Applied Microbiology and Biotechnology (2018) 102:3607–3621

https://doi.org/10.1007/s00253-018-8880-1

BIOTECHNOLOGICAL PRODUCTS AND PROCESS ENGINEERING

Magnetic graphene oxide inlaid with silver nanoparticles as antibacterial

and drug delivery composite
Mohammad Kooti 1 & Azar Naghdi Sedeh 1 & Hossein Motamedi 2 & Seyedeh Elham Rezatofighi 2

Received: 8 August 2017 / Revised: 14 February 2018 / Accepted: 16 February 2018 / Published online: 6 March 2018
# Springer-Verlag GmbH Germany, part of Springer Nature 2018

Abstract
A three-component composite consisted of graphene oxide, cobalt ferrite, and silver nanoparticles has been prepared by a facile
method and fully characterized. The antibacterial activity of this composite has been greatly enhanced after being combined with
ciprofloxacin drug. This clearly showed the occurrence of a strong synergistic effect between ciprofloxacin and the Ag NPs in the
composite. The ciprofloxacin-conjugated composite was found to be a potent antimicrobial agent while having rather low
cytotoxicity and high stability. The studies based on field emission scanning electron microscopy (FESEM) analysis and zeta
potential measurement have revealed that the composite sticks to the bacterial cell wall causing irreversible cell damage. This
multifunctional magnetic nanocomposite was also examined as drug delivery system for ciprofloxacin in solutions with different
pH. It was observed that the release of ciprofloxacin in this system is pH-sensitive with gradual and controlled manner.
Mechanisms for the synergistic effect and drug release behavior, as well as explanation for the antibacterial action, of the
nanocomposite were also demonstrated.

Keywords Silver nanoparticles . Graphene oxide . Antibacterial . Drug delivery . Synergistic effect

Introduction
Silver nanoparticles (Ag NPs) have attracted a lot of interest,
mainly because of their well-documented antimicrobial and
disinfectant properties (Ghodake et al. 2013). It is also known
that Ag NPs have much higher biocidal activity than their bulk
counterparts (Choi and Hu 2008). The emergence of
multidrug-resistant bacteria has increased the attention to Ag
NPs as an alternative to the conventional antibiotics in recent
years. More importantly, silver nanoparticles have shown a
broad spectrum antibacterial activity to kill a variety of bacte-
ria existing in daily life including some of those antibiotic-
Electronic supplementary material The online version of this article
(https://doi.org/10.1007/s00253-018-8880-1) contains supplementary
material, which is available to authorized users.
* Mohammad Kooti
m_kooti@scu.ac.ir
1
Department of Chemistry, Faculty of Science, Shahid Chamran
University of Ahvaz, Ahvaz 6135743135, Iran
2
Department of Biology, Shahid Chamran University of Ahvaz,
Ahvaz, Iran
resistant types (Kasithevar et al. 2017). Ag NPs are also found
to be superior to the conventional water disinfectants
(Chamakura et al. 2011). The traditional disinfectants, such
as free chlorine, may produce harmful disinfection byproducts
(DBPs); many of which are considered carcinogens, muta-
gens, and teratogens (Li et al. 2008). Ag NPs exhibit slow
but long-term and persistent bactericidal effect, thus were pro-
posed to inactivating of microorganisms present in drinking
water to avoid the production of hazardous DBPs (Nangmenyi
et al. 2011).
It has been also confirmed that the bactericidal effect of Ag
NPs is strongly size dependent and increases by the decrease
of dimensions (Sadeghi et al. 2012). However, Ag NPs with
small size (< 30 nm) tend to aggregate and therefore their
antibacterial efficiency will inevitably be decreased. The ag-
gregation problem can be minimized by incorporation of Ag
NPs into appropriate organic or inorganic matrices. This will
preserve the high surface to mass ratio of Ag NPs required for
efficient antibacterial activity.
Many supporting inert solids have been utilized to control
the particle size of Ag NPs and enhancing their stability of
(Liu et al. 2013). However, nanocomposites consisting of Ag
NPs and one magnetic component have recently been exten-
sively studied for their ease of separation and recycling
3608
purposes (Kooti et al. 2014). It is known that all silver-based
materials are toxic to aqua system and therefore must be re-
moved from solution after disinfection process ( Beer et al.
2012). In this respect, combination of Ag NPs with a magnetic
system can provide an efficient means for the separation of Ag
NP-based materials from disinfected solution (Chi et al.
2012). Having a composite consisted of Ag NPs with a mag-
netic component can offer two interesting advantages. First, it
could be easily manipulated by applying a magnetic field and
second, Ag NPs could be delivered to specific target, which is
an important factor in designing drug delivery systems. In this
case, the availability of Ag NPs will be increased in specific
position while their concentration in the bulk solution is
reduced.
Although Ag NPs have been immobilized on numerous
single or two-component supporting materials, depositing of
these nanoparticles on graphene oxide (GO) or reduced
graphene oxide (rGO) sheets can potentially provide a new
way to develop powerful antibacterial agents (Tian et al.
2014). Graphene oxide with its unique and interesting proper-
ties can be a promising support for Ag NPs. More interesting-
ly, however, supporting of Ag NPs on GO pre-coated with a
magnetic material will give magnetic antibacterial composite
that can broaden the horizons of using GO and Ag NPs in
water treatment as well as in medicine for drug delivery pur-
poses (Wang et al. 2016a, b).
It was noted that the antibacterial activity of
GO@CoFe2O4@Ag is greatly increased after incorporation
with ciprofloxacin drug, which clearly revealed the emergence
of a strong synergistic effect in this system. According to the
results of the present work and other previous studies, a mech-
anism for the antibacterial action of GO@CoFe2O4@Ag is
proposed. The synergistic effect, which leads to the enhance-
ment of bactericidal activity of the composite after being com-
bined with ciprofloxacin, was also demonstrated.
On the other hand, drug delivery systems have become an
interesting subject for researchers of different discipline in
recent years. The aim of using novel drug delivery systems
is to improve the therapeutic efficiency of drugs, which is
mainly achieved by minimizing their side effects and increas-
ing their bioavailability in specific site. Among the various
drug delivery systems, nanoparticles based, especially those
having magnetic property, are more attracting and have been
widely studied (Goyal et al. 2016). Therefore, the introduced
GO@CoFe2O4@Ag nanocomposite was examined as drug
delivery agent for ciprofloxacin in simulated physiological
solutions. It was observed that this composite releases the
loaded ciprofloxacin in a gradual and controlled manner to
provide adequate amount of the drug for an extended period
of time.
We herein, report the preparation of GO@CoFe2O4@Ag
nanocomposite via a step-wise and simple procedure, much
easier than the solvothermal method reported by Zhan et al.
Appl Microbiol Biotechnol (2018) 102:3607–3621
for the synthesis of similar composite (Ma et al. 2015). In the
present method, graphene oxide sheets were first coated with
CoFe 2 O 4 and then Ag NPs were inlaid on the top of
GO@CoFe2O4. In the previously reported method of synthe-
sizing Ag-CoFe2O4-GO composite, however, Ag NPs and
CoFe2O4 were prepared simultaneously and doped on the sur-
face of GO. The Ag NPs are, therefore, distributed in the
whole composite, whereas in present method, the Ag NPs
are mainly located in the outer layer of the three-component
GO@CoFe2O4@Ag composite. This interesting multifunc-
tional composite deserves extended investigation and the pres-
ent contribution aims to study and explore some of its impor-
tant aspects.
Materials and methods
Preparation of GO@ CoFe2O4@Ag composite
This magnetic nanocomposite was fabricated by a fast and
straightforward method. Graphene oxide was first pre-
pared by oxidation of graphite powder according to
Hummers’ method (Hummers and Offeman 1958), with
a minor modification of removing NaNO3 from the reac-
tion, as suggested by Shi et al. (Chen et al. 2013). In the
second step, 0.2 g of the pre-synthesized GO was coated
with cobalt ferrite nanoparticles using co-precipitation
method to give GO@CoFe2O4. Silver nanoparticles were
subsequently deposited onto the surface of GO@CoFe2O4
via chemical reduction to obtain GO@CoFe 2 O 4 @Ag
nanocomposite.
Drug loading
In this experiment, 0.1 g GO@CoFe2O4@Ag nanocom-
posite was dispersed in 20 mL of deionized water contain-
ing 0.01 g of ciprofloxacin (CFX). The mixture was stirred
with constant rate of 80 rpm at 37 °C in an incubator for
24 h to obtain GO@CoFe2O4@Ag-CFX, which was sepa-
rated by applying an external magnetic field. The amount
of the ciprofloxacin remained in the supernatant was deter-
mined by UV-vis spectrophotometer at wavelength of
283 nm, using a calibration curve (liner in the range of
1–25 ppm, y = 0.0966x + 0.0336, R 2 = 0.9973). The
adsorbed ciprofloxacin on the composite was then obtained
by subtracting the amount of drug remained in the super-
natant liquid from the initial used amount. The obtained
results showed that about 42.4% of the used ciprofloxacin
was adsorbed by 0.1 g of the composite. A schematic rep-
resentation of the step-by-step preparation of GO@
CoFe2O4@Ag-CFX is shown in Fig. 1.
Appl Microbiol Biotechnol (2018) 102:3607–3621 3609

OH
CoFe2O4 O-

COOH
COOH O COO-
COO- O
OH O
O-
O-
H2SO4 Fe(NO3)3, Co(NO3)2

KMnO4 COOH PVP, OH-

COO-
COOH
O COO-
O O- O
OH
COOH
COO-

Graphite
Graphite oxide
Ag (NPs)
O-
O-

O COO- COO-
O
COO- COO-
O- O-
Ciprofloxacin Ag+, PVP
RT OH-, Glucose
COO-
COO- COO-
COO-
O- O
O-
Ciprofloxacin
COO- COO-

Fig. 1 Schematic representation for the synthesis of GO@CoFe2O4@Ag composite and its impregnation with ciprofloxacin

In vitro ciprofloxacin release Surface charge measurement

To investigate the release of ciprofloxacin from
GO@CoFe2O4@Ag-CFX, 0.1 g of this nanocomposite was
immersed in 100 mL of phosphate buffer saline (PBS) with
pH = 7.4. The suspension was stirred at 80 rpm and 37 °C in
an incubator. Samples were withdrawn at different time inter-
vals, and the amount of released drug in the buffer solution
was measured by UV-visible spectrophotometer at 283 nm.
All the measurements were carried out in triplicate, and aver-
age value was recorded. The release of this drug from the
composite was also studied in solutions with pH = 4 and
pH = 9 to determine the effect of pH on its release behavior.
In order to find possible effects of GO@CoFe2O4@Ag and
GO@ CoFe2O4@Ag-CFX on cell membrane potential of bac-
teria, zeta potential charge measurement was performed. The
details are presented in the supplementary material file.
Field emission scanning electron microscope analysis
For this purpose, E. coli cells were treated as mentioned in the
Surface charge measurement section and following 24-h incu-
bation, the bacterial cells were then studied using FESEM. An
untreated bacterial culture was also used as control.

Antibacterial tests Fluorescent staining assay

Antibacterial susceptibility tests were carried out on the as-
synthesized magnetic composites, based on Kirby-Bauer
disc diffusion method (CLSI 2012). Sterile blank discs
(6.4 mm diameter) were saturated with different concentra-
tions of GO@CoFe2O4@Ag alone and combined with cip-
rofloxacin (10, 20, 40, and 80 mg/mL) in sterile water as
the final effective dose per each disc was 0.4, 0.8, 1.6, and
3.2 mg. The antibacterial effect of ciprofloxacin alone was
also tested using the same above-mentioned method. The
details are included in the supplementary material file.
To observe the effects of GO@CoFe2O4@Ag and GO@
CoFe2O4@Ag-CFX on bacteria by fluorescence microscope,
E. coli and Staphylococcus aureus were cultured, centrifuged
at 5000 rpm for 10 min (1.5 mL OD600 nm 0.5), and washed
three times with PBS. The pellets were resuspended in 1.5 mL
of PBS, and bacterial cells were treated with 100 μL of 20 μg
GO@CoFe2O4@Ag@ and GO@CoFe2O4@Ag-CFX for 1 h.
Untreated bacteria served as a control in 10 mL of PBS. The
bacteria were stained at dark condition for 15 min with 100 μL
of fluorescent dyes including 1 mg/mL acridine orange (AO,
3610
Merck) and 1 mg/mL ethidium bromide (EB, Merck) in PBS.
Staining smear was then prepared from stained bacteria and
observed under the fluorescence microscope (Olympus,
Japan).
Cytotoxicity assay
A-549 cell line (ATCC), a popular cell line used in
nanotoxicology studies (Mahmoudi et al. 2011), was cultured
in RPMI1640, supplemented with 10% (v/v) fetal bovine se-
rum, 1% penicillin/streptomycin. For the evaluation of cyto-
toxic properties of the GO@CoFe 2 O 4 @Ag and GO@
CoFe2O4@Ag-CFX, MTT [3-(4, 5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide] assay was performed. The
results are given in the supplementary material file.
Apoptosis and necrosis analysis
HeLa cells were incubated with GO@CoFe2O4@Ag-CFX
composite at concentrations of 0, 0.01, 0.1, and 10 μg/mL
for 24 h. The cells were washed with PBS and then collected
and centrifuged at 3000 rpm. The cells were resuspended in
100 μL of PBS with 1% FBS and then 2 μL of Annexin V-
FITC and 0.5 μL of propidium iodide (PI) were added to the
cells, and they were incubated at room temperature for 30 min
in the dark. The samples were analyzed using a Gallios multi-
color flow cytometer instrument (Beckman Coulter, Inc.,
Fullerton, CA). Cells which were PI and Annexin V negative
were considered healthy, the cells which were PI negative and
Annexin V positive were considered early apoptotic, cells
which were PI positive and Annexin V negative were consid-
ered necrotic, and those which were positive for both PI and
Annexin V were considered late apoptotic.
Minimum inhibitory concentration and minimum
bactericidal concentration
Minimum inhibitory concentration (MIC) and minimum bac-
tericidal concentration (MBC) of GO@ CoFe2O4@Ag alone
and combined with ciprofloxacin were surveyed against
E. coli, P. aeruginosa, B. subtilis, and S. aureus. MIC testing
of antimicrobials was conducted via a macro broth dilution
procedure using standard NCCLS method. The obtained re-
sults are included in the supplementary material file.
Results
Characterization of the synthesized composites
The UV-vis spectrum of GO@CoFe2O4@Ag showed the
peaks expected for GO and Ag NPs (Cuong et al. 2010). In
the UV-vis spectrum of GO@CoFe2O4@Ag-CFX, however,
Appl Microbiol Biotechnol (2018) 102:3607–3621
another band is also observed which is attributed to ciproflox-
acin. FT-IR and Raman spectra of graphene oxide are in ac-
cordance with the previously reported results (de Faria et al.
2014). Figure 2 presents the FT-IR spectra of ciprofloxacin
and GO@CoFe2O4@Ag-CFX composite. Almost all the
main peaks of ciprofloxacin, except the peak belonging to
the carboxylic group, as well as those of GO are observed in
the FT-IR spectrum of the composite, indicating successful
immobilization of this drug onto GO@CoFe2O4@Ag.
The PXRD of all the synthesized samples are shown in Fig.
S1. In the PXRD pattern of GO, a peak at 11° was observed
which is due to the stacking of the GO layers. The PXRD
pattern of GO@CoFe2O4@Ag composite shows that all the
peaks belong to CoFe2O4 (JCPDS card no. 22-1086) and Ag
NPs (JCPDS cards 4-0783). The crystallite size of CoFe2O4
and Ag NPs are 16 and 15 nm, respectively, as measured by
using Debye-Scherrer formula (Cullity 1978). This observa-
tion confirms that Ag NPs are successfully immobilized on the
surface of the magnetic graphene oxide to give the final three-
component GO@CoFe2O4@Ag composite. No diffraction
peak of GO was observed in the XRD pattern of
GO@CoFe2O4@Ag, which is due to the destruction of the
stacking graphene sheets on coating with CoFe2O4 and Ag
NPs. This observation has been also reported for other similar
composite (Fu et al. 2012).
The morphologies of the samples were investigated by
field emission scanning electron microscopy (FESEM). As it
is clearly seen from Fig. 3a, GO presents sheet-like morphol-
ogy with smooth surface and wrinkled edges indicating very
good exfoliation of graphite during oxidation process. The
FESEM of GO@CoFe2O4@Ag (Fig. 3d) shows that the
GO@CoFe2O4 sheets are decorated with clusters of Ag NPs
which appeared as white spots in the image.
The magnetic properties of the as-prepared samples were
determined using a vibrating sample magnetometer (VSM)
with a peak field of 8 kOe. Magnetic hysteresis loops for
cobalt ferrite and GO@CoFe2O4@Ag-CFX are shown in
Fig. S3, and magnetic properties of all the studied materials
are given in Table 1. The magnetization saturation (Ms) and
remanent magnetization (Mr) values of CoFe2O4 decreased
after being anchored on GO sheets and subsequent immobili-
zation of Ag NPs and ciprofloxacin. This can be due to the low
content of the magnetic CoFe2O4 in the composites. Although
the Ms of GO@CoFe2O4@Ag-CFX is significantly decreased
compared with bare CoFe2O4, its magnetism is still high
enough that it could be moved by a permanent magnet.
The composition of GO@CoFe2O4@Ag was confirmed by
energy dispersive X-ray spectroscopy (EDX). The EDX spec-
trum shows the existence of main elements (O, Fe, Co, and
Ag) expected to be in this three-component composite (see
Fig. S3). Furthermore, the silver content of the composite
was 6.3% (w/w), as measured by inductively coupled plasma
atomic emission spectroscopy (ICP-AES) analysis.
Appl Microbiol Biotechnol (2018) 102:3607–3621
Fig. 2 FT-IR spectra of
ciprofloxacin (a) and
GO@CoFe2O4@Ag-CFX (b)
Antibacterial study
After synthesis and characterization of GO@CoFe2O4@Ag
composite, its antibacterial activity, alone and impregnated with
ciprofloxacin, was examined against some bacteria using disc
diffusion method. The tested bacteria were of gram-positive
and gram-negative species, including Staphylococcus aureus
(ATCC 6538), Bacillus subtilis (ATCC 6633), Escherichia coli
(ATCC 25922), and Pseudomonas aeruginosa (ATCC 9027).
The bactericidal activities of these materials and that of ciproflox-
acin alone were evaluated by measuring the diameter of inhibi-
tion zone (DIZ). The measured DIZ around each of these sam-
ples, measured in millimeters, along with DIZ of some standard
drugs against test strains is presented in Table 2. The DIZ size
indicates the extent of susceptibility of the microorganisms to a
specific antibacterial agent, and the DIZ size of strain vulnerable
to drug is larger than that of resistant strain.
The antibacterial assay results of GO@CoFe2O4@Ag re-
vealed that this composite is a potent bacterial growth inhib-
itor against both gram-negative and gram-positive bacteria.
Interestingly, the bactericidal activity of GO@CoFe2O4@Ag
against all test strains was dramatically enhanced after incor-
poration with ciprofloxacin. As it is seen from data of Table 2,
the DIZ values for GO@CoFe2O4@Ag-CFX, in most cases,
increased by more than twice compared to the DIZ values of
GO@CoFe2O4@Ag and also much greater than that of cipro-
floxacin drug alone. This observation clearly showed a syner-
gistic effect between GO@CoFe2O4@Ag composite and
3611
ciprofloxacin. The enhancement of antibacterial effect of
GO@CoFe2O4@Ag composite after combination with cipro-
floxacin is more pronounced for gram-negative bacteria. The
DIZ values for S. aureus and B. subtillis (both gram-positive)
are 32 and 30, whereas these values are 39 and 34 for the
gram-negative E. coli and P. aeruginosa, respectively, all mea-
sured with 40 mg/mL dose (see Table 2). The differential
susceptibility of gram-negative and gram-positive bacteria to-
wards the as-made nanocomposites is possibly related to their
cell wall structure. Although both of the examined composites
show somewhat higher bactericidal effect on gram-negative as
compared with gram-positive bacteria, the difference, howev-
er, is not significant. This observation indicates that
GO@CoFe2O4@Ag and GO@CoFe2O4@Ag-CFX compos-
ites, especially the latter, can be considered as excellent anti-
bacterial agents with broad spectrum of bactericidal activity.
Zeta potential measurement and FESEM analysis
In order to find a mechanism for the antibacterial action of the
introduced magnetic composites, two sets of experiments, i.e.,
zeta potential measurement and FESEM analysis, have been
performed on treated and untreated bacterium. The measured
zeta potential for E.coli was − 13.2 mv, while for treated E. coli
with GO@CoFe2O4@Ag and GO@CoFe2O4@Ag-CFX was −
14.1 and − 13.7 mv respectively. According to these zeta values
(Fig. S4), it can be stated that the membrane charge of treated
bacteria remained almost the same as untreated bacterial cells.
3612 Appl Microbiol Biotechnol (2018) 102:3607–3621

Fig. 3 FESEM images of GO (a, b), GO@CoFe2O4 (c), and GO@CoFe2O4@Ag (d)

Table 1 Magnetic parameters for the synthesized magnetic materials That is, no increase in the zeta potential happened following
treatment of E. coli with each of these nanocomposites. This
Sample (Ms) emu/g (Mr) emu/g (Hc) Oe
finding suggests that the composites have no significant effect
CoFe2O4 58.022 28.310 796 on the phospholipids of bacterial cytoplasmic membrane, and
GO@CoFe2O4 47.493 16.119 796 this part of bacterial cell is not their main target site.
GO@CoFe2O4@Ag 37.357 13.789 795 FESEM image of E.coli in control culture shows the typical
GO@CoFe2O4@Ag-CFX 16.366 5.502 480 rod-shaped bacterial cells and even bacterial division is clearly
seen (Fig. 4). Figure 4b–d shows the images resulted from
Appl Microbiol Biotechnol (2018) 102:3607–3621 3613

Table 2 Antibacterial activity of GO@CoFe2O4@Ag and GO@CoFe2O4@Ag-CFX nanocomposites (diameter of the inhibition zone is measured in
mm)

Compounds Microorganism

Gram-positive Gram-negative

S. aureus B. subtillis E. coli P. aeruginosa

C (mg mL−1) 10 20 40 80 10 20 40 80 10 20 40 80 10 20 40 80
GO@CoFe2O4@Ag 14 17 15 15 8 14 11 11 11 13 11 10 12 16 15 13
GO@CoFe2O4@Ag-CFX 30 30 32 35 27 30 30 30 32 38 39 40 32 34 34 34
Kanamycin (30 μg) 12 23 25 0
Gentamycin (10 μg) 16 21 20 20
Penicillin (10 μg) 26 13 12 0
Streptomycin (10 μg) 12 22 16 0
Erythromycin (15 μg) 25 27 0 0
Vancomycin (30 μg) 17 23 14 0
Ciprofloxacin (20 μg) 20 19 27 19

bacterial treatment with GO@CoFe2O4@Ag, and Fig. 4e is
the FESEM image of bacterial cells treated with
GO@CoFe2O4@Ag-CFX.
Stability tests
The stability of the as-prepared GO@CoFe2O4@Ag composite
was checked by ICP analysis. As it can be found from Fig. 5a,
the amounts of Co2+ and Ag+ released from the composite are
very low after 12 h following the treatment of target bacteria with
GO@CoFe2O4@Ag. In the case of Co2+, maximum concentra-
tion was about 18 ppm, and for Ag+, less than 5 ppm was re-
corded. These data indicate that almost all of the composites were
remained unchanged even after 12 h of incubation, and dissoci-
ation of the metals is negligible. It can be concluded that the
composite forms stable bonds with peptidoglycan structure of
bacterial cells and that is why it remains almost unchanged which
can therefore efficiently affect bacterial cells.

Fig. 4 FESEM images of untreated E. coli (a), treated with GO@CoFe2O4@Ag (b, c, d), and treated with GO@CoFe2O4@Ag-CFX (e), composite
treatment at 10 mg/ml for 12 h
3614
Fig. 5 Ag+ and Co2+ released from GO@CoFe2O4@Ag-CFX (10 mg/
mL) after different times of E. coli treatment (a) and from free composite
in PBS (b)
Another stability test was also performed using atomic ab-
sorption spectroscopy (AAS). For this test, 0.1 g of the com-
posite was dispersed in 100 mL of PBS and stirred at room
temperature. Samples were taken out and analyzed by AAS.
The amounts of Co2+ and Ag+ released from this composite
are presented in Fig. 5b which indicate very low concentra-
tions of the dissociated metals after 24 h of stirring. The slow
releasing of Co2+ and Ag+ from this composite is a desirable
characteristic because it provides the possibility of using lower
concentrations of it for treating bacterial cells. Moreover, the
negligible leaching of Ag NPs is another benefit of this anti-
bacterial agent because Ag NPs are hazardous for the aqua
systems.
Fluorescence microscopic results
Bacterial treatment with either of the as-made composites
caused bacterial death as shown in the images of Fig. 6, ob-
tained from fluorescent microscopic study. Reduction of green
fluorescent (AO) and increase in red fluorescent (EB) con-
firms bacterial death and loss of viability following the treat-
ments with these nanocomposites.
MIC and MBC results
The MIC and MBC indices of GO@CoFe2O4@Ag against
E. coli were 2.5 mg/mL and in the case of S. aureus were
Appl Microbiol Biotechnol (2018) 102:3607–3621
1.25 mg/mL. When GO@CoFe2O4@Ag composite was con-
jugated with ciprofloxacin, its inhibitory effect increased sig-
nificantly for E. coli, and both the MIC and MBC indices
reduced to 1.25 mg/mL, while no change was observed for
these indices against S. aureus. This reduction in MIC and
MBC also predicates that a synergistic effect between cipro-
floxacin and GO@CoFe2O4@Ag happened. This antibiotic
prevents DNA supercoiling and so leads to bacterial lysis.
Therefore, it is possible that GO@CoFe2O4@Ag-CFX targets
other bacterial structures such as cell wall or cell membrane
that in synergistic effect kill affected bacteria.
The results of the MIC and MBC tests after 48 and 72 h
showed that the composite has high bactericidal and bacteri-
ostatic activity (Table 3). The passage of time was found to
have no significant impact on the MIC index. This means that
the inhibitory power of the examined composite does not be-
come quickly inactive, and it can inhibit the bacteria with an
initial MIC for at least 72 h. The MBC index was two-fold for
all bacteria after 72 h compared to 48 h. This indicates that the
composite partially loses its inhibitory effects with passage of
time. This may be due to inactivation of the composite, active
release from the cell, or its adsorption on the bacterial cell
surface. The inhibitory power of the composite was approxi-
mately the same for all tested gram-positive and -negative
bacteria. Therefore, the main target for this composite is not
the cell structure or if it is so, the metabolic pathways can be
also involved. In general, as much as the MIC and MBC of an
antibacterial agent are lower, its undesirable side effects will
be diminished and it can be used with more safety. Both these
composites are potent bactericidal agents which prevent ap-
pearance of antibiotic-resistant bacteria and can be used more
confidently for bacterial eradication. Finally, there was no sig-
nificant difference between the effects of this composite
against gram-positive and -negative bacteria in terms of MIC
and MBC tests.
MTT assay
Both GO@CoFe2O4@Ag and GO@CoFe2O4@Ag-CFX
demonstrated growth inhibitory effect in a dose-dependent
manner with IC50 values of 912 and 402 μg/mL respectively.
Both these composites, however, did not affect the viability of
A-549 cells at concentrations below 10 and 0.01 μg/mL, 48 h
post-treatment. No change in the cell morphology compared
to the control was observed upon microscopic examination of
the cell monolayers, and the results are given in Fig. 7.
Apoptosis and necrosis analysis
For the quantification of live, necrotic, early, and late apopto-
tic cells, flow cytometry was performed. The percentage of
dead cells is revealed in the upper left and right quadrants of
the flow cytometry images, whereas the percentage of live
Appl Microbiol Biotechnol (2018) 102:3607–3621 3615

Fig. 6 Fluorescent microscope

images of S. aureus (a) and E. coli
(b) after treatment with NPs, and
antibiotic +NP for 60 min,
respectively. The untreated
bacterial cells were used as the
control. The bacterial cells that
appeared green (AO)/red (EB)
when visualized under
fluorescence microscopy were
considered live/dead with intact/
damaged cell walls respectively

cells are shown in the lower left and right quadrants. As com-
pared to the control cells, irrespective of NP doses, a lower
number of necrotic, early, and late apoptotic cells were found
in each tested sample. This confirmed that the studied NPs in
this work have not led to a delay in cell division, and the cells
were being continuously proliferated. The observed results
suggest that this composite did not induce apoptosis in HeLa
cells; however, the percentage of necrotic cells gradually in-
creases in response to increase of the composite dose. This
finding is in agreement with the MTT assay and shows that the
3616
Table 3 MIC and MBC results of
GO@CoFe2O4@Ag-CFX Bacteria 24 h
composite
MIC mg/ml
S. aureus 1.25
B. subtilis 0.5
E. coli 1.25
P. aeruginosa 0.5
composite has very low cytotoxicity to the tested eukaryotic
cells. A graphical representation of the obtained data is given
in Fig. 8.
Release of ciprofloxacin
Loading of ciprofloxacin was achieved by simple mixing of
this antibiotic with GO@CoFe2O4@Ag composite at ambient
conditions. The release behavior of loaded ciprofloxacin was
studied under neutral, acidic, and basic media to demonstrate
the pH responsiveness of this drug carrier system. The amount
of the released drug was determined by measuring the inten-
sity of ciprofloxacin spectrum at 283 nm. The drug release
profile of this composite is depicted in Fig. 9, obtained at 0
to 24 h. It was observed that the initial burst drug release of
38% occurred in the first 4 h, and from 4 to 24 h, a constant
release is reached. The release pattern observed for this drug
delivery system seemed to be acceptable in cases where suf-
ficient concentration of drug is needed for improving the me-
dicinal effectiveness (Wang et al. 2008). As it seen from the
release profile, higher concentration of ciprofloxacin is re-
leased in acidic and alkaline media compared with solution
of pH = 7.4. The greater extent of ciprofloxacin release from
the nanocomposite in acidic and basic solutions can be ex-
plained on the bases of the pH-dependent structure of this
drug.
Fig. 7 Viability percent of the cells treated with different concentrations
of GO@CoFe2O4@Ag and GO@CoFe2O4@Ag-CFX ranging from 0.01
to 1000 μg/mL by MTT assay. Each experiment was repeated three times,
and data are presented as mean ± standard deviation
Appl Microbiol Biotechnol (2018) 102:3607–3621
48 h 72 h
MBC mg/ml MIC mg/ml MBC mg/ml MIC mg/ml MBC mg/ml
1.25 0.5 1 0.5 2
0.5 0.5 0.5 0.5 1
1.25 0.5 1 1 2
0.5 0.5 0.5 0.5 1
Discussion
The three-component GO@ CoFe2O4@Ag composite was
prepared by an easy and fast method via precipitation of
CoFe2O4 on GO sheets. The surface of graphene oxide sheets
are negatively charged because of the presence of several
functional groups, such as carbonyl and hydroxyl. On addition
of Fe3+ and Co2+ to the GO suspension, the metal cations are
efficiently adsorbed by the functional groups of GO. The
adsorbed metals can then be converted to CoFe2O4 NPs by
addition of alkaline solution. These magnetic NPs are precip-
itated on both sides of the GO sheets and can stabilize these
sheets resulting in destruction of their regular stacking. Ag
NPs were subsequently inlaid on the surface of these already
magnetized sheets by chemical reduction of AgNO3 solution.
Ciprofloxacin was finally immobilized on the GO@
CoFe2O4@Ag composite under mild conditions.
Mechanism for the antibacterial action
of the nanocomposites
The results of zeta potential measurement and FESEM analy-
sis indicate that both composites have a significant effect on
bacterial structure and mainly on the cell wall. Cell deforma-
tion, cell shape change from rod to coccoid or irregular shapes,
loss of cell wall integrity, releasing of cytoplasmic content,
ectopic septum formation, swelling of bacterial cell, and final-
ly cell lysis are important changes that happened as a result of
bacterial treatment with these composites. This means that the
examined composites can affect enzymes involved in cell wall
synthesis, i.e., penicillin binding proteins (PBPs), so
transglycosylation and transpeptidation reaction will be
prevented and cell wall will undergo structural changes.
Furthermore, these changes might be due to the activity of
autolytic enzymes which cause partial disintegration of pepti-
doglycan in different parts of the bacterial cell wall.
Localization of proteins involved in cell replication is an im-
portant subject that conducts septum formation. Lateral devi-
ation of septum in some treated cells suggest that
GO@CoFe2O4@Ag and GO@CoFe2O4@Ag-CFX can inter-
fere with localization of proteins in the mid of bacterial cell.
Another possible effect of these antibacterial agents can be the
interaction with bacterial cytoskeleton, and consequently, the
Appl Microbiol Biotechnol (2018) 102:3607–3621
Fig. 8 Flow cytometric cytotoxicity assay performed by dual parameter
analysis of PI and Annexin-V dyes. HeLa cells were treated with a 0, b
0.01, c 0.1, and d 10 μg/mL of nanoparticles. The percentage of necrotic
bacterial shape has been changed from rod to coccoid or ir-
regular shapes. Briefly, it can be concluded that
GO@CoFe2O4@Ag and GO@CoFe2O4@Ag-CFX have no
significant effect on cytoplasmic membrane while their main
Fig. 9 Ciprofloxacin release profile at different pH
3617
cells (upper left and right quadrants) gradually increased in response to
increases of the composite dose
effects are on cell wall. With regard to this fact that there is a
high similarity between eukaryotic and prokaryotic cytoplas-
mic membranes, any compound that can affect cytoplasmic
membrane of bacteria may have some adverse effects on eu-
karyotic membranes. While those which affect cell wall show,
to some extent, selectivity against bacteria because there is not
any similar structure in eukaryotic cells. The selectivity of
GO@CoFe2O4@Ag and GO@CoFe2O4@Ag-CFX, there-
fore, is a considerable index for these compounds which pro-
vide more safety.
Mechanism for the drug release behavior
of the nanocomposite
Ciprofloxacin has two functional groups, one with pKa1 =
6.18 (–COOH) and the other with pKa2 = 8.76 (–NH2). Each
of these two functional groups ionized at specific pH. It is
expected that different charged ciprofloxacin species are
3618
produced by varying of the pH. Ciprofloxacin at pH ranges
around 7 exists mainly as zwitterions having both positive and
negative charges; whereas in acidic and basic media, it is
found as cationic and anionic species respectively
(Vasudevan et al. 2009). The possible forms of ciprofloxacin
at three different pH conditions are depicted in Fig. 10. It has
been also reported, recently, that ciprofloxacin was adsorbed
to magnetite (Fe3O4) through coordination of its carboxylic
functional group (–COOH) with Fe (III) ions of this oxide
(Rakshit et al. 2013). Since CoFe2O4 is structurally similar
to Fe3O4, it is likely that in GO@CoFe2O4@Ag-CFX, the
ciprofloxacin molecule is coordinated to Fe(III) and Co(II)
metals via its caboxylic group. Comparing the FT-IR spectra
of free and the immobilized ciprofloxacin gives strong proof
for its mode of interaction with the nanocomposite. It is clearly
seen in Fig. 2 that the peak at 1708 cm−1, assigned to the
carboxylic group of free ciprofloxacin, disappeared in the IR
spectrum of the ciprofloxacin loaded on the composite. This
observation indicates the coordination of the drug to the
metals in the composite which is in accordance with other
previously reported results (Gu and Karthikeyan 2005).
As mentioned above, ciprofloxacin is mainly positively
charged in acidic solution and therefore would have lower
tendency for coordination to the surface metals of the com-
posite. Moreover, at lower pH, the surface of CoFe2O4 will
have positive charges and its interaction with ciprofloxacin is
decreased. Under acidic pH, another factor might be involved
for higher release of the drug compared to neutral and basic
pH and it is the possible dissociation of the ferrite. At higher
pH, on the other hand, ciprofloxacin exists as anionic species
and the surface of CoFe2O4, as shown in Fig. 10, is also
surrounded with negative charges. At such conditions, the
binding strength of ciprofloxacin with the composite will be
Fig. 10 Ciprofloxacin structures in different pH (a) and the surface charges of CoFe2O4 NPs at acidic and basic solutions (b)
Appl Microbiol Biotechnol (2018) 102:3607–3621
reduced leading to higher release of the drug. Therefore, in
both acidic and alkaline solutions, the possibility of coordina-
tion of ciprofloxacin to the metals is decreased and the release
of this drug from the composite will be higher than its release
at pH around 7.
The other possibility of the interaction of ciprofloxacin
with the composite can be through the formation of hydrogen
bonds between electronegative atoms of this drug with some
of the GO functional groups. This type of interaction will be
also less probable at acidic and alkaline solutions compared to
neutral medium. According to the explanation given above,
the pH-dependent release profile of ciprofloxacin from the
drug carrier composite (Fig. 9) seems to be logical and accept-
able. The release fashion of ciprofloxacin in this study is in
agreement with other previously reported observations
(Demirci et al. 2014).
Evaluation of antibacterial activity
of the as-fabricated composites
Among the tested bacteria, P. areuginosa and S. aureus were
significantly inhibited by the examined GO@CoFe2O4@Ag
and GO@CoFe2O4@Ag-CFX composites. These bacteria are
responsible for nosocomial infections (hospital-acquired
infections) and can cause severe and life-threatening infec-
tions. These fatal bacterial infections are mainly caused by
the development of strains resistant to all existing antibiotics.
There is an urgent medical need, therefore, for new treatments
against these sever bacterial infections. So, finding new anti-
bacterial agents against these species is of utmost importance,
especially in the case of S. aureus, for which only few effec-
tive antibiotics are presently available for the treatment of its
infection. Hence, control and eradication of this pathogen in
Appl Microbiol Biotechnol (2018) 102:3607–3621
the environment is advantageous and any effort for synthesis
of new antibacterial agents against this species is of great
value.
The multifunctional GO@CoFe2O4@Ag composite can
serve as potential drug delivery carrier for ciprofloxacin and
probably other antibiotics having carboxylic functional
groups. This composite becomes a much more effective anti-
bacterial agent after being combined with ciprofloxacin. The
low toxicity and high stability of the ciprofloxacin-conjugated
composite can ensure its oral administration as a safe and
effective alternative to traditional antibacterial drugs.
One of the main points in the medical applications of var-
ious nanoparticles is the safety of these materials; therefore,
MTT assay and apoptosis and necrosis analysis were used to
analyze the cytotoxicity of the nanocomposite. All the tests
confirmed good cytocompatibility of the nanocomposite for
eukaryotic cells even at concentrations up to 10 μg/mL. The
studied composite is, therefore, ideal for biomedical applica-
tions such as magnetically targeted drug delivery. Chen et al.
(2011) reported that Fe 3 O 4 -GO composite has low
cytotoxicity and is suitable for use in medicine including
magnetic resonance imaging, cell separation, and drug
delivery. Wang et al. (2016a, b) also found that MnFe2O4-
GO composite is biocompatible with normal cells and are
ideal for biomedical application purposes. However, these
studies were performed in vitro and as is normal for a drug
delivery composite system, clinical safety evaluation and clin-
ical rational evaluation are essential, therefore, it is necessary
that these composites be tested in vivo conditions.
Furthermore, GO@CoFe2O4@Ag can be considered as a
potential alternative to conventional water disinfectants be-
cause it leaves no hazardous byproducts in water. More im-
portantly, it can be easily removed from disinfected media by
a permanent magnet after disinfection process. This is one of
the important features of the as-made antibacterial composite
because there is a great concern about Ag NP toxicity. Silver
kills both harmful and useful bacteria and therefore efficient
separation of Ag NPs-based material from media is highly
recommended (Vazquez-Muñoz et al. 2017). In this regard,
the magnetic silver-based antibacterial agents, such as the
composites introduced in this work, are much superior to
non-magnetic counterparts.
Proposed mechanism for the synergistic effect
One of the interesting findings of this study is observing the
synergistic effect between ciprofloxacin drug and
GO@CoFe2O4@Ag. Although the synergy between Ag NPs
and some antibiotics has been studied by a number of re-
searchers in recent years, the main cause of this effect has not
been fully uncovered yet (Habash et al. 2014). Apparently, no
clear single mechanism can be given for the enhancement of
antibacterial activity of a certain antibiotic that occurred after
3619
its combination with Ag NPs. The emergence of this interesting
effect and the way by which Ag NPs and an antibiotic cooperate
can be of different nature. In a recent study on this subject, Yu
et al. observed that enoxacin, kanamycin, neomycin, and tetra-
cycline combined with Ag NPs show synergistic growth inhibi-
tion on some drug-resistant bacteria whereas ampicillin and pen-
icillin do not (Deng et al. 2016). In the reported study, the syn-
ergistic effect was suggested to be due to the enhancement of Ag
NPs binding to bacteria which is promoted by an antibiotic. It is
noteworthy to mention that any proposed mechanism for the
appearance of synergy between Ag NPs and an antibiotic might
not be applicable for the Ag NPs immobilized on a specific
support, such as the composite introduced in the present work.
In the following discussion, however, a plausible mechanism
was proposed for the observed synergy between the Ag NPs
containing composite and ciprofloxacin. Ciprofloxacin belongs
to the second generation of flouroquinolone family which pre-
vents DNA supercoiling and so leads to bacterial lysis. This
antibiotic can bind to Ag NPs and other metals in the composite
through its functional groups as discussed in the foregoing sec-
tions. It is unlikely that all the active sites of the antibiotic are
engaged with the metal atoms of the composite, and some of its
functional groups remained intact. The enhancement of the an-
tibacterial activity of GO@CoFe2O4@Ag composite, after com-
bination with ciprofloxacin, may be therefore attributed to the
interaction of the antibiotic molecule with bacterial genetic ma-
terial. Ciprofloxacin, which is either on the surface of the com-
posite or free in solution, can interact with cell wall through its
functional groups and thus increasing the penetration of Ag NPs
containing composite into the bacterium. The ciprofloxacin-
impregnated composite can then react with the DNA of the cells
and prevents supercoiling, which finally results in large damage
to bacterial cells.
According to the obtained results, it is expected that conju-
gation of ciprofloxacin with the as-made GO@CoFe2O4@Ag
nanocomposite can prevent development of resistance by mi-
crobes and enhance the antimicrobial activity of the antibiotic.
Hence, the dosage of antibiotics against multidrug-resistance
pathogens will be minimized and such bactericidal agents can
be used more confidently with fewer side effects for bacterial
eradication.
Our findings in this study have shown that the
GO@CoFe2O4@Ag impregnated with ciprofloxacin can be
considered as a promising antibacterial candidate in the med-
ical field. The method described for the fabrication of this
composite is simple and cost effective to produce stable, safe,
and highly efficient Ag NP-based antibacterial agent.
In summary, Ag NP-immobilized magnetic graphene oxide
(GO@CoFe2O4@Ag), alone or impregnated with ciprofloxa-
cin, has shown potent bactericidal activity and the main target
of the antibacterial agents was almost confirmed to be the
bacterial cell wall. These magnetic bactericidal materials can
prevent the spread of antibiotic-resistant bacteria and may be
3620
used more confidently for bacterial eradication. Moreover, the
as-prepared nanocomposites are stable, nontoxic, and can be
easily manipulated under magnetic field. Therefore, they can
be efficiently separated from the disinfected media, if used as
water disinfectant, to avoid any possible contamination to the
environment. The as-prepared multifunctional composite was
also found to be a promising candidate for drug delivery ap-
plications which shows a prolonged and gradual release of the
loaded ciprofloxacin in acidic, neutral, and basic media.
Acknowledgments The authors would like to thank Prof. A. V.
Fernandez, Faculty of Biology, University of Barcelona, for the provided
help to perform flow cytometry tests. The valuable help of Prof. Jaume
Comas, head of the flow cytometry unit, Scientific and Technological
Centers, University of Barcelona, in analyzing the cytometry data is also
highly acknowledged.
Funding information The authors wish to acknowledge the support of
this work (grant No. 1396) by the Research Council of Shahid Chamran
University of Ahvaz, Iran.
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
Compliance with ethics requirements This article does not contain any
studies with human or animal subjects
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