Magnetic Properties and Biological Activity Evaluation of Iron Oxide Nanoparticles

).Analysisofhysteresisloopsdata atroomtemperatureformagneticIO-NPssampleindicatedthattheIO-NPsweresuperparamagneticatroomtemperature.hecal-culatedsaturationmagnetizationformagneticironoxidewasM s = 18.1emu/g. he antimicrobial activity of the obtained PMC-NPs was tested against Gram-negative ( Pseudomonas aeruginosa 1397, Escherichia coli ATCC 25922), Gram-positive ( Enterococcus faecalis ATCC 29212, Bacillus subtilis IC 12488) bacterial as well as fungal ( Candida krusei 963) strains. he obtained results suggested that the antimicrobial activity of IO-NPs is dependent on the metallic ions concentrations and on the microbial growth state, either planktonic or adherent. he obtained IO-NPs exhibited no cytotoxic efect on HeLa cells at the active antimicrobial concentrations.


Introduction
During the last decade, there has been an increasing interest in developing and understanding of matter at nanometric scale [1,2]. he progress made in the area of nanotechnology in the last years has allowed the scientists to develop and characterize materials with outstanding properties and nanometric sizes [3]. he ield of nanomaterials has been the centre of attention in the scientiic community due to the unique and remarkable properties exhibited by materials when studied at nanometric scale.Recent studies state that inorganic materials are the core of future developments in industrial and technological applications [4].During the past few years, the most studied materials were the ones with magnetic behaviour due to their promising applications in areas such as magnetic data storage [5], gas sensors [6][7][8], tunnelling magneto-resistance (TMR) [9,10], ferroluid, targeted drug delivery, contrast agents in magnetic resonance imaging [11], hyperthermia, magnetic ield assisted radionuclide therapy [12]a sw e l la sm a g n e t i c a l l ys e p a r a b l e absorbents [13].Amongst materials with magnetic properties, iron oxide nanoparticles are most suitable for biomedical applications due to their proven biocompatibility.Magnetite and maghemite, the most common iron oxide nanoparticles, have been the subject of intensive studies because at nanometric scale they exhibit unique physical and chemical particularities [14].
Due to the increased resistance to existing antibiotics of various microorganisms, many researchers have turned towards engineered nanoparticles for inding a solution.he ability of pathogenic bacteria and fungi to resist antimicrobial agents, by genetic mechanisms and by phenotypic resistance due to the bioilm growing state, has emerged i nt h er e c e n ty e a r sa n db e c a m eam a j o rh e a l t hp r o b l e m .Recent studies reported various metal nanoparticles to exhibit antimicrobial efects [15].Iron oxide nanoparticles, due to their small size, superparamagnetic properties and controllable parameters, can interact directly with various microorganisms without posing a risk for human body.hese nanoparticles can interact directly with the microbial cells causing damage to the envelope and thus rendering the bacterial cell defenceless or, in some cases, disrupting the metabolic functions and causing the destruction of the cell [16].
Magnetic iron oxide nanoparticle are increasingly being d e v e l o p e df o ru s ei nr e s e a r c ha n db i o m e d i c a la p p l i c a t i o n s , such as targeted transport of antimicrobial drug. he fundamental understanding on the inluence of magnetic iron oxide nanoparticle on cellular growth and functions is very important for further biological applications.he comparative study on growth of diferent bacteria the inluence of magnetic iron oxide were revealed the efect of magnetic iron oxide nanoparticle on bacterial growth.
his study focuses on the investigation of the antimicrobial behavior of iron oxide nanoparticles (IO-NPs) prepared by an adapted co-precipitation method [17][18][19][20][21]a n do nt h e analysis of M-H hysteresis loops at room temperature.Transmission electron microscopy studies have been conducted to obtain information about size, structure and morphology of the fabricated IO-NPs. he antimicrobial activity of the IO-NPs was investigated by qualitative and quantitative assays and their cytotoxic efect was assessed on HeLa cells.In this study, the IO-NPs were tested in aqueous suspension against a large spectrum of microbial strains, both by antimicrobial susceptibility standard methods (agar difusion and minimal inhibitory concentration assay) as well as by methods aimedtoinvestigatetheeiciencyofIO-NPsagainstbioilm embedded cells, which are much more resistant to diferent stress factors, including antimicrobials, than their planktonic counterparts.

Synthesis of Iron
Oxide Ferroluid and Characterization. he synthesis of iron oxide ferroluid was carried out as reported in other papers [22].Transmission electron microscopy (TEM) images for these samples were recorded using a FEI Tecnai 12 equipped with a low dose digital camera from Gatan. he specimen for TEM imaging was prepared by ultramicrotome to get thin section of about 60 nm. he powder was embedded in an epoxy resin (polaron 612) before microtomy.he magnetic properties of the samples were measured using a superconducting quantum interference device (MPMS magnetometer) at room temperature.
2.3.Cytotoxicity Assay.Quantiication of cell viability was performed using propidium iodide (PI) and luorescein diacetate (FdA).Briely, 5×1 0 4 HeLa cells were seeded in each well of a plate with 24 wells and ater 24 h were treated with 10 LofFe 2+ /Fe 3+ of 0.03 inal concentration. he efects on cell viability were evaluated ater 24 h by adding 100 LPI (0.1 mg/mL) and 100 L FdA (0.1 mg/mL) and luorescence was quantiied using Observer D1 Carl Zeiss microscope.All cells from several ields were counted and cell viability was established by the ratio between viable (green) and dead cells (red) [23].

he In Vitro Antibacterial and Antibioilm Activity
Assessment of the Antimicrobial and Antipathogenic Activity of the New Oxides. he in vitro qualitative screening of the antimicrobial activity was carried out by an adapted agar difusion technique using a bacterial suspension of 0.5 McFarland density obtained from 24 hour cultures [24,25]. he antimicrobial activity of the IO-NPs was tested against Gram-negative (Pseudomonas aeruginosa 1397, Escherichia coli ATCC 25922), Gram-positive (Enterococcus faecalis ATCC 29212, Bacillus subtilis (IC 12488) bacterial as well as fungal (Candida krusei 963) strains. he microbial strains identiication was conirmed by aid of VITEK II automatic system.VITEK cards for identiication and susceptibility testing (GNS-522) were inoculated and incubated according to the manufacturer's recommendations.IO-NPs were tested starting with a concentration of 10 mg/mL.
Qualitative Screening of the IO-NPs Antimicrobial Activity.A volume of 10 L of each tested IO-NPs sample of 10 mg/mL concentration was spotted on Muller Hinton agar for bacteria and Yeast Peptone Glucose (YPG) agar for fungi, previously seeded with the microbial inocula of 0.5 MacFarland density. he inoculated plates were incubated for 24 h at 37 ∘ C. he antimicrobial activity was assessed by measuring the growth inhibition zones diameters expressed in mm [26].Following the results of the qualitative screening, only the microbial strains proving to be susceptible have been further tested in the quantitative assay.
he quantitative assay of the minimal inhibitory concentration (MIC, g/mL) was based on liquid medium two-fold microdilutions and performed in 96 multi-well plates.For this purpose, serial binary dilutions of the tested compounds (ranging between 5 and 0.01 g/mL) were performed in a 200 L volume of nutrient broth (for bacteria)/YPG (for fungi) and each well was seeded with 20 L microbial inocula of 0.5 McFarland density. he plates were incubated for 24 h at 37 ∘ C, the antimicrobial efect being assessed by measuring the absorbance of the obtained cultures at 620 nm [26]. he lower absorbance value was, the more intensive the antimicrobial efect is.
he antibioilm activity of the IO-NPs was tested by the microtiter method.For this purpose, the microbial strains have been grown in the presence of two-fold serial dilutions ofthetestedcompounds(rangingbetween5and0.01g/mL),performed in liquid nutrient broth/YPG, distributed in 96well plates and incubated for 24 hours at 37 ∘ C. At the end of the incubation period, the plastic wells were emptied, washed three times with phosphate bufered saline (PBS), ixed with cold methanol and stained with 1% violet crystal solution for 30 minutes. he bioilm formed on plastic wells was resuspended in 30% acetic acid. he intensity of the colored suspensions was assessed by measuring the absorbance at 492 nm [26][27][28].he lower absorbance value was, the more intensive the antibioilm efect was considered.

Results and Discussion
Figure 1 shows the XRD patterns of the iron oxide particles.he 2 v a l u e so ft h epe a k sa r ec o m p a r e dw i t ht h es t a n d a r d data for -Fe 2 O 3 .Difraction peaks at (220), (311), (400), (422), ( 511) and (440) are readily recognized from the XRD pattern in the sample.he XRD peaks of iron oxide nanoparticles (Figure 1) can be indexed into the spinel cubic l a t t i c et y p ew i t hal a t t i c eo f0 .8 3 5n m .hi sv a l u eo fl a t t i c e parameter is in agreement with the values of the standard data (JCPDS no.4-755).hereinementofXRDspectraindicatedthat no other phases except the maghemite are detectable.he XRD showed a inite broadening of the difraction lines.he particle size calculated using the Scherer [29]f o r m u l aw a s estimated at around 7.7 nm. Figure 2(a) shows TEM picture of iron oxide nanoparticles (IO-NPs), clearly showing that the product is entirely composed of crystals with a relatively uniform, spherical morphology.Grain size distribution was determined by measuring the mean diameter, ,o fa b o u t5 0 0p a r t i c l e s on the micrographs (Figure 2(c)).TEM images indicate a very uniform size distribution of iron oxide nanoparticles. he average grain size of the monodisperse nanoparticles is 7 ± 0.2 nm. Figure 2(b) also shows the selected area electron difraction (SAED) pattern recorded from an area containing a large number of nanoparticles and high-resolution TEM picture. he rings in the SAED pattern can be indexed as the (220), (311), (400), (422), ( 511) and (440) relections of the cubic maghemite in agreement with the XRD results.
he magnetic properties of the prepared IO-NPs were monitored by measuring the hysteresis loops at a temperature of 300 K. Magnetization curves (M-H loop) for the magnetic IO-NPs measured at room temperature were presented in Figure 3.At the maximum applied magnetic ield of 10 kOe the magnetization did not reach saturation no hysteresis loops can be observed (no remanent magnetization and zero coercivity ields) [30,31].his behavior indicated that the IO-NPs were superparamagnetic at room temperature with a calculated saturation magnetization of 18.1 emu/g. he iron oxide nanoparticles sample shows a saturation magnetization at lower than the reported values for the saturation magnetization of bulk maghemite (-Fe 2 O 3 ) [32].his trend could be attributed to the particles sizes efect. he magnetic nanoparticles are so small that they may be considered to have a single magnetic domain [33].In his studies, Hergt et al., showed that the saturation magnetizations, Ms, decreases with decreasing particle size [34].Analysis the hysteresis loops of the IO-NPs, at 300 K conirms average size and particle size distribution of IO-NPs already obtained from TEM and XRD analyses.Combination of magnetic properties and antimicrobial features of iron oxide nanoparticles, designates these nanoparticles for exploitation in biomedical applications, such as targeted transport of antimicrobial drug.With regard to their unique magnetic and antibacterial properties, the iron oxide nanoparticles can be exploited at lower concentrations so that their toxic efect on human cells to be minimized.
A great advantage of the tested compound is that they are not cytotoxic at the active concentrations, as reveal by the cytotoxicity assay, demonstrating no cytotoxic efect of the IO-NPs (Figure 4). he microbial species of clinical interest oten involved in bioilm associated diseases are belonging to a very large spectrum, from the Gram positive (S.epidermidis and S. aureus), and Gram negative (P. a e r u g i n o s a , E. coli)bacterial strains to diferent members of the Candida genus [35]. he understanding of microbial infections related to the bioilm development on tissues or indwelling devices was possible by using diferent qualitative and quantitative in vitro speciic assays.Inourstudywehavealsousedbothassaysforstudying the susceptibility of microbial cells grown in suspension, known as planktonic cells, as well as of those developed in bioilms, called adherent or sessile cells.
he antimicrobial activity of the IO-NPs has been studied on strains belonging to common bacterial pathogens, that is, the Gram-negative, P. a e r u g i n o s a , E. coli,G r a m -p o s i t i v eE.faecalis, B. subtilis, and a yeast strain of C. krusei.
Ater the qualitative screening, the microbial strains which proved to be susceptible to the tested nanoparticles have been investigated in the quantitative assay for establishing the MIC value.he highest tested concentration was of 5 mg/mL, because at higher concentrations, the colored iron oxide suspension would have interfered with the reading  of the absorbance values at 620 nm. he quantitative assay showed that the IO-NPs generally stimulated the growth of all microbial cells in suspension from the highest tested concentrations of 5 mg/mL to 0.039 mg/mL, as demonstrated b ytheabsorbancemeasuremen tsa t620nmoftheobtained cultures (Figure 5).Ab a c t e r i c i d a le f e c th a sb e e no b s e r v e da tl o wI O -N P s concentrations, that is, from 0.039 to 0.01 mg/mL against E. faecalis ATCC 29212 and P. a e r u g i n o s a 1397, from 0.02 to 0.01 against E. coli ATCC 25922. he IO-NPs, irrespective to the tested concentration exhibited no inhibitory efect on C. krusei 963 and B. subtilis IC 12488 growth.
Metal oxide nanoparticles, including magnetite, have been proved to have inherent antimicrobial properties, which are enhanced, or occur, when the materials are in the nanometer size and in relation to surface area [36].Since nanoparticles can be smaller in size than bacterial pores, they have a unique ability of crossing the cell membrane, disrupting its function or interfering with nucleic acid or protein synthesis [37].Our study demonstrated that IO-NPs are rather exhibiting a stimulatory efect on the microbial growth especially at high concentrations.he stimulatory efect of iron oxide nanoparticles on the microbial growth was also reported by other authors, that is, on E. coli [38]orE.coli, P. a e r u g i n o s a and E. faecalis, C. albicans [39].hese results could be explained by the ability of the microbial strains to use the iron oxide as a metabolic source of iron, which is k n o w nt op o s i t i v e l yr e g u l a t et h em i c r o b i a lg r o w t hr a t ea n d other physiological processes [40].
However, at lower concentrations ranging from 39 to 10 g/mL, they inhibited the growth of E. faecalis, P. a e r u ginosa and E. coli strains.Our results come into agreement with other studies showing that iron oxide nanoparticles substantially inhibited the growth of E. coli and S. aureus at concentrations comparable with those used in our study, from 10 to 100 g/mL [41].Fe 2 O 3 nanoparticles have been shown to exhibit the least bactericidal activity, when compared with other metal oxides, like ZnO or CuO [42]. he antibacterial activity of composite nanoparticles containing iron oxide, zinc oxide and zinc ferrite phases has been shown to be proportional with the weight ratio [Zn]/[Fe] of the composite nanoparticle, on Staphylococcus aureus and Escherichia coli,sothelowesttheironconcentration,themost intensive the antimicrobial activity [37].
Studies in the last decades on microbial adherence to dif er en ts ubs tra taledt oth eco nc l usio ntha tth es urvi valo f microorganisms in the natural habitats, including medical ecosystems, is dependent on their capacity to adhere to diferent surfaces/substrata and form bioilms.A bioilm is a sessile microbial community composed of cells embedded in a matrix of extracellular polymeric substances attached to a substratum or interface.he matrix is primarily of microbialoriginandthecellsencasedinthismatrixpresenta modiied phenotype, being metabolically more eicient and well protected, exhibiting resistance to diferent stress factors, including host defense mechanisms and antibiotics [43].
he quantitative assay of the inhibitory efect of IO-NPs on bioilms developed on the inert substratum by diferent m i c r o b i a ls t r a i n s ,q u a n t i i e db yt h ea b s o r b a n c em e a s u r ements at 490 nm are presented in Figure 6. he highest tested concentration (i.e., 5 mg/mL) exhibited a strong stimulatory efect on the bioilm development by all tested microbial strains, this efect drastically decreasing in case of the other tested concentrations (Figure 6). he tested IO-NPs exhibited only a slight antibioilm activity for the same strains susceptible in the planktonic state, that is, E. faecalis 29212 (0.625 to 0.01 mg/mL), P. a e r u g i n o s a 1 3 9 7 (2.5 to 0.01 mg/mL), and E. coli ATCC 25922 (0.156 to 0.01 mg/mL) strains (Figure 6).Comparing the range of active concentrations of IO-NPs against planktonic and respectively adherent strains, it could be observed that the antibioilm activity of the obtained nanoparticles is maintained on a wider range of concentrations.

Conclusions
Iron oxide nanoparticles were synthesized by a facile and rapid coprecipitation method.he prepared IO-NPs were characterized by various techniques.he TEM images show Figure6:hequantitativeassayoftheinhibitoryefectofIO-NPs on bioilms developed on the inert substratum by diferent microbial strains, quantiied by the absorbance measurements at 490 nm.
well-crystallized magnetic IO-NPs with average size of 7 ± 0.2 nm and narrow size distributions without particle agglomeration. he hysteresis loop of IO-NPs demonstrates a superparamagnetic behavior.he IO-NPs exhibited a strong stimulatory efect directly correlated with high concentrations of the tested nanoparticles, both on the growth of microbial strains in suspension and in adherent state, probably due to the potential of these strains to use the iron oxide as a source of iron ions required for the microbial metabolism and pathogenicity.However, the tested iron oxide nanoparticles inhibited on a certain range of concentrations the ability of E. faecalis, P. a e r u g i n o s a , and E. coli strains to grow, both in suspension and in bioilm state, results demonstrating their potential use in the design of antibacterial and antibioilm materials and surfaces, for biomedical applications, exhibiting also the great advantage of reduced cytotoxicity on eukaryotic host cells and tissues.he combination of magnetic properties and antimicrobial features designates the iron oxide nanoparticles to be possibly used in applications such as clinical infections and local antimicrobial therapy.On the other hand, these nanoparticles couldbeusedfortargetedtransportofanantimicrobialagent and its subsequent removal by means of an external magnetic ield.

Figure 2 :Figure 3 :
Figure 2: Bright ield TEM picture showing a homogeneous distribution of iron oxide nanoparticles (a), SAED pattern from a region including a large number of nanoparticles (b), and size distribution of IO-NPs (c).

Figure 4 :Figure 5 :
Figure 4: Fluorescence microscopy image revealing the absence of the cytotoxic efect on HeLa cells (no dead cell stained in red) ater 24 h treatment with 200 L of iron oxide (×100).