Synthesis, characterizations of superparamagnetic fe3o4–ag hybrid nanoparticles and their application for highly effective bacteria inactivation

We suggest that the enhancement in bactericidal activity of Fe3O4–Ag hybrid NPs might be likely from main factors such as: i enhanced surface area property of hybrid nanopar-ticles; ii t

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Article

Journal of Nanoscience and Nanotechnology

Vol 16, 5902–5912, 2016 www.aspbs.com/jnn

Synthesis, Characterizations of Superparamagnetic

Highly Effective Bacteria Inactivation

Le Minh Tung1 ∗, Nguyen Xuan Cong2, Le Thanh Huy2 3, Nguyen Thi Lan2, Vu Ngoc Phan2, Nguyen Quang Hoa4, Le Khanh Vinh5, Nguyen Viet Thinh1, Le Thanh Tai1, Duc-The Ngo6,

Kristian Mølhave6, Tran Quang Huy7, and Anh-Tuan Le2 ∗

1Department of Physics, Tien Giang University, My Tho City, Tien Giang Province 86000, Vietnam

2Department of Nanoscience and Nanotechnology, Advanced Institute for Science and Technology (AIST),

Hanoi University of Science and Technology (HUST), Hai Ba Trung District, Hanoi 10000, Vietnam

3Faculty of Chemistry and Environment Technology, Hung Yen University of Technology and Education, Hung Yen 16000, Vietnam

4Department of Physics, Hanoi University of Science, 334 Nguyen Trai, Thanh Xuan, Ha Noi 10000, Vietnam

5Institute of Physics at Ho Chi Minh City, Vietnam Academy of Science and Technology (VAST), Ho Chi Minh 70000, Vietnam

6Department of Micro-and Nanotechnology, Technical University of Denmark Building 345Ø,

Ørsted Plads, Kgs Lyngby 2800, Denmark

7National Institute of Hygiene and Epidemiology (NIHE), 1-Yersin Street, Hai Ba Trung District, Hanoi 10000, Vietnam

In recent years, outbreaks of infectious diseases caused by pathogenic micro-organisms pose a

serious threat to public health In this work, Fe3O4-Ag hybrid nanoparticles were synthesized by

sim-ple chemistry method and these prepared nanoparticles were used to investigate their antibacterial

properties and mechanism against methicilline-resistantStaphylococcus aureus (MRSA) pathogen

The formation of dimer-like nanostructure of Fe3O4–Ag hybrid NPs was conﬁrmed by X-ray

diffrac-tion and High-resoludiffrac-tion Transmission Electron Microscopy Our biological analysis revealed that the

Fe3O4–Ag hybrid NPs showed more noticeable bactericidal activity than that of plain Fe3O4 NPs

and Ag-NPs We suggest that the enhancement in bactericidal activity of Fe3O4–Ag hybrid NPs

might be likely from main factors such as: (i) enhanced surface area property of hybrid

nanopar-ticles; (ii) the high catalytic activity of Ag-NPs with good dispersion and aggregation stability due

to the iron oxide magnetic carrier, and (iii) large direct physical contacts between the bacterial cell

membrane and the hybrid nanoparticles The superparamagnetic hybrid nanoparticles of iron oxide

magnetic nanoparticles decorated with silver nanoparticles can be a potential candidate to

effec-tively treat infectious MRSA pathogen with recyclable capability, targeted bactericidal delivery and

minimum release into environment

Keywords: Composite Materials, Magnetic Materials, Chemical Synthesis, Electron Microscopy

1 INTRODUCTION

In recent years, several outbreaks of infectious diseases

were reported, leading to a signiﬁcant threat on global

economies and public health The outbreak of infectious

diseases has not only occurred in developing countries

with low levels of hygiene and sanitation, but has also been

recognized in developed countries.1 In addition, the

inap-propriate and unmethodical uses of bactericidal antibiotic

∗ Authors to whom correspondence should be addressed.

drugs in humans as well as in veterinary and agricultural medicine have led to a rapid increase in new strains of drug-resistant micro-organisms Especially in developing countries, the combination of patients with poor immune system and cross-infection in several hospitals and health care systems has given rise to nosocomial infections with multi-drug-resistant pathogens.2 The global challenge is facing, the outbreak infections mediated by these highly resistant pathogens can cause uncontrolled epidemics of bacterial diseases that can no longer be treated and hence seriously risk expenses for the society community.1 2

5902 J Nanosci Nanotechnol 2016, Vol 16, No 6 1533-4880/2016/16/5902/011 doi:10.1166/jnn.2016.11029

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To eliminate outbreak of infectious pathogens,

disinfec-tion methods should be done properly to eliminate these

pathogens from infected environmental areas, and effective

treatments should also be carried for patients in hospitals

and in the community.1 2Interactions between

nanomate-rials and bio-systems have been intensively researched.3–6

Among them, nano-antimicrobials have been studied for

treatment of these pathogens.7 8 Among existing

nano-antimicrobials, silver nanoparticles (Ag-NPs) have been

recognized as a promising candidate to ﬁght against

resis-tant pathogens.9 10 However, the Ag-NPs showed two

major shortcomings for application in practice: ﬁrst,

Ag-NPs exhibit a highly toxic potential to human cells

and ecology when the use of high concentrations; and

second, due to the protection offered by the bioﬁlm

mode of growth, the Ag-NPs cannot eradicate bacterial

bioﬁlms.11–13

In order to overcome these challenges, hybrid

nanoparticles of silver nanoparticles and magnetic oxide

nanoparticles (i.e., Fe3O4, Fe2O3) were studied for

enhanc-ing antibacterial activity and high compatibility with

human cells.13 14 For example, Prucek et al.13 produced

two types of magnetic composite nanoparticles including

Ag@Fe3O4 and Fe2O3@Ag Both synthesized

nanocom-posites exhibited signiﬁcant antibacterial and

antifun-gal activities against ten tested bacterial strains and

four candida species At the observed minimum

inhi-bition concentration, these nanocomposites did not

dis-play acute cytotoxicity against mice embryonal ﬁbroblast

Chen et al.14 reported one-pot synthesis of Ag@Fe2O3

core–shell and Ag–Fe2O3 heteromer nanoparticles They

also indicated that the Ag@Fe2O3 core–shell

nanoparti-cles exhibited superior antibacterial property compared to

heteromer Ag–Fe2O3nanoparticles and plain Ag

nanopar-ticles The authors proposed that the slow diffusion of

silver ion out of the Fe2O3 shell resulting in enhanced

antibacterial activity Although some previous results15–18

have been achieved for improving the antibacterial activity

of hybrid nanoparticles by combining the silver

nanopar-ticles and magnetic oxide nanoparnanopar-ticles, but the possible

mechanism for enhanced bactericidal activity of hybrid

nanoparticles has not been fulﬁlled The other results19–34

also have proved that the magnetic oxide nanoparticles are

a promising carrier for enhancing biological activity of

hybrid nanoparticles through tuning its surface and

inter-face However, there is a challenge when synthesizing the

hybrid nanoparticles and applying them in practice is the

aggregation instability of hybrid nanoparticles caused by

magnetic and electrostatic interactions.13 28 29

To prevent their aggregation, several chemical routes

have been made such as the use of polymer matrix as an

effective linker, immobilization of Ag-NPs on the surface

of magnetic silica composite Fe3O4–SiO2–Ag15or of

mag-netic carbon composite Fe3O4@C@Ag,16 or synthesizing

Ag–Fe3O4 core–shell nanoparticles at high temperature.17

In this work, we report a facile synthesis of Ag-NPs,

Fe3O4-NPs and their Ag–Fe3O4 hybrid nanoparticles by modiﬁed coprecipitation and photochemical method To the best our knowledge, there is no report on photo-chemical growth of Ag nanocrystals on Fe3O4 seed with oleic acid as a stabilizer and glucose as reducing agent The photochemical method is a promising alternative for decoration of Ag nanocrystals on the nanosized carri-ers for various practical applications This method shows many advantages over reported previous methods such

as green synthesis, reaction occurring at room temper-ature, long-term stable dispersions and small particles sizes A comparative study of antibacterial activity of

prepared nanoparticles was conducted against

Staphylo-coccus aureus bacteria, which is a the most frequently

drug-resistant pathogen in Vietnam We provide more insights in mechanism of bactericidal activity of these nanoparticles against drug-resistant bacteria in the light of ultrastructural studies

2 EXPERIMENTAL PROCEDURES 2.1 Chemicals

The analytical-grade silver nitrate (AgNO3, 99.9%), sodium hydroxide (NaOH), ammonium hydroxide (NH3, 25%), ferrous chloride tetrahydrate (FeCl2·4H2O,≥99%), ferric chloride hexahydrate hydrogen (FeCl3 · 6H2O

≥99%), oleic acid ≥99% and glucose that were used

in this study were purchased from Shanghai Chemical Reagent Co Ltd

2.2 Synthesis of Fe3O4 Nanoparticles by Co-Precipitation Method

The superparamagnetic Fe3O4 nanoparticles were synthe-sized a coprecipitation method as described previously.35 Brieﬂy, 5.41 g (0.02 mol) FeCl3· 6H2O and 1.99 g (0.01 mol) FeCl2· 4H2O were dissolved in the water and stirred under air in 10 min In order to obtain inverse spinel phase, 20 mL of 0.5 M NaOH solution was added slowly to the solution of iron salts The color of solu-tion changed immediately from orange to dark brown after addition of NaOH indicating the formation of superpara-magnetic Fe3O4 nanoparticles The precipitation reaction was then stirred at temperature about 30 C for 30 min The product of Fe3O4 nanoparticles was separated from solution by external magnetic ﬁeld and washed several times by deionized water and acetone

2.3 Synthesis of Ag Nanoparticles by Tollens Process The silver nanoparticles were prepared by using a modi-ﬁed Tollens process as reported elsewhere.36Brieﬂy, 1.7 g (10 mmol) of AgNO3 was dissolved in 100 mL of deion-ized water The AgNO3solution was then precipitated with 0.62 g (15.5 mmol) of sodium hydroxide (Aldrich,>99%).

The obtained precipitate, which is composed of Ag2O,

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was ﬁltered and dissolved in 100 mL of aqueous ammonia

(0.4% w/w, 23 mmol) until a transparent solution of silver

ammonium complex [Ag(NH32]+(aq) formed Up to 2.5 g

(8.9 mmol) of oleic acid was then added dropwise into the

complex, and the resulting solution was gently stirred for

2 h at room temperature until the complete homogeneity

of the reaction mixture was achieved The reduction

pro-cess of the silver complex solution by the addition of 2 g

(11.1 mmol) of glucose was initiated with UV irradiation

A UV lamp ( = 365 nm, 35 W) was used as a light source

to stimulate the reduction process

2.4 Synthesis of Fe3O4–Ag Hybrid Nanoparticles by

Modiﬁed Photochemical Method

A schematic process for synthesis of Fe3O4–Ag hybrid

nanoparticles was shown in Figure 1 First, 30 mg of the

Fe3O4 nanoparticles were added to 100 mL of deionized

water together with 3 mL of acid oleic In order to well

disperse the Fe3O4nanoparticles, the solution was

contin-uously stirred at 80 C in 20 hour Subsequently, 10 mL

of silver ammonium complex [Ag(NH32]+aqwas added to

the complex of oleic acid-coated Fe3O4nanoparticles and

the resulting solution was gently stirred for 4 h at room

temperature until the complete homogeneity of the

reac-tion mixture was achieved After 12 h of UV irradiareac-tion,

the colloidal solution of Fe3O4–Ag hybrid nanoparticles

was obtained

2.5 Characterization Techniques

Transmission electron microscopy (TEM, JEOL-JEM

1010) was conducted to investigate the morphology and

size distribution of as-prepared Fe3O4 and Ag

nanoparti-cles Also, high resolution TEM (Tecnai, G20, 200 kV,

FEI) was used to determine the structure of Fe3O4–Ag

hybrid nanoparticles The samples for TEM/HRTEM

char-acterization were prepared by placing a drop of

col-loidal solution on a formvar-coated copper grid that

was dried at room temperature The composition of

the Fe3O4–Ag hybrid nanoparticles was characterized by

energy-dispersive X-ray (5410 LV JEOL) The crystalline

structure of the prepared Fe3O4, Ag nanoparticles and

Fe3O4–Ag hybrid nanoparticles was analyzed by X-ray

diffraction (XRD, Bruker D5005) using CuK radiation

Figure 1 Two-step protocol for formation of Fe 3 O 4 –Ag hybrid

nanoparticles.

The background was subtracted using linear interpolation method The UV-vis absorbance spectra were recorded using a HP 8453 spectrophotometer, and the absorp-tion spectrum of all suspension samples in 10 mm path length quartz cuvettes was 300 nm to 900 nm Magnetiza-tion curves of Fe3O4 nanoparticles and Fe3O4–Ag hybrid nanoparticles were measured by vibrating system magne-tometers (VSM, MicroSense)

2.6 Evaluation of Antibacterial Activity

• Bacterial strain and culture medium

Bacterial strain chosen for this study was Gram-positive

Staphylococcus aureus (ATCC 43300) This strain was

provided from the Department of Virology at the National Institute of Hygiene and Epidemiology (NIHE) in Hanoi The growth of cell cultures was executed in a Luria-Bertani (LB) medium (1% tryptone, 0.5% yeast extract and 1% NaCl, pH 7) Next, the culture medium containing bacteria was kept in an incubator for 24 h at the tempera-ture 37C; then the content of bacterial culture in it was

108CFU/ml, where the CFU is the colony forming unit

• Paper-disc diffusion method

The paper-disc diffusion method was used to evaluate the antibacterial activity of the studied samples against tested bacteria Using the spread plate method, nutrient agar plates were inoculated with 100 l of bacterial

sus-pension containing 105CFU Sterile Whatman No 1 ﬁlter paper discs with a diameter of 5 mm each, loaded with

Control plates were maintained with discs containing dis-tilled water These plates were incubated at 37C for 24 h and the zone of inhibition (ZOI) was measured by sub-tracting the disc diameter from the total inhibition zone diameter

• Standard microdilution method

The standard microdilution method was applied to quantitatively compare the bactericidal activity of stud-ied samples.36First, the nutrient agar medium was heated

to 50 C to get a uniform distribution Next, 10 ml of each Fe3O4, Ag and Fe3O4–Ag nanoparticles solution was added into Petri plates containing 25 ml of nutrient agar medium Total volume in each Petri plate was kept 35 ml and the mixing solution was solidiﬁed with agar after

15 min After that 100 l of a suspension of S aureus

bac-teria was pipetted and spread on the surface of agar medium containing Fe3O4, Ag and Fe3O4–Ag nanoparticles The Petri plates were incubated at 37C for 24 h in a shak-ing incubator (150 rpm) to encourage bacterial cell growth The intensity of bacterial growth on agar plates with Fe3O4,

Ag and Fe3O4–Ag nanoparticles was monitored by naked eye and stereo microscope (ZMS800, Nikon) All the tests

were compared with the S aureus growth intensity on the

agar plate in the absence of studied nanoparticles

In order to determine the bactericidal activity of Fe3O4,

Ag and Fe3O4–Ag nanoparticles we used colony count-ing method for determincount-ing the number of CFU which has

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been grown after addition of S aureus suspension of less

concentration (103 CFU) in agar medium mixed with all

studied nanoparticles Control solutions were treated

sim-ilarly but without exposure to the nanoparticles for

com-parative purpose Colonies were counted and compared to

those in control plates to calculate the changes in the

bac-terial growth inhibition All experiments were performed

under sterile conditions and in triplicate

The percentage reduction ratio of the bacteria has been

expressed as:

whereR is the percentage reduction ratio, A–the number

of bacterial colonies in the Petri plates without

nanopar-tiles and B–the number of colonies in the Petri plates

con-taining Fe3O4, Ag and Fe3O4–Ag nanoparticles

2.7 Ultrathin Sectioning Sample Preparation of

MRSA Bacteria Cells

In order to obtain further understanding of the

bacte-ricidal and interaction mechanism of the nanoparticles

samples, ultrathin sectioning technique was carried out

to observe the ultrastructural changes of bacterial cells

destroyed by action of the Ag-NPs and Fe3O4–Ag hybrid

nanoparticles.36After the S aureus bacteria were exposed

to the Ag-NPs and Fe3O4–Ag hybrid nanoparticles, the

samples were collected and ﬁxed by 2.5%

glutaralde-hyde in cacodylate buffer (0.1 M) for 30 min at room

temperature Washing the ﬁxed samples by cacodylate

buffer three times for 10 min each, then transferred to 1%

OsO4/cacodylate buffer for 1 h The samples were then

dehydrated by using a series of alcohol with 50, 70, 80,

90 and 100% (two times× 5 min), and then propylene

oxide (three times× 5 min) The samples were inﬁltrated

and ﬁnally embedded in Epon 812 at 60C for 24 h The

polymerized samples were sectioned into ultrathin slices

60–90 nm in thickness, and placed on the collodion-coated

copper grids (300 meshes) The analyses of

ultrastruc-tural changes of interior of the bacteria cells were

con-ducted by transmission electron microscopy (TEM, JEM

1010, JEOL)

3 RESULTS AND DISCUSSION

3.1 Microstructure Analysis

3.1.1 Synthesis of Ag Nanoparticles by Tollens Process

In our work, silver nanocrystals were synthesized using

Tollens reaction by reduction of silver ions by glucose with

the presence of oleic acid as a stabilizer and UV

irradia-tion A fundamental reduction reaction involving Tollens

process under UV irradiation is follows:37 38

32++ RCHOH h

−→ Ag0+ 2NH3+ H++ R ˙COH nAg0→ Agn0

It was shown that, in Tollens reaction, the mean diameter, particle size distribution, and aggregate stability of silver nanocrystals were strongly dependent on the conditions of synthesis such as the temperature, ammonia concentration, and the change in the pH of the medium during the reduc-tion process Furthermore, the addireduc-tion of surfactants into the reaction medium could control the particle size and uniform aqueous dispersion of NPs.36–38

In order to control the stable dispersions and sizes of nanoparticles, we also employed the UV irradiation of the reagent mixture at the stage of reducing the silver ammo-nium complex with glucose.39It was shown that activating the nanoparticles formation under the UV treatment with

a wavelength of 365 nm made it possible to carry out the reduction process at room temperature while obtaining the stable dispersion of NPs.39 40 Our previous works36–38 revealed that the use of oleic acid as a surfactant, glu-cose as a reducing agent with simultaneous UV-irradiation can effectively produce long-term stable dispersion of the silver NPs in aqueous medium (>1 year) with narrow

average size distribution In addition, this synthesis route

is environmentally friendly because of use of non-toxic chemicals

Figures 2(a), (b) shows a TEM image of the Ag-NPs and corresponding size distribution of Ag-NPs calculated from TEM image It can be seen that the Ag-NPs were well-formed and well-dispersed Almost no aggregates

of Ag-NPs were observed through TEM investigation Figure 2(b) shows the histogram of the size distribution

of silver NPs obtained from the TEM image The average size of Ag-NPs was about 4.5 nm with a relatively nar-row distribution The crystallinity of the Ag-NPs was con-ﬁrmed by powder X-ray diffraction (see Fig 3) It showed three broad peaks assigned to 111, 200, and 220 planes of

a face centered cubic lattice of bulk silver (JCPDS card,

No 004-0783) This fact conﬁrmed that the synthesized NPs consisted of pure silver with high crystallinity Our calculation from the XRD patterns according to Scherrer expression revealed that, the oleic acid-stabilized Ag-NPs had average-sized particles (∼6–7 nm) This result is fully consistent with the calculation from TEM image Further-more, the broadening of the full width at half maximum (FWHM) of the XRD pattern revealed the nanoscale sizes

of Ag-NPs.37 38

3.1.2 Synthesis of Fe3O4 Nanoparticles by Co-Precipitation Method

Magnetic iron oxide NPs (Fe3O4-NPs) were synthesized using co-precipitation reaction A fundamental reaction for formation of iron oxide NPs is as follows:

Fe2++ 2Fe3 ++ 8NaOH → Fe3O4+ 4H2O

It revealed that in co-precipitation reaction the particles size and shape of iron oxide nanocrsytals were strongly dependent on synthesis conditions such as the mol ratio of

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Figure 2 TEM images and size distribution of (a), (c) Ag NPs and (b), (d) Fe 3 O 4 NPs.

Fe2+/Fe3+, concentration of sodium hydroxide and pH of

solution.19

Figures 2(c), (d) shows a TEM image of as-prepared

Fe3O4-NPs and a histogram of size distribution of Fe3O4

-NPs calculated from TEM image It can be seen that the

average size of Fe3O4-NPs obtained about 19.2 nm The

XRD analysis was conduct to examine the crystallinity of

Fe3O4-NPs as shown in Figure 3 It revealed that there

are eight broad peaks assigned to 111, 220, 311, 222,

400, 422, 511 and 440 planes of a spinel structural

lat-tice of magnetite (JCPDS card, No 11-0614) Our

cal-culation from the XRD patterns according to Scherrer

expression indicated that the as-prepared Fe3O4-NPs had

Figure 3 XRD patterns of Ag NPs, Fe 3 O 4 NPs and Fe 3 O 4 –Ag hybrid

nanoparticles.

average-sized particles (∼17–18 nm) This result is con-sistent with the calculation from TEM image

3.1.3 Synthesis of Fe3O4–Ag Hybrid Nanoparticles by Modiﬁed Photochemical Method

In order to obtain Fe3O4–Ag hybrid nanoparticles, the iron oxide nanoparticles were also used as seed for the growth

of silver nanoparticles on their surface through the pho-tochemical reaction of Ag in the Fe3O4 nanoparticle seed solution As it can be seen from Figure 3 that seven peaks are observed in the XRD pattern of Fe3O4–Ag hybrid nanoparticles Four of them can be indexed well to the fcc inverse spinel structure of Fe3O4 phase They are marked with crystal planes of (311), (220), (400) and (200) of magnetite (JCPDS card, No 11-0614), respectively The reflections positioned at 2theta= 38.1, 44.3 and 64.4 correspond to (111), (220) and (311) fcc crystal planes of bulk silver (JCPDS card, No 004-0783), respectively High-resolution transmission electron microscopy (HRTEM) along with EDS and FFT analyses was employed to confirm the heterodimer nature of Fe3O4–Ag nanostructure Figure 4 shows the bright field TEM image along with its EDS spectrum and HRTEM images of nanostructure Figure 4(a) indicates that formed Fe3O4–Ag nanostructures have quasi-spherical particles and nearly uniform distribution The selected area electron diffraction (SAED) shows polycrystalline structure (see the inset of Fig 4(a)) The EDS spectrum (Fig 4(b)) of bright field TEM image reveals that the nanostructure consisted of main composition of Fe, O and Ag elements indicating the high purity of sample However, it is not possible

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Figure 4 TEM images and EDS elemental analysis of Fe 3 O 4 –Ag hybrid nanoparticles.

to determine the elemental ratio of Fe to Ag from EDS

spectrum because they are recorded at independent

loca-tions The HRTEM images (Figs 4(c) and (d)) conﬁrmed

the exact nature of Fe3O4 and Ag nanoparticles By using

Figure 5 High-resolution TEM images of Fe3O4–Ag dimer nanoparticles.

FFT analysis in Gatan Digital Micrographs software we conﬁrmed the formation of both Ag(111) and Fe3O4(311) nanoparticles with high crystallinity Furthermore, the het-erodimer nature of Fe3O4–Ag nanostructure is evidenced

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Figure 6 UV-vis spectra of Fe 3 O 4 NPs, Ag NPs and Fe 3 O 4 –Ag hybrid

nanoparticles.

from HRTEM analysis as shown in Figure 5 The Ag

nanocrystals with particle sizes about ∼10 nm are stably

formed on the Fe3O4 seeds

3.2 UV-Vis Analysis

Figure 6 shows (a) the UV-vis spectra of Fe3O4, Ag-NPs,

and Fe3O4–Ag hybrid nanoparticles Obviously, the

Ag-NPs and Fe3O4–Ag hybrid nanoparticles samples display

strong absorption peaks at 428 and 437 nm, respectively,

because of the surface plasmon resonance (SPR) effect

of Ag-NPs In addition, there is no SPR band

observa-tion for Fe3O4 sample The appearance of characteristic

surface plasmon band at 437 nm indicates the

forma-tion of Ag-NPs on Fe3O4 seeds The SPR phenomenon

occurs when the incident light interacts with valence

elec-trons at the outer band of Ag-NPs, leading to oscillation

of electrons along with the frequency of the

electromag-netic source.42 However, the absorption band of Fe3O4–

Ag sample (∼437 nm) is shifted to longer wavelength

as compared to Ag-NPs sample alone (∼428 nm) The

shifting of the absorption peak toward longer wavelength

Figure 7 Magnetization curves of (a) Fe3O4NPs and (b) Fe3O4–Ag hybrid nanoparticles.

indicates the formation of larger Ag nanoparticles with different shapes and sizes.36 37 This is consistent with results from TEM analysis, the size of Ag-NPs on Fe3O4 seeds (dTEM∼10 nm) is slightly larger than that of Ag-NPs (dTEM∼5 nm) as calculation of size distribution from TEM images (see Figs 2 and 5) The surface plasmon band shifts are strongly dependent on particle size, shape, chemical surrounding, and adsorbed species on the surface and dielectric medium, whereas the plasmon peak and full width at half maximum depends on the extent of colloid aggregation.38 39

3.3 Magnetization Analysis The magnetic properties of Fe3O4 NPs and Fe3O4–Ag hybrid NPs were checked by magnetization measurements Figure 7 depicts the magnetization curves measured at room temperature of (a) Fe3O4 NPs and (b) Fe3O4–Ag hybrid NPs samples It can be seen that the Fe3O4 NPs sample were superparamagnetic and saturation magnetiza-tion value (Ms) was about ∼55.35 emu/g The interaction between the nanoscale Ag and Fe3O4leads to a change on magnetization behavior of Fe3O4–Ag NPs Obviously, the

Fe3O4–Ag hybrid NPs also exhibited superparamagnetic behavior and the Ms value was ∼52,98 emu/g (calcula-tion was corrected for only amount of Fe3O4 phase) This result indicates that the formation of silver nanoscrytals on

Fe3O4 seeds does not affect superparamagnetic property

of sample In addition, the existence of silver nanoscrytals can prevent agglomeration of the Fe3O4 NPs Noticeably, due to high value of magnetization the Fe3O4–Ag hybrid NPs can be easily removed from solutions and recycled by exerting an external magnetic ﬁeld Thus, the Fe3O4–Ag hybrid NPs showed high potential to be developed into a new class of recyclable antibacterial agents

3.4 Antibacterial Activity Analysis The antibacterial activities of Fe3O4, Ag-NPs, and Fe3O4–

Ag hybrid nanoparticles were initially assessed with the

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paper-disc diffusion method, which is widely used for

quick antibiotic susceptibility determinations Figure 8

dis-plays the photographs of result of antibacterial test for

all studied samples using paper-disc diffusion method

As shown in Figure 8(a), the Ag-NPs exhibit signiﬁcant

inhibitory effects on S aureus bacteria Obviously, the

zone of inhibition (ZOI) increases with increasing

concen-tration of Ag-NPs from Ag1 sample (∼10 g/mL) to Ag4

sample (∼50 g/mL) A similar tendency was found for

Fe3O4–Ag sample as shown in Figure 8(b) The Fe3O4–Ag

(c)

Figure 8 Antibacterial activity of Fe 3 O 4 NPs, Ag NPs and Fe 3 O 4 –Ag

hybrid nanoparticles.

hybrid NPs show noticeable inhibitory effects on S aureus

bacteria, however, there is no inhibitory effect for bare

Fe3O4sample (see Fig 8(b)) This ﬁnding suggests that the antibacterial activity of the hybrid nanoparticles samples might be resulting from the Ag nanocrystals decorated on

Fe3O4seeds The evaluation of ZOI for all studied samples was summarized in Figure 8(c)

In order to obtain more precise and comprehensive data for the antibacterial activities of three nanoparticles

sam-ples against S aureus, we used standard microdilution

method for determining the number of growth bacterial colonies in these samples Figure 9 shows the agar plates treated (a) without nanoparticles and with the (b) Fe3O4, (c) Ag-NPs, and (d) Fe3O4–Ag hybrid nanoparticles sam-ples It can be seen that in the absence of nanoparticles (Fig 9(a)), a drastic growth of bacteria was observed With the presence of Ag-NPs and Fe3O4–Ag NPs (Figs 9(c), (d)), the bacterial growth was completely inhibited, how-ever for the case of Fe3O4 NPs sample (Fig 9(b)), no signiﬁcant inhibitory effect was also found The MIC val-ues were also determined from microdilution method It is noted that the MIC values for hybrid nanoparticles sam-ples are corrected for the effective concentration of Ag

in the heterodimer Fe3O4–Ag Our obtained results reveal that the MIC value of Ag-NPs sample (∼8.2 g/mL) is slightly lower than that of Fe3O4–Ag hybrid NPs sample (∼9.7 g/mL) Moreoever, the percentage reduction ratio

of the bacteria for both Ag-NPs and Fe3O4–Ag NPs sam-ples obtained nearly 100% as calculation using Eq (1), whereas percentage reduction ratio of bacteria for Fe3O4 samples was about 3.4% This result suggests that Fe3O4 nanoparticles could play a minor role in bactericidal activ-ity of hybrid nanoparticles

Figure 9 Agar plates treated (a) without nanoparticles (control), (b) with Fe3O4NPs, (c) Ag NPs, and (d) Fe3O4–Ag hybrid nanoparticles.

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3.5 Proposed Bactericidal Mechanism of Ag-NPs and

Fe3O4–Ag Hybrid NPs

To provide more rudimentary insights into the

interac-tion and bactericidal mechanism between the Ag-NPs and

Fe3O4–Ag hybrid nanoparticles with tested S aureus

bac-teria, the ultrastructural and morphological analyses by

using electron microscopic technique were conducted The

colloidal solutions of studied samples were dropped onto

the surface of S aureus bacteria grown on agar plates.

After 0 min, 15 min, and 30 min, S aureus cells were

taken out and underwent the sectioning method for TEM

observation

Figure 10 displays the cross-sectional TEM images

showing different stages of interaction of (a) Ag-NPs

sam-ple and (b) Fe3O4–Ag hybrid nanoparticles with S aureus

bacteria after 0 min, 15 min, and 30 min It was

estab-lished for the case of bare Ag-NPs that the antibacterial

activity of the Ag-NPs is a complex process, in which the

silver NPs and their ions caused by the release of silver

ions Ag+can produce free radicals, resulting in induction

of oxidative stress (i.e., reactive oxygen species; ROS).43

The produced ROS can irreversible damage bacteria by

multiple mechanisms (such as direct attachment to cell

Figure 10 The cross-sectional TEM images showing different stages of interaction of (a) Ag-NPs sample and (b) Fe 3 O 4 –Ag hybrid nanoparticles

with S aureus bacteria after 0 min, 15 min, and 30 min.

membrane and disruption of membrane integrity, changes

in membrance permeability, interaction with proteins and disruption of their regular function, interference with DNA replication and causing DNA damage), and ﬁnally result-ing in cell bacterial death.39 43

As shown in Figure 10 (left part), at different magniﬁca-tions and secmagniﬁca-tions, TEM results showed that some Ag-NPs

bindings around both the S aureus cell membranes as well

as inside the cells were found Obviously, the Ag-NPs ﬁrst attached to the surface of the cell membrane, and then penetrated further inside the bacteria It should be noted that only Ag-NPs with sufﬁciently small diameters pene-trated into the cells (usually< 10 nm) After 30 min, the

cytoplasm was completely destroyed as the silver NPs pen-etrated the cell [see the left part of Fig 10] This analysis

probes how S aureus bacterial cells can be destroyed by

the Ag-NPs

In presence of Fe3O4–Ag hybrid nanoparticles [see the

right part of Fig 10], the surface of S aureus

bacte-rial cells was significantly modified In first stage, lots

of hybrid nanoparticles were attached to the surface of cells membrane The strong release of Ag+and Fe2+ions made the hybrid nanoparticles positively charged Because

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bacteria cell walls are negatively charged hence they attract

each other by electrostatic interaction, leading to large

density of hybrid nanoparticles attached on the surface of

cells membrane The enhanced surface area of Fe3O4–Ag

nanoparticles produces large contact area between the

bac-terial cell membrane and nanoparticles.44 The Fe3O4–Ag

hybrid nanoparticles dissolve the outer envelope of

bacte-rial cell wall and thereby leakage of cellular constituents,

resulting in cell death In our present case, due to large

sizes of Fe3O4–Ag hybrid nanoparticles (25–30 nm), they

can not penetrate into the cells membrane as the case of

Ag-NPs (small sizes∼ 5 nm)

More noticeably, the increase in bacterial resistance

to antimicrobial agents poses a serious problem in the

treatment of infectious diseases as well as in

epidemiolog-ical practice One of the major shortcomings of

antibac-terial drugs and nanoparticles is their failure to ﬁght with

bacteria (i.e., S aureus) because they have capality to

produce biofilms Biofilms are known as a significant

prob-lem because bioﬁlm formation protects pathogenic

bacte-ria against antibiotic drugs and is one of the main reasons

for outbreak of infectious diseases The results of

ultra-structural studies clearly demonstrates that the as-prepared

Ag-NPs and Fe3O4–Ag hybrid nanoparticles have

consid-erable capability to penetrate into bioﬁlms of S aureus

bacteria and disrupt the integrity and viability of the

bac-terial membrane, resulting in cells death.45The Fe3O4–Ag

hybrid nanoparticles can be high potential as new

antibac-terial agents to ﬁght with infectious pathogens due to many

advantages over conventional silver nanoparticles such as

recyclable capability to reduce the release of nanoparticles

in environment, noticeable antibacterial activity and high

biomedical compatibility

4 CONCLUSIONS

In this work, we established facile chemistry methods

for synthesis of Fe3O4–Ag hybrid NPs The Ag

nano-crystals were growth by photochemical reaction on the

Fe3O4 seeds to form a heterodimer nature of Fe3O4–

Ag hybrid nanoparticles A detailed comparative study

of antibacterial activity and mechanism of these NPs

against the MRSA pathogen was conducted The Fe3O4–

Ag hybrid NPs exhibited more signiﬁcant antibacterial

efﬁcacy against MRSA pathogen than that of the plain

Fe3O4 NPs and Ag-NPs The interaction and bactericidal

mechanism between the Fe3O4–Ag hybrid nanoparticles

with tested S aureus bacteria was veriﬁed by adapting

ultrastructural studies The obtained results suggested that

the Fe3O4–Ag hybrid nanoparticles could be a promising

antimicrobial material for practical applications in

treat-ment and prevention of infectious diseases

Acknowledgment: This research is funded by Vietnam

National Foundation for Science and Technology

Develop-ment (NAFOSTED) under grant number 103.99-2012.10

The authors would like to acknowledge the Center for Electron Nanoscopy, Technical University of Denmark (DTU CEN) for HRTEM works Also, the technical sup-ports for biological measurements at National Institute

of Hygiene and Epidemiology (NIHE) in Vietnam are acknowledged

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with S aureus bacteria after min, 15 min, and 30 min.

membrane and disruption... The evaluation of ZOI for all studied samples was summarized in Figure 8(c)

In order to obtain more precise and comprehensive data for the antibacterial activities of three nanoparticles. .. Fe3O4, (c) Ag-NPs, and (d) Fe3O4–Ag hybrid nanoparticles sam-ples It can be seen that in the absence of nanoparticles (Fig 9(a)), a drastic growth of bacteria was observed

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