Article Copyright © 2016 American Scientific Publishers All rights reserved Printed in the United States of America Journal of Nanoscience and Nanotechnology Vol 16, 5902–5912, 2016 www.aspbs.com/jnn Synthesis, Characterizations of Superparamagnetic Fe3O4 –Ag Hybrid Nanoparticles and Their Application for Highly Effective Bacteria Inactivation Le Minh Tung1 ∗ , Nguyen Xuan Cong2 , Le Thanh Huy2 , 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 ∗ Department of Physics, Tien Giang University, My Tho City, Tien Giang Province 86000, Vietnam Department 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 Faculty of Chemistry and Environment Technology, Hung Yen University of Technology and Education, Hung Yen 16000, Vietnam Department of Physics, Hanoi University of Science, 334 Nguyen Trai, Thanh Xuan, Ha Noi 10000, Vietnam Institute of Physics at Ho Chi Minh City, Vietnam Academy of Science and Technology (VAST), Ho Chi Minh 70000, Vietnam Department of Micro-and Nanotechnology, Technical University of Denmark Building 345Ø, Ørsted Plads, Kgs Lyngby 2800, Denmark National Institute of Hygiene and Epidemiology (NIHE), 1-Yersin Street, Hai Ba Trung District, Hanoi 10000, Vietnam Delivered by Ingenta to: Chalmers Tekniska Hogskola (Chalmers University of Technology) IP: 5.101.218.42 On: Thu, 12 May 2016 18:01:07 In recent years, outbreaks of infectious diseases caused by pathogenic micro-organisms pose a Copyright: American Scientific Publishers serious threat to public health In this work, Fe3 O4 -Ag hybrid nanoparticles were synthesized by simple chemistry method and these prepared nanoparticles were used to investigate their antibacterial properties and mechanism against methicilline-resistant Staphylococcus aureus (MRSA) pathogen The formation of dimer-like nanostructure of Fe3 O4 –Ag hybrid NPs was confirmed by X-ray diffraction and High-resolution Transmission Electron Microscopy Our biological analysis revealed that the Fe3 O4 –Ag hybrid NPs showed more noticeable bactericidal activity than that of plain Fe3 O4 NPs and Ag-NPs We suggest that the enhancement in bactericidal activity of Fe3 O4 –Ag hybrid NPs might be likely from main factors such as: (i) enhanced surface area property of hybrid nanoparticles; (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 effectively treat infectious MRSA pathogen with recyclable capability, targeted bactericidal delivery and minimum release into environment Keywords: Composite Materials, Magnetic Materials, Chemical Synthesis, Electron Microscopy 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 inappropriate and unmethodical uses of bactericidal antibiotic ∗ Authors to whom correspondence should be addressed 5902 J Nanosci Nanotechnol 2016, Vol 16, No 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 1533-4880/2016/16/5902/011 doi:10.1166/jnn.2016.11029 Tung et al Synthesis, Characterizations of Superparamagnetic Fe3 O4 –Ag Hybrid Nanoparticles To eliminate outbreak of infectious pathogens, disinfecIn this work, we report a facile synthesis of Ag-NPs, tion methods should be done properly to eliminate these Fe3 O4 -NPs and their Ag–Fe3 O4 hybrid nanoparticles by modified coprecipitation and photochemical method To pathogens from infected environmental areas, and effective treatments should also be carried for patients in hospitals the best our knowledge, there is no report on photoand in the community.1 Interactions between nanomatechemical growth of Ag nanocrystals on Fe3 O4 seed with rials and bio-systems have been intensively researched.3–6 oleic acid as a stabilizer and glucose as reducing agent Among them, nano-antimicrobials have been studied for The photochemical method is a promising alternative for treatment of these pathogens.7 Among existing nanodecoration of Ag nanocrystals on the nanosized carriantimicrobials, silver nanoparticles (Ag-NPs) have been ers for various practical applications This method shows recognized as a promising candidate to fight against resismany advantages over reported previous methods such tant pathogens.9 10 However, the Ag-NPs showed two as green synthesis, reaction occurring at room tempermajor shortcomings for application in practice: first, Agature, long-term stable dispersions and small particles NPs exhibit a highly toxic potential to human cells sizes A comparative study of antibacterial activity of and ecology when the use of high concentrations; and prepared nanoparticles was conducted against Staphylosecond, due to the protection offered by the biofilm coccus aureus bacteria, which is a the most frequently mode of growth, the Ag-NPs cannot eradicate bacterial drug-resistant pathogen in Vietnam We provide more biofilms.11–13 insights in mechanism of bactericidal activity of these In order to overcome these challenges, hybrid nanoparticles against drug-resistant bacteria in the light of nanoparticles of silver nanoparticles and magnetic oxide ultrastructural studies nanoparticles (i.e., Fe3 O4 , Fe2 O3 ) were studied for enhancing antibacterial activity and high compatibility with EXPERIMENTAL PROCEDURES human cells.13 14 For example, Prucek et al.13 produced 2.1 Chemicals two types of magnetic composite nanoparticles including The analytical-grade silver nitrate (AgNO3 , 99.9%), Ag@Fe3 O4 and Fe2 O3 @Ag Both synthesized nanocomsodium hydroxide (NaOH), ammonium hydroxide (NH3 , posites exhibited significant antibacterial and antifun25%), ferrous chloride tetrahydrate (FeCl2 · 4H2 O, ≥99%), gal activities against ten tested bacterial strains and ferric hexahydrate (FeCl3 · 6H2 O four candida species At the to: observed minimum inhi- Hogskolachloride Delivered by Ingenta Chalmers Tekniska (Chalmers Universityhydrogen of Technology) ≥99%), oleic acid ≥99% and glucose that were used IP: 5.101.218.42 bition concentration, these nanocomposites did notOn: dis-Thu, 12 May 2016 18:01:07 in this study were purchased from Shanghai Chemical American Scientific Publishers play acute cytotoxicity against mice Copyright: embryonal fibroblast Reagent Co Ltd Chen et al.14 reported one-pot synthesis of Ag@Fe2 O3 core–shell and Ag–Fe2 O3 heteromer nanoparticles They 2.2 Synthesis of Fe3 O4 Nanoparticles by also indicated that the Ag@Fe2 O3 core–shell nanopartiCo-Precipitation Method cles exhibited superior antibacterial property compared to The superparamagnetic Fe3 O4 nanoparticles were syntheheteromer Ag–Fe2 O3 nanoparticles and plain Ag nanoparsized a coprecipitation method as described previously.35 ticles The authors proposed that the slow diffusion of Briefly, 5.41 g (0.02 mol) FeCl3 · 6H2 O and 1.99 g silver ion out of the Fe2 O3 shell resulting in enhanced 15–18 (0.01 mol) FeCl2 · 4H2 O were dissolved in the water and antibacterial activity Although some previous results stirred under air in 10 In order to obtain inverse have been achieved for improving the antibacterial activity spinel phase, 20 mL of 0.5 M NaOH solution was added of hybrid nanoparticles by combining the silver nanoparslowly to the solution of iron salts The color of soluticles and magnetic oxide nanoparticles, but the possible tion changed immediately from orange to dark brown after mechanism for enhanced bactericidal activity of hybrid 19–34 addition of NaOH indicating the formation of superparananoparticles has not been fulfilled The other results magnetic Fe3 O4 nanoparticles The precipitation reaction also have proved that the magnetic oxide nanoparticles are was then stirred at temperature about 30 C for 30 a promising carrier for enhancing biological activity of The product of Fe3 O4 nanoparticles was separated from hybrid nanoparticles through tuning its surface and intersolution by external magnetic field and washed several face However, there is a challenge when synthesizing the times by deionized water and acetone hybrid nanoparticles and applying them in practice is the aggregation instability of hybrid nanoparticles caused by 2.3 Synthesis of Ag Nanoparticles by Tollens Process magnetic and electrostatic interactions.13 28 29 The silver nanoparticles were prepared by using a modiTo prevent their aggregation, several chemical routes fied Tollens process as reported elsewhere.36 Briefly, 1.7 g have been made such as the use of polymer matrix as an (10 mmol) of AgNO3 was dissolved in 100 mL of deioneffective linker, immobilization of Ag-NPs on the surface of magnetic silica composite Fe3 O4 –SiO2 –Ag15 or of magized water The AgNO3 solution was then precipitated with netic carbon composite Fe3 O4 @C@Ag,16 or synthesizing 0.62 g (15.5 mmol) of sodium hydroxide (Aldrich, >99%) The obtained precipitate, which is composed of Ag2 O, Ag–Fe3 O4 core–shell nanoparticles at high temperature.17 J Nanosci Nanotechnol 16, 5902–5912, 2016 5903 Synthesis, Characterizations of Superparamagnetic Fe3 O4 –Ag Hybrid Nanoparticles 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(NH3 ]+ (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 h at room temperature until the complete homogeneity of the reaction mixture was achieved The reduction process of the silver complex solution by the addition of 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 Tung et al The background was subtracted using linear interpolation method The UV-vis absorbance spectra were recorded using a HP 8453 spectrophotometer, and the absorption spectrum of all suspension samples in 10 mm path length quartz cuvettes was 300 nm to 900 nm Magnetization curves of Fe3 O4 nanoparticles and Fe3 O4 –Ag hybrid nanoparticles were measured by vibrating system magnetometers (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 2.4 Synthesis of Fe3 O4 –Ag Hybrid Nanoparticles by provided from the Department of Virology at the National Modified Photochemical Method Institute of Hygiene and Epidemiology (NIHE) in Hanoi A schematic process for synthesis of Fe3 O4 –Ag hybrid The growth of cell cultures was executed in a Luriananoparticles was shown in Figure First, 30 mg of the Bertani (LB) medium (1% tryptone, 0.5% yeast extract Fe3 O4 nanoparticles were added to 100 mL of deionized and 1% NaCl, pH 7) Next, the culture medium containing water together with mL of acid oleic In order to well bacteria was kept in an incubator for 24 h at the temperadisperse the Fe3 O4 nanoparticles, the solution was continture 37 C; then the content of bacterial culture in it was uously stirred at 80 C in 20 hour Subsequently, 10 mL 108 CFU/ml, where the CFU is the colony forming unit of silver ammonium complex [Ag(NH3 ]+aq was added to • Paper-disc diffusion method the complex of oleic acid-coated Fe3 O4 nanoparticles and The paper-disc diffusion method was used to evaluate the resulting solution was gently stirred for h at room the antibacterial activity of the studied samples against temperature until the complete homogeneity of the reactested bacteria Using the spread plate method, nutrient tion mixture was achieved After 12 h of UV irradiation, agar plates were inoculated with 100 l of bacterial susthe colloidal solution of Fe3 O4 –Ag hybrid nanoparticles pension containing 105 CFU Sterile Whatman No filter was obtained Delivered by Ingenta to: Chalmers Tekniska Hogskola (Chalmers University of Technology) discs with18:01:07 a diameter of mm each, loaded with IP: 5.101.218.42 On: Thu, paper 12 May 2016 10 L ofPublishers samples, were placed on the inoculated plates 2.5 Characterization Techniques Copyright: American Scientific Control plates were maintained with discs containing disTransmission electron microscopy (TEM, JEOL-JEM tilled water These plates were incubated at 37 C for 24 h 1010) was conducted to investigate the morphology and and the zone of inhibition (ZOI) was measured by subsize distribution of as-prepared Fe3 O4 and Ag nanopartitracting the disc diameter from the total inhibition zone cles Also, high resolution TEM (Tecnai, G20, 200 kV, diameter FEI) was used to determine the structure of Fe3 O4 –Ag • Standard microdilution method hybrid nanoparticles The samples for TEM/HRTEM charThe standard microdilution method was applied to acterization were prepared by placing a drop of colquantitatively compare the bactericidal activity of studloidal solution on a formvar-coated copper grid that ied samples.36 First, the nutrient agar medium was heated was dried at room temperature The composition of to 50 C to get a uniform distribution Next, 10 ml of the Fe3 O4 –Ag hybrid nanoparticles was characterized by each Fe3 O4 , Ag and Fe3 O4 –Ag nanoparticles solution was energy-dispersive X-ray (5410 LV JEOL) The crystalline added into Petri plates containing 25 ml of nutrient agar structure of the prepared Fe3 O4 , Ag nanoparticles and medium Total volume in each Petri plate was kept 35 ml Fe3 O4 –Ag hybrid nanoparticles was analyzed by X-ray and the mixing solution was solidified with agar after diffraction (XRD, Bruker D5005) using CuK radiation 15 After that 100 l of a suspension of S aureus bac( = 154 nm) at a step of 0.02 (2 ) at room temperature teria was pipetted and spread on the surface of agar medium containing Fe3 O4 , Ag and Fe3 O4 –Ag nanoparticles The Petri plates were incubated at 37 C for 24 h in a shaking incubator (150 rpm) to encourage bacterial cell growth The intensity of bacterial growth on agar plates with Fe3 O4 , Ag and Fe3 O4 –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 Fe3 O4 , Ag and Fe3 O4 –Ag nanoparticles we used colony countFigure Two-step protocol for formation of Fe3 O4 –Ag hybrid nanoparticles ing method for determining the number of CFU which has 5904 J Nanosci Nanotechnol 16, 5902–5912, 2016 Tung et al Synthesis, Characterizations of Superparamagnetic Fe3 O4 –Ag Hybrid Nanoparticles 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 similarly but without exposure to the nanoparticles for comparative purpose Colonies were counted and compared to those in control plates to calculate the changes in the bacterial growth inhibition All experiments were performed under sterile conditions and in triplicate The percentage reduction ratio of the bacteria has been expressed as: A−B × 100% (1) R= A where R is the percentage reduction ratio, A–the number of bacterial colonies in the Petri plates without nanopartiles and B–the number of colonies in the Petri plates containing Fe3 O4 , Ag and Fe3 O4 –Ag nanoparticles 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 reduction process Furthermore, the addition 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 ammonium complex with glucose.39 It 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, glucose as a reducing agent with simultaneous UV-irradiation 2.7 Ultrathin Sectioning Sample Preparation of can effectively produce long-term stable dispersion of the MRSA Bacteria Cells silver NPs in aqueous medium (>1 year) with narrow In order to obtain further understanding of the bacteaverage size distribution In addition, this synthesis route ricidal and interaction mechanism of the nanoparticles is environmentally friendly because of use of non-toxic samples, ultrathin sectioning technique was carried out chemicals to observe the ultrastructural changes of bacterial cells Figures 2(a), (b) shows a TEM image of the Ag-NPs destroyed by action of the Ag-NPs and Fe3 O4 –Ag hybrid and corresponding size distribution of Ag-NPs calculated nanoparticles.36 After the S aureus bacteria were exposed from TEM image It can be seen that the Ag-NPs were to the Ag-NPs and Fe3 O4 –Ag hybrid nanoparticles, the well-formed and well-dispersed no aggregates Delivered by Ingenta to: Chalmers Tekniska Hogskola (Chalmers University ofAlmost Technology) samples were collected and fixed by 2.5% glutaraldeof Ag-NPs were observed through TEM investigation IP: 5.101.218.42 On: Thu, 12 May 2016 18:01:07 hyde in cacodylate buffer (0.1 M) Copyright: for 30 American at room Scientific Figure 2(b) shows the histogram of the size distribution Publishers temperature Washing the fixed samples by cacodylate of silver NPs obtained from the TEM image The average buffer three times for 10 each, then transferred to 1% size of Ag-NPs was about 4.5 nm with a relatively narOsO4 /cacodylate buffer for h The samples were then row distribution The crystallinity of the Ag-NPs was condehydrated by using a series of alcohol with 50, 70, 80, firmed by powder X-ray diffraction (see Fig 3) It showed 90 and 100% (two times × min), and then propylene three broad peaks assigned to 111, 200, and 220 planes of oxide (three times × min) The samples were infiltrated a face centered cubic lattice of bulk silver (JCPDS card, and finally embedded in Epon 812 at 60 C for 24 h The No 004-0783) This fact confirmed that the synthesized polymerized samples were sectioned into ultrathin slices NPs consisted of pure silver with high crystallinity Our 60–90 nm in thickness, and placed on the collodion-coated calculation from the XRD patterns according to Scherrer copper grids (300 meshes) The analyses of ultrastrucexpression revealed that, the oleic acid-stabilized Ag-NPs tural changes of interior of the bacteria cells were conhad average-sized particles (∼6–7 nm) This result is fully ducted by transmission electron microscopy (TEM, JEM consistent with the calculation from TEM image Further1010, JEOL) more, the broadening of the full width at half maximum (FWHM) of the XRD pattern revealed the nanoscale sizes of Ag-NPs.37 38 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 irradiation A fundamental reduction reaction involving Tollens process under UV irradiation is follows:37 38 Ag NH3 + h ˙ + RCHOH −→ Ag0 + 2NH3 + H+ + RCOH nAg0 → Agn J Nanosci Nanotechnol 16, 5902–5912, 2016 3.1.2 Synthesis of Fe3 O4 Nanoparticles by Co-Precipitation Method Magnetic iron oxide NPs (Fe3 O4 -NPs) were synthesized using co-precipitation reaction A fundamental reaction for formation of iron oxide NPs is as follows: Fe2+ + 2Fe3+ + 8NaOH → Fe3 O4 + 4H2 O 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 5905 Synthesis, Characterizations of Superparamagnetic Fe3 O4 –Ag Hybrid Nanoparticles Figure Tung et al TEM images and size distribution of (a), (c) Ag NPs and (b), (d) Fe3 O4 NPs Fe2+ /Fe3+ , concentration of sodium hydroxide and pH of average-sized particles (∼17–18 nm) This result is consistent with the calculation from TEM image solution.19 Figures 2(c), (d)byshows a TEM image of as-prepared Delivered Ingenta to: Chalmers Tekniska Hogskola (Chalmers University of Technology) Fe3 O4 -NPs and a histogram of IP: size5.101.218.42 distribution of Fe On: 12 May 2016 18:01:07 3.1.3 Synthesis of Fe3 O4 –Ag Hybrid Nanoparticles by O4Thu, Publishers NPs calculated from TEM image ItCopyright: can be seenAmerican that the Scientific Modified Photochemical Method average size of Fe3 O4 -NPs obtained about 19.2 nm The In order to obtain Fe3 O4 –Ag hybrid nanoparticles, the iron XRD analysis was conduct to examine the crystallinity of oxide nanoparticles were also used as seed for the growth Fe3 O4 -NPs as shown in Figure It revealed that there of silver nanoparticles on their surface through the phoare eight broad peaks assigned to 111, 220, 311, 222, tochemical reaction of Ag in the Fe3 O4 nanoparticle seed 400, 422, 511 and 440 planes of a spinel structural latsolution As it can be seen from Figure that seven peaks tice of magnetite (JCPDS card, No 11-0614) Our calare observed in the XRD pattern of Fe3 O4 –Ag hybrid culation from the XRD patterns according to Scherrer nanoparticles Four of them can be indexed well to the fcc expression indicated that the as-prepared Fe3 O4 -NPs had inverse spinel structure of Fe3 O4 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 Fe3 O4 –Ag nanostructure Figure shows the bright field TEM image along with its EDS spectrum and HRTEM images of nanostructure Figure 4(a) indicates that formed Fe3 O4 –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 Figure XRD patterns of Ag NPs, Fe3 O4 NPs and Fe3 O4 –Ag hybrid the high purity of sample However, it is not possible nanoparticles 5906 J Nanosci Nanotechnol 16, 5902–5912, 2016 Tung et al Figure Synthesis, Characterizations of Superparamagnetic Fe3 O4 –Ag Hybrid Nanoparticles TEM images and EDS elemental analysis of Fe3 O4 –Ag hybrid nanoparticles to determine the elemental ratio of Fe to Ag from EDS FFT analysis in Gatan Digital Micrographs software we confirmed the formation of both Ag(111) and Fe3 O4 (311) spectrum because they are recorded at independent locaDelivered by Ingenta to: Chalmers Tekniska Hogskola (Chalmers University of Technology) nanoparticles with high crystallinity Furthermore, the hettions The HRTEM images (Figs 4(c) and (d)) confirmed IP: 5.101.218.42 On: Thu, 12 May 2016 18:01:07 erodimer nature of Fe3 O4 –Ag nanostructure is evidenced using Scientific the exact nature of Fe3 O4 and Ag nanoparticles Copyright: By American Publishers Figure High-resolution TEM images of Fe3 O4 –Ag dimer nanoparticles J Nanosci Nanotechnol 16, 5902–5912, 2016 5907 Synthesis, Characterizations of Superparamagnetic Fe3 O4 –Ag Hybrid Nanoparticles Tung et al 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 Fe3 O4 seeds (dTEM ∼10 nm) is slightly larger than that of AgNPs (dTEM ∼5 nm) as calculation of size distribution from TEM images (see Figs 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 Fe3 O4 NPs and Fe3 O4 –Ag hybrid NPs were checked by magnetization measurements Figure UV-vis spectra of Fe3 O4 NPs, Ag NPs and Fe3 O4 –Ag hybrid nanoparticles Figure depicts the magnetization curves measured at room temperature of (a) Fe3 O4 NPs and (b) Fe3 O4 –Ag from HRTEM analysis as shown in Figure The Ag hybrid NPs samples It can be seen that the Fe3 O4 NPs nanocrystals with particle sizes about ∼10 nm are stably sample were superparamagnetic and saturation magnetizaformed on the Fe3 O4 seeds tion value (Ms ) was about ∼55.35 emu/g The interaction between the nanoscale Ag and Fe3 O4 leads to a change on magnetization behavior of Fe3 O4 –Ag NPs Obviously, the 3.2 UV-Vis Analysis Fe3 O4 –Ag hybrid NPs also exhibited superparamagnetic Figure shows (a) the UV-vis spectra of Fe3 O4 , Ag-NPs, behavior and the Ms value was ∼52,98 emu/g (calculaand Fe3 O4 –Ag hybrid nanoparticles Obviously, the Agtion was corrected for only amount of Fe3 O4 phase) This NPs and Fe3 O4 –Ag hybrid nanoparticles samples display result indicates that the formation of silver nanoscrytals on strong absorption peaks at 428 and 437 nm, respectively, Fe3 O4 seeds does notUniversity affect superparamagnetic property becauseDelivered of the surface plasmon (SPR) effect Hogskola by Ingenta to:resonance Chalmers Tekniska (Chalmers of Technology) In addition, the existence of silver nanoscrytals 5.101.218.42 On: Thu, of 12sample May 2016 18:01:07 of Ag-NPs In addition, there IP: is no SPR band observaCopyright: American Scientific Publishers can prevent agglomeration of the Fe3 O4 NPs Noticeably, of characteristic tion for Fe3 O4 sample The appearance due to high value of magnetization the Fe3 O4 –Ag hybrid surface plasmon band at 437 nm indicates the formaNPs can be easily removed from solutions and recycled by tion of Ag-NPs on Fe3 O4 seeds The SPR phenomenon exerting an external magnetic field Thus, the Fe3 O4 –Ag occurs when the incident light interacts with valence elechybrid NPs showed high potential to be developed into a trons at the outer band of Ag-NPs, leading to oscillation new class of recyclable antibacterial agents of electrons along with the frequency of the electromagnetic source.42 However, the absorption band of Fe3 O4 – 3.4 Antibacterial Activity Analysis Ag sample (∼437 nm) is shifted to longer wavelength as compared to Ag-NPs sample alone (∼428 nm) The The antibacterial activities of Fe3 O4 , Ag-NPs, and Fe3 O4 – shifting of the absorption peak toward longer wavelength Ag hybrid nanoparticles were initially assessed with the Figure 5908 Magnetization curves of (a) Fe3 O4 NPs and (b) Fe3 O4 –Ag hybrid nanoparticles J Nanosci Nanotechnol 16, 5902–5912, 2016 Tung et al Synthesis, Characterizations of Superparamagnetic Fe3 O4 –Ag Hybrid Nanoparticles hybrid NPs show noticeable inhibitory effects on S aureus bacteria, however, there is no inhibitory effect for bare Fe3 O4 sample (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 Fe3 O4 seeds 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 samples against S aureus, we used standard microdilution method for determining the number of growth bacterial colonies in these samples Figure shows the agar plates treated (a) without nanoparticles and with the (b) Fe3 O4 , (c) Ag-NPs, and (d) Fe3 O4 –Ag hybrid nanoparticles samples 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 Fe3 O4 –Ag NPs (Figs 9(c), (d)), the bacterial growth was completely inhibited, however for the case of Fe3 O4 NPs sample (Fig 9(b)), no significant inhibitory effect was also found The MIC values were also determined from microdilution method It is noted that the MIC values for hybrid nanoparticles samples are corrected for the effective concentration of Ag in the heterodimer Fe3 O4 –Ag Our obtained results reveal that the MIC value of Ag-NPs sample (∼8.2 g/mL) is slightly lower than that of Fe3 O4 –Ag hybrid NPs sample (∼9.7 g/mL) Moreoever, the percentage reduction ratio Delivered by Ingenta to: Chalmers Tekniska Hogskola (Chalmers University of Technology) of the bacteria for both Ag-NPs and Fe O –Ag NPs samIP: 5.101.218.42 On: Thu, 12 May 2016 18:01:07 Copyright: American Scientific Publishers ples obtained nearly 100% as calculation using Eq (1), whereas percentage reduction ratio of bacteria for Fe3 O4 samples was about 3.4% This result suggests that Fe3 O4 nanoparticles could play a minor role in bactericidal activity of hybrid nanoparticles paper-disc diffusion method, which is widely used for quick antibiotic susceptibility determinations Figure displays 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 concentration of Ag-NPs from Ag1 sample (∼10 g/mL) to Ag4 sample (∼50 g/mL) A similar tendency was found for Fe3 O4 –Ag sample as shown in Figure 8(b) The Fe3 O4 –Ag (c) Figure Antibacterial activity of Fe3 O4 NPs, Ag NPs and Fe3 O4 –Ag hybrid nanoparticles J Nanosci Nanotechnol 16, 5902–5912, 2016 Figure Agar plates treated (a) without nanoparticles (control), (b) with Fe3 O4 NPs, (c) Ag NPs, and (d) Fe3 O4 –Ag hybrid nanoparticles 5909 Synthesis, Characterizations of Superparamagnetic Fe3 O4 –Ag Hybrid Nanoparticles 3.5 Proposed Bactericidal Mechanism of Ag-NPs and Fe3 O4 –Ag Hybrid NPs To provide more rudimentary insights into the interaction and bactericidal mechanism between the Ag-NPs and Fe3 O4 –Ag hybrid nanoparticles with tested S aureus bacteria, 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 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 sample and (b) Fe3 O4 –Ag hybrid nanoparticles with S aureus bacteria after min, 15 min, and 30 It was established 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 Tung et al 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 resulting in cell bacterial death.39 43 As shown in Figure 10 (left part), at different magnifications and sections, 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 penetrated into the cells (usually < 10 nm) After 30 min, the cytoplasm was completely destroyed as the silver NPs penetrated 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 Fe3 O4 –Ag hybrid nanoparticles [see the right part of Fig 10], the surface of S aureus bacterial 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 Delivered by Ingenta to: Chalmers Tekniska Hogskola (Chalmers University of Technology) IP: 5.101.218.42 On: Thu, 12 May 2016 18:01:07 Copyright: American Scientific Publishers Figure 10 The cross-sectional TEM images showing different stages of interaction of (a) Ag-NPs sample and (b) Fe3 O4 –Ag hybrid nanoparticles with S aureus bacteria after min, 15 min, and 30 5910 J Nanosci Nanotechnol 16, 5902–5912, 2016 Tung et al Synthesis, Characterizations of Superparamagnetic Fe3 O4 –Ag Hybrid Nanoparticles bacteria cell walls are negatively charged hence they attract The authors would like to acknowledge the Center for each other by electrostatic interaction, leading to large Electron Nanoscopy, Technical University of Denmark density of hybrid nanoparticles attached on the surface of (DTU CEN) for HRTEM works Also, the technical supcells membrane The enhanced surface area of Fe3 O4 –Ag ports for biological measurements at National Institute nanoparticles produces large contact area between the bacof Hygiene and Epidemiology (NIHE) in Vietnam are terial cell membrane and nanoparticles.44 The Fe3 O4 –Ag acknowledged hybrid nanoparticles dissolve the outer envelope of bacterial cell wall and thereby leakage of cellular constituents, References and Notes resulting in cell death In our present case, due to large World Health Organization, Global report for research on infectious sizes of Fe3 O4 –Ag hybrid nanoparticles (25–30 nm), they diseases of poverty (2012) can not penetrate into the cells membrane as the case of K S Kaye MD and D Kaye MD, Current Infectious Disease Reports 2, 391 (2000) Ag-NPs (small sizes ∼ nm) W Nakanishi, K Minami, L K Shrestha, Q Ji, J Hill, and More noticeably, the increase in bacterial resistance K Ariga, Nano Today 9, 378 (2014) to antimicrobial agents poses a serious problem in the S Gai, C Li, P Yang, and J Lin, Chem Rev 114, 2343 (2014) treatment of infectious diseases as well as in epidemiolog5 K Ariga, K Kawakami, M Ebara, Y Kotsuchibashi, Q Ji, and ical practice One of the major shortcomings of antibacJ Hill, New J Chem 38, 5149 (2014) N Ahmad, H Younus, A Chughtai, and F Verpoort, Chem Soc terial drugs and nanoparticles is their failure to fight with Rev 44, (2015) 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Huy, N V Quy, University and A T Le, Adv Nat Sci.: Nanosci Nano3 O4 –Ag Hogskola Deliveredresulting by Ingenta to:death Chalmers Tekniska (Chalmers of Technology) technol 4, 033001 (2013) hybrid nanoparticles can be highIP: potential as new antibac5.101.218.42 On: Thu, 12 May 2016 18:01:07 13 R Prucek, J Tucek, M Kilianova, A Panacek, L Kvitek, J Filip, terial agents to fight with infectious pathogens dueAmerican to many Scientific Copyright: Publishers M Kolar, K Tomankova, and R Zboril, Biomaterials 32, 4704 advantages over conventional silver nanoparticles such as (2011) recyclable capability to reduce the release of nanoparticles 14 Y.Chen, N Gao, and J Jiang, Small 9, 3242 (2013) in environment, noticeable antibacterial activity and high 15 X Zhang, H Niu, J Yan, and Y Cai, Colloids and Surfaces A: Physicochem Eng Aspects 375, 186 (2011) biomedical compatibility CONCLUSIONS In this work, we established facile chemistry methods for synthesis of Fe3 O4 –Ag hybrid NPs The Ag nanocrystals were growth by photochemical reaction on the Fe3 O4 seeds to form a heterodimer nature of Fe3 O4 – Ag hybrid nanoparticles A detailed comparative study of antibacterial activity and mechanism of these NPs against the MRSA pathogen was conducted The Fe3 O4 – Ag hybrid NPs exhibited more significant antibacterial efficacy against MRSA pathogen than that of the plain Fe3 O4 NPs and Ag-NPs The interaction and bactericidal mechanism between the Fe3 O4 –Ag hybrid nanoparticles with tested S aureus bacteria was verified by adapting ultrastructural studies The obtained results suggested that the Fe3 O4 –Ag hybrid nanoparticles could be a promising antimicrobial material for practical applications in treatment and prevention of infectious diseases Acknowledgment: This research is funded by Vietnam National Foundation for Science and Technology Development (NAFOSTED) under grant number 103.99-2012.10 J Nanosci Nanotechnol 16, 5902–5912, 2016 16 M Zhu, C Wang, D Meng, and G Diao, J 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Copyright: American Scientific Publishers 5912 J Nanosci Nanotechnol 16, 5902–5912, 2016 ... NPs, and (d) Fe3 O4 –Ag hybrid nanoparticles 5909 Synthesis, Characterizations of Superparamagnetic Fe3 O4 –Ag Hybrid Nanoparticles 3.5 Proposed Bactericidal Mechanism of Ag-NPs and Fe3 O4 –Ag Hybrid. .. activity of Fe3 O4 , Ag and Fe3 O4 –Ag nanoparticles we used colony countFigure Two-step protocol for formation of Fe3 O4 –Ag hybrid nanoparticles ing method for determining the number of CFU which... stages of interaction of (a) Ag-NPs sample and (b) Fe3 O4 –Ag hybrid nanoparticles with S aureus bacteria after min, 15 min, and 30 It was established for the case of bare Ag-NPs that the antibacterial