIC values of 26 nm silver
nanoparticles. The different Ag-related MIC values reflecting
nanoparticles, the Ag-related MIC values of nanocomposites are also listed.
different antibacterial and antifungal activity of these nano-
composites can be explained by different sizes of silver nano- Tested bacteria and yeasts MIC of nanocomposite/ MIC of silver
MIC of silver (mg/L) nanoparticles (mg/L)
particles present in the respective nanocomposite. The
nanocomposite of the Ag@Fe3O4 type consists of silver nano- Ag@Fe3O4/Ag g-Fe2O3@Ag/Ag Ag NPs (26 nm)
particles with an average size of z5 nm exhibiting thus a high Enterococcus faecalis 125/7.2 125/13.2 6.8
antimicrobial activity compared to aqueous dispersion of 26 nm CCM 4224
Staphylococcus aureus 31.3/1.8 62.5/6.6 3.4
silver nanoparticles, whereas g-Fe2O3@Ag nanocomposite is
CCM 3953
composed of silver nanoparticles with an average size of z30 nm Escherichia coli 15.6/0.9 15.6/1.6 1.7
showing thus lower antimicrobial activity than that of aqueous CCM 3954
dispersion of 26 nm silver nanoparticles. Thus, it turns out that Pseudomonas 15.6/0.9 62.5/6.6 3.4
antibacterial activity of silver nanoparticles depends on their size as aeruginosa
CCM 3955
published earlier [37e39]. Pseudomonas aeruginosa 15.6/0.9 31.3/3.3 1.7
Taking into account the acquired results, it is evident that silver 532
nanoparticles incorporated into a nanocomposite practically do not Staphylococcus epidermidis 31.3/1.8 31.3/3.3 1.7
loose their antimicrobial properties as they are comparable with 1879
Staphylococcus epidermidis 31.3/1.8 31.3/3.3 3.4
that of silver nanoparticles themselves [37]. Similarly, synthesized
2901
iron oxide nanoparticles preserve their magnetic properties when Staphylococcus aureus 62.5/3.6 125/13.2 6.8
forming a nanocomposite. This is important from the viewpoint of (4591MRSA)
application potential of these nanocomposites in, for example, Enterococcus faecium 62.5/3.6 125/13.2 3.4
medicine. Combination of magnetic properties of iron oxide (1324 VRE)
Klebsiella pneumoniae 31.3/1.8 31.3/3.3 3.4
nanoparticles and antimicrobial features of silver predestinates (2486 ESBL)
these nanocomposites for exploitation in human medicine where Candida albicans I 1.9/0.1 1.9/0.2 0.2
they can be used for targeted transport of antimicrobial drug and/or Candida albicans II 1.9/0.1 1.9/0.2 0.2
for its removal by an external magnetic field. With regard to their Candida tropicalis 5 3.9/0.2 31.3/3.3 0.4
Candida parapsilosis 6 7.8/0.5 31.3/3.3 0.8
unique magnetic and antibacterial properties, they can be exploited
4712 R. Prucek et al. / Biomaterials 32 (2011) 4704e4713
at their low concentrations so that their toxic effect on human cells
is minimized. In literature, no nanocomposite with such a high
antibacterial activity has been reported so far. Dallas et al. [40]
described a synthetic procedure of magnetic nanocomposite
based on g-Fe2O3 and silver nanoparticles incorporated into
multifunctional phosphotriazine matrix, however, the lowest MIC
value of this nanocomposite were found to be z80 mg/L depending
on the bacteria type. In our case, both investigated nanocomposites
exhibit MIC values as low as 15 mg/L for tested bacteria, concluding
that the Ag@Fe3O4 and g-Fe2O3@Ag nanocomposites possess an
antimicrobial activity several times higher. Moreover, both kinds of
nanocomposites work with the biocompatible polyacrylate linker,
which extends their applicability for a targeted delivery in
biomedical and disinfection areas.
3.4. Cytotoxicity assay
Cytotoxic activity of the synthesized nanocomposites was tested
by means of an MTT test against mice embryonal fibroblasts.
Cytotoxicity was monitored for the Ag@Fe3O4 and g-Fe2O3@Ag
nanocomposites 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.
Table 3
Comparison of antimicrobial activity and cytotoxicity of synthesized Ag@Fe3O4 and
g-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 430
g-Fe2O3@Ag 1.9e125 292
a verification. Cytotoxity was determined on the basis of viability of
cells in dependence on the nanocomposite concentration. In this
way, we obtained dependence of toxic activity of tested compound
on its concentration. Once this dependence was known, we eval-
uated 24 h LC50 toxic index. The dependence of viability of cells on
nanocomposite concentration is depicted in Fig. 7. As one can see,
the Ag@Fe3O4 and g-Fe2O3@Ag nanocomposites exhibit different
limits of toxic activity against tested fibroblasts. For the Ag@Fe3O4
nanocomposite, we observed a weak (pale) cytotoxicity activity at
a concentration above 125 mg/L; a significant cytotoxicity activity
was observable for the highest tested concentration (i.e., 500 mg/L).
The determined 24 h LC50 toxic index was equal to 430 mg/L for
Ag@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% at
a concentration of 250 mg/L and higher. As far as the 24 h LC50 toxic
index of the g-Fe2O3@Ag nanocomposite is concerned, we found it
to be equal to 292 mg/L, a somewhat lower than that for Ag@Fe3O4
nanocomposite. The different values of the 24 h LC50 toxic index
are explainable taking into account different amount of silver in the
respective nanocomposite; a higher cytotoxicity activity of the
g-Fe2O3@Ag nanocomposite is governed by a higher amount of
present silver (10.5% w/w in comparison to 5.8% for the Ag@Fe3O4
nanocomposite). This is supported by a fact that Fe3O4 and g-Fe2O3
did not exhibit cytotoxicity activity against tested fibroblast;
a slight cytotoxicity activity of iron oxide phases was observed at
the highest concentration of 500 mg/L of Fe3O4 and g-Fe2O3;
however, 70% of cells have been surviving in this case. It thus
follows that the 24 h LC50 toxic indices of Fe3O4 and g-Fe2O3 are
expected to be equal to values much higher than 500 mg/L.
When comparing the MIC and 24hod LC50 values of synthesized
nanocomposites, one can conclude that both nanocomposites
effectively destroy microorganisms at concentrations far below the
LC50 values (see Table 3), i.e., concentrations that are not toxic for
mammal (eukaryotic) cells. In most cases, this difference is of an
order which is appealing from the viewpoint of exploitation of
these nanocomposites in various biomedical applications where
both high antimicrobial activity and low cytotoxicity of
a compound is highly required. From this aspect, our results
demonstrate that the Ag@Fe3O4 nanocomposite is a more effective
antimicrobial agent since, regardless of its lower content of silver
and thus its lower cytotoxicity, it effectively destroys pathogenic
microorganisms due to a smaller size of its silver nanoparticles
compared to that found for the g-Fe2O3@Ag nanocomposite.
4. Conclusion
In this work, we synthesized and characterized in details two
types of magnetic nanocomposites exhibiting high antimicrobial
activities e Ag@Fe3O4 and g-Fe2O3@Ag. Molecules of polyacrylate
with a relative molar mass of 8000 were exploited as a spacer
among iron oxide and silver nanoparticles, synthesized via in situ
chemical reduction by maltose. In the case of the Ag@Fe3O4
nanocomposite, ultrasmall silver nanoparticles (z5 nm) were
caught at the surfaces of Fe3O4 magnetic cores (z70 nm) and their
weight content was determined to be equal to 5.8%. The
 R. Prucek et al. / Biomaterials 32 (2011) 4704e4713
g-Fe2O3@Ag nanocomposite revealed a higher content (10.5%) of
larger silver nanocores (20e40 nm), which are surrounded by
ultrasmall g-Fe2O3 nanomagnets (z5 nm). Both studied nano-
composites possess eminent magnetic properties (e.g., high value
of magnetization achievable at relatively low applied fields,
superparamagnetic and soft magnetic behavior at room tempera-
ture from the viewpoint of SQUID measurements, suppression of
interparticle magnetic interactions) since they are very easily
controlled by a low external magnetic field. Both synthesized
nanocomposites 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 of
magnetic properties of iron oxide nanoparticles and antimicrobial
features of nanosilver thus predestinates these nanocomposites to
be possibly used in disinfection and biomedical applications where
they can be exploited for a targeted transport of an antimicrobial
agent and its subsequent removal by means of an external magnetic
field.
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 and
CZ.1.05/2.1.00/01.0030 of the Ministry of Education, Youth and Sports
of the Czech Republic). This work has been supported by the Ministry
of Education, Youth and Sports of the Czech Republic (Projects
No. 1M6198959201, MSM6198959218 and MSM6198959223),
the Academy of Sciences of the Czech Republic (Project
No. KAN115600801) and the Czech Science Foundation (Project
No. GAP304/10/1316).
References
[1] Chen DY, Jiang MJ, Li NJ, Gu HW, Xu QF, Ge JF, et al. Modification of magnetic
silica/iron oxide nanocomposites with fluorescent polymethacrylic acid for
cancer targeting and drug delivery. J Mater Chem 2010;20:6422e9.
[2] Kluchova K, Zboril R, Tucek J, Pecova M, Zajoncova L, Safarik I, et al. Super-
paramagnetic maghemite nanoparticles from solid-state synthesis e their
functionalization towards peroral MRI contrast agent and magnetic carrier for
trypsin immobilization. Biomaterials 2009;30:2855e63.
[3] Corr SA, Rakovich YP, Gun’ko YK. Multifunctional magnetic-fluorescent
nanocomposites for biomedical applications. Nanoscale Res Lett 2008;3:
87e104.
[4] Fujiwara Y, Kobayashi Y, Kita K, Kakehashi R, Noro M, Katayama J, et al. Ag
nanoparticle catalyst for electroless Cu deposition and promotion of its
adsorption onto epoxy substrate. J Electrochem Soc 2008;155:D377e82.
[5] Kohler JM, Abahmane L, Wagner J, Albert J, Mayer G. Preparation of metal
nanoparticles with varied composition for catalytical applications in micro-
reactors. Chem Eng Sci 2008;63:5048e55.
[6] Lin YC, Lin CH. Catalytic and photocatalytic degradation of ozone via utiliza-
tion of controllable nano-Ag modified on TiO2. Environ Prog 2008;27:
496e502.
[7] Mohan YM, Lee K, Premkumar T, Geckeler KE. Hydrogel networks as nano-
reactors: a novel approach to silver nanoparticles for antibacterial applica-
tions. Polymer 2007;48:158e64.
[8] Wang AQ, Liu JH, Lin SD, Lin TS, Mou CY. A novel efficient AueAg alloy catalyst
system: preparation, activity, and characterization. J Catal 2005;233:186e97.
[9] Lee D, Cohen RE, Rubner MF. Antibacterial properties of Ag nanoparticle
loaded multilayers and formation of magnetically directed antibacterial
microparticles. Langmuir 2005;21:9651e9.
[10] Lee HJ, Yeo SY, Jeong SH. Antibacterial effect of nanosized silver colloidal
solution on textile fabrics. J Mater Sci 2003;38:2199e204.
[11] Pal S, Tak YK, Song JM. Does the antimicrobial activity of silver nanoparticles
depend on the shape of the nanoparticle? a study of the gram-negative
bacterium Escherichia coli. Appl Environ Microbiol 2007;73:1712e20.
4713
[12] Sondi I, Salopek-Sondi B. Silver nanoparticles as antimicrobial agent: a case
study on E. coli as a model. J Colloid Interface Sci 2004;275:177e82.
[13 Lok CN, Ho CM, Chen R, He QY, Yu WY, Sun HZ, et al. Proteomic analysis of the
mode of antibacterial action of silver nanoparticles. J Proteome Res 2006;5:
916e24.
[14] Cao YWC, Jin RC, Mirkin CA. Nanoparticles with Raman spectroscopic
fingerprints for DNA and RNA detection. Science 2002;297:1536e40.
[15] Freeman RG, Grabar KC, Allison KJ, Bright RM, Davis JA, Guthrie AP, et al. Self-
assembled metal colloid monolayers an approach to SERS substrates. Science
1995;267:1629e32.
[16] Kneipp K, Wang Y, Kneipp H, Perelman LT, Itzkan I, Dasari R, et al. Single
molecule detection using surface-enhanced Raman scattering (SERS). Phys
Rev Lett 1997;78:1667e70.
[17] Prucek R, Panacek A, Fargasova A, Ranc V, Masek V, Kvitek L, et al. Re-crystalli-
zation of silver nanoparticles in highly concentrated NaCl environment e a new
substrate for surface enhanced IR-visible Raman spectroscopy. CrystEngComm;
2011. doi:10.1039/c1030ce00776e.
[18] Nie SM, Emory SR. Probing single molecules and single nanoparticles by
surface-enhanced Raman scattering. Science 1997;275:1102e6.
[19] Garrell RL. Surface enhanced Raman spectroscopy. Anal Chem 1989;61:
A401e11.
[20] Jain PK, Huang XH, El-Sayed IH, El-Sayed MA. Noble metals on the nanoscale:
optical and photothermal properties and some applications in imaging,
sensing, biology, and medicine. Acc Chem Res 2008;41:1578e86.
[21] Hu KW, Huang CC, Hwu JR, Su WC, Shieh DB, Yeh CS. A new photothermal
therapeutic agent: core-free nanostructured AuxAg1x dendrites. Chem Eur J
2008;14:2956e64.
[22] Xing GC, Jiang J, Ying JY, Ji W. Fe3O4eAg nanocomposites for optical limiting:
broad temporal response and low threshold. Opt Express 2010;18:6183e90.
[23] Travan A, Pelillo C, Donati I, Marsich E, Benincasa M, Scarpa T, et al. Non-
cytotoxic silver nanoparticle-polysaccharide nanocomposites with antimi-
crobial activity. Biomacromolecules 2009;10:1429e35.
[24] Nadagouda MN, Varma RS. Synthesis of thermally stable carboxymethyl
cellulose/metal biodegradable nanocomposite films for potential biological
applications. Biomacromolecules 2007;8:2762e7.
[25] Pinto RJB, Marques P, Neto CP, Trindade T, Daina S, Sadocco P. Antibacterial
activity of nanocomposites of silver and bacterial or vegetable cellulosic fibers.
Acta Biomater 2009;5:2279e89.
[26] Reddy KR, Lee KP, Gopalan AY, Kang HD. Organosilane modified magnetite
nanoparticles/poly(aniline-co-o/m-aminobenzenesulfonic acid) composites:
synthesis and characterization. React Funct Polym 2007;67:943e54.
[27] Masotti A, Pitta A, Ortaggi G, Corti M, Innocenti C, Lascialfari A, et al. Synthesis
and characterization of polyethylenimine-based iron oxide composites as
novel contrast agents for MRI. Magn Reson Mater Phys Biol Med 2009;22:
77e87.
[28] Park MJ, Park J, Hyeon T, Char K. Effect of interacting nanoparticles on the
ordered morphology of block copolymer/nanoparticle mixtures. J Polym Sci
Polym Phys 2006;44:3571e9.
[29] Zhang Y, Liu JY, Yang F, Zhang YJ, Yao Q, Cui TY, et al. A new strategy for
assembling multifunctional nanocomposites with iron oxide and amino-
terminated PAMAM dendrimers. J Mater Sci Mater Med 2009;20:2433e40.
[30] Bao J, Chen W, Liu TT, Zhu YL, Jin PY, Wang LY, et al. Bifunctional AueFe3O4
nanoparticles for protein separation. ACS Nano 2007;1:293e8.
[31] Liu XM, Li YS. One-step facile fabrication of Ag/gamma-Fe2O3 composite
microspheres. Mater Sci Eng C Biomimetic Supramol Syst 2009;29:1128e32.
[32] Chen M, Kim YN, Lee HM, Li C, Cho SO. Multifunctional magnetic silver
nanoshells with sandwichlike nanostructures. J Phys Chem C 2008;112:
8870e4.
[33] Cui YL, Hu DD, Fang Y, Ma JB. Preparation and mechanism of Fe3O4/Au core/
shell super-paramagnetic microspheres. Sci China Ser B Chem 2001;44:
404e10.
[34] Cornell RM, Schwertmann U. The iron oxides: structure, properties, reactions,
occurrence and uses. Weinheim, Germany: Wiley-VCH Publishers; 2003.
[35] Maeda Y. High coercivity g-Fe2O3 fine particles. The Electronics and Tele-
communications Laboratories, NNT, vol. 179. E.C.L. Techn; 1978. p. 1e7.
[36] Dormann JL, Fiorani D, Tronc E. Magnetic relaxation in fine-particle systems.
In: Prigogine I, Rice SA, editors. Advances in chemical Physics, vol. 98. New
York: John Wiley & Sons; 1997. p. 283e494.
[37] Panacek A, Kvitek L, Prucek R, Kolar M, Vecerova R, Pizurova N, et al. Silver
colloid nanoparticles: synthesis, characterization, and their antibacterial
activity. J Phys Chem B 2006;110:16248e53.
[38] Baker C, Pradhan A, Pakstis L, Pochan DJ, Shah SI. Synthesis and antibacterial
properties of silver nanoparticles. J Nanosci Nanotechnol 2005;5:244e9.
[39] Morones JR, Elechiguerra JL, Camacho A, Holt K, Kouri JB, Ramirez JT, et al.
The bactericidal effect of silver nanoparticles. Nanotechnology 2005;16:
2346e54.
[40] Dallas P, Tucek J, Jancik D, Kolar M, Panacek A, Zboril R. Magnetically
controllable silver nanocomposite with multifunctional phosphotriazine
matrix and high antimicrobial activity. Adv Funct Mater 2010;20:2347e54.