Abstract
The glycerol iron oxide nanoparticles (GIO-NPs) were obtained by an adapted coprecipitation method. The X-ray diffraction (XRD) studies demonstrate that GIO-NPs were indexed into the spinel cubic lattice with a lattice parameter of 0.835 nm. The refinement of XRD spectra indicated that no other phases except maghemite were detected. The adsorption of glycerol on iron oxide nanoparticles was investigated by Fourier transform infrared (FTIR) spectroscopy. On the other hand, this work implicated the use of GIO-NPs in antibacterial studies. The results indicate that, in the case of P. aeruginosa 1397 biofilms, at concentrations from 0.01 mg/mL to 0.625 mg/mL, the glycerol iron oxide inhibits the ability of this strain to develop biofilms on the inert substratum.
1. Introduction
In the previous decades the nanotechnology progress has made possible the understanding of matter at a fundamental level.Due to the outstanding properties exhibited by various materials when investigated at the nanometric scale, the scientific community focused on developing new ways of synthesizing engineered nanoparticles. Nowadays, the most investigated nanomaterials are the inorganic ones due to their novel and improved physical, chemical, and biological properties [1–6]. Because they exhibit very good electric, optical, electronic, magnetic, and biological properties, these nanoparticles have been widely studied for their use in pharmaceutical, medical, and environmental applications.
Nanoparticles with magnetic behavior are one of the most interesting materials due to their broad-wide possible applications in fields such as environment, medical applications, electronics, media recording devices and Iron oxides nanoparticles, mostly magnetite and maghemite, have been the subject of numerous studies concerning their use in magnetic resonance imaging (MRI), cell labeling, and hyperthermia. Being the only types of magnetic nanoparticles with proven biological properties and good biocompatibility, iron oxides are still in the researchers priorities even though these nanoparticles have been intensively studied and characterized during the years [7, 8].
Magnetite with the general formula Fe3O4 and its oxidized form maghemite (γ-Fe2O3) are suitable to be used in biomedical application due to their biological properties, superparamagnetic behavior, and controllable parameters [4–6]. Due to their unique properties attempts were made to introduce these nanoparticles in other fields of applications [9, 10]. Recent studies have been focused on finding new and improved antibacterial agents. The search for new materials having inhibitory effect on the growth of various microorganisms reached the area of nanomaterials [11]. Nanoparticles were chosen because they can interact with microbial cells directly by disrupting/penetrating the cell envelope, produce secondary products, dissolved heavy metal ions, interrupting transmembrane electron transfer, or indirectly being the carrier for other substances with antibacterial properties [12–14]. Studies show that metals have proven to affect bacterial growth kinetics and have a high influence against bacterial strains growth [15].
2. Materials and Methods
2.1. Materials
Ferrous chloride tetrahydrate (FeCl2·4H2O), ferric chloride hexahydrate (FeCl3·6H2O), natrium hydroxide (NaOH), and clorhidric acid (HCl), nitric acid (HNO3), and ferric nitrate nonahydrate (FeNO3·9H2O) were purchased from Merck. Glycerol (99.5%) was purchased from Sigma. Deionized water was used in the synthesis of nanoparticles and for rinsing of clusters.
2.2. Synthesis of Glycerol Iron Oxide Ferrofluid
Glycerol iron oxide nanoparticles were prepared by coprecipitation according to [16–19]. Glycerol solution (60 mL in 200 mL of NaOH (2 mol·L−1) was agitated under vigorous stirring for about 2 h. Ferrous chloride tetrahydrate (FeCl2·4H2O) in 2 M HCl and ferric chloride hexahydrate (FeCl3·6H2O) were mixed at room temperature (Fe2+/Fe3+ = ). The mixture was dropped into glycerol solution under vigorous stirring. The precipitate of magnetite (black precipitate immediately formed) was converted into γ-Fe2O3 particles by repeated treatment with HNO3 (2 mol·L−1) and ferric nitrate nonahydrate (0.3 mol·L−1) solutions according to [19, 20]. The acidic precipitate was isolated by decantation on a magnet, separated by centrifugation (12000 rpm), washed, and dispersed in deionized water at . The final ion concentration was 0.38 mol·L−1.
2.3. Sample Characterization
The X-ray diffraction measurements for the iron oxide samples were recorded using a Bruker D8 Advance diffractometer, with nickel filtered Cu ( Å) radiation, and a high efficiency one-dimensional detector (Lynx Eye type) operated in integration mode. The diffraction patterns were collected in the range 20°–70°, with a step of 0.02° and 34 s measuring time per step. Transmission electron microscopy (TEM) images for these samples were recorded using an FEI Tecnai 12 equipped with a low-dose digital camera from Gatan. The specimen for TEM imaging was prepared by ultramicrotomy to get thin section of about 60 nm. The powder was embedded in an epoxy resin (polaron 612) before microtomy. TEM modes used were Bright Field (BF) and Selected Area Diffraction (SAD). The functional groups present in the prepared nanoparticles and thin films were identified by FTIR using a Spectrum BX spectrometer. To obtain the nanoparticles spectra, 1% of the nanopowder was mixed and ground with 99% KBr. Tablets of 10 mm diameter were prepared by pressing the powder mixture at a load of 5 tons for 2 min. The spectrum was recorded in the range of 500 to 4000 cm−1 with 4 cm−1 resolution.
2.4. The In Vitro Antibacterial and Antibiofilm Activity
Assessment of the Antimicrobial and Antipathogenic Activity of the New Oxides. The in vitro qualitative screening of the antimicrobial activity was carried out by an adapted agar diffusion technique using a bacterial suspension of 0.5 McFarland density obtained from 24-hour cultures. The antimicrobial activities of the GIO-NPs were determined against microbial clinical and ATCC reference strains, that is, P. aeruginosa 1397 and E. faecalis ATCC 29212 strains The microbial strains identification was confirmed by aid of VITEK II automatic system. VITEK cards for identification and susceptibility testing (GNS-522) were inoculated and incubated according to the manufacturer’s recommendations. The results were interpreted by using software version AMS R09.1. GIO-NPs were suspended in dimethyl sulfoxide to a final concentration of 10 mg/mL. A volume of 10 μL of each tested sample was distributed directly on the solid medium previously seeded with the microbial inocula. The inoculated plates were incubated for 24 hrs at 37°C. Antimicrobial activity was assessed by measuring the growth inhibition zones diameters expressed in mm [21–23]. Following the results of the qualitative screening, only the microbial strains proving to be susceptible have been further tested in the quantitative assay.
The Quantitative Assay of the Minimal Inhibitory Concentration (MIC, μg/mL). It was based on liquid medium twofold microdilutions and performed in 96 multiwell plates. In this purpose, serial binary dilutions of the tested compounds (ranging between 5 and 0.01 μg/mL) were performed in a 200 μL volume of nutrient broth/YPG and each well was seeded with 20 μL microbial inocula of 0.5 McFarland density. The plates were incubated for 24 hours at 37°C for bacterial strains, and the antibacterial effect was assessed by measuring the absorbance of the obtained culture at 620 nm [21–23].
The Antibiofilm Activity of the GIO-NPs. It was tested by the microtiter method. For this purpose, the microbial strains have been grown in the presence of twofold serial dilutions of the tested compounds performed in liquid nutrient broth/YPG, distributed in 96-well plates, and incubated for 24 hours at 37°C for bacterial strains. At the end of the incubation period, the plastic wells were emptied, washed three times with phosphate buffered saline (PBS), fixed with cold methanol, and stained with 1% violet crystal solution for 30 minutes. The biofilm formed on plastic wells was resuspended in 30% acetic acid. The intensity of the colored suspensions was assessed by measuring the absorbance at 492 nm [24, 25].
3. Results and Discussions
Figure 1 shows the powder X-ray diffraction (XRD) pattern from the as-prepared sample glycerol iron oxide nanoparticles (GIO-NPs). The peaks can be indexed fcc cubic maghemite structure (ICSD card no. 01-083-0112) and lattice constants 0.835 nm. No impurity peaks were observed, demonstrating a high purity of the final product. The width of the peaks, however, is relatively broad compared with the XRD pattern of bulk maghemite, indicating a small crystal size. The average sizes of the nanoparticles were computed using Scherer’s formula [26]: where is the averaged length of coherence domains (that is of perfectly ordered crystalline domains) taken in the direction normal to the lattice plane that corresponds to the diffraction line taken into account, is the line broadening due to the small crystallite size, is the wavelength of X-rays (1.5416 Å), is the Bragg angle, and is a constant related to crystallite shape and to the definition of (integral breadth or full width at half maximum). A crystallite size of GIO-NPs was estimated at around 4.2 nm.
Figure 2(a) presents a magnified TEM image of GIO-NPs, clearly showing that the product is entirely composed of crystals with a relatively uniform, spherical morphology. Figure 2(b) shows selected area electron diffraction (SAED) pattern recorded from an area containing a large number of nanoparticles. The rings in the SAED pattern can be indexed as the (220), (311), (400), (422), (511), and (440) reflections of the cubic maghemite in agreement with the XRD results. The average grain size of the nanoparticles was nm.
(a)
(b)
The attachment of the glycerol on the iron oxide particles surface was investigated by FT-IR spectroscopy. Figure 3 illustrates the FT-IR spectra of GIO-NPs. According to [27] maximum of 1554 cm−1 is attributed to COO− band. The bands in the region 1048–1384 cm−1 are assigned to primary alcohol and the band at 1459 cm−1 represented secondary alcohol in good accord with [27].
The bands at 1516 cm−1 and 1537 cm−1 correspond to C–O–H vibration modes. The bands observed at 2337 cm−1 and 2364 cm−1 are assigned to C–O vibration modes. The bands at 2855 cm−1 and 2940 cm−1 are attributed to C–H vibration modes. The spectral ranges 1000–1200 cm−1 were due to ν(C–C) and ν(C–O) stretching vibration with contributions from δ(C–O–H) motion [28]. The spectrum of GIO-NPs contains characteristic OH stretching ν(OH) and HOH bending (δ OH) vibrational bands at 3440 cm−1 and 1600 cm−1 due to the adsorbed water molecules in the sample [29]. Since magnetite has an inverse spinel type structure, it shows the characteristic vibrational bands: –O– (ν1≈ 620–550 cm−1), –O (ν2≈ 470 cm−1), and (ν3≈ 350–400 cm−1), where and correspond to the metal occupying tetrahedral and octahedral positions respectively [30, 31]. The bands observed at 620 cm−1 and 580 cm−1 corresponds to the stretching vibration ν(Fe–O) of tetrahedral iron atom [32].
Inorganic metal oxides may serve as effective disinfectants, due to their relatively nontoxic profile, chemical stability, and efficient antibacterial activity [33]. The insertion of the prosthetic medical devices for different exploratory or therapeutical purposes, especially in severe pathological conditions, represents a risk factor for the occurrence of chronic infections in developed countries, being characterized by slow onset, middle intensity symptoms, chronic evolution, and resistance to antibiotic treatment [34]. The microbial species of clinical interest, often involved in biofilm-associated diseases, are belonging to a very large spectrum, from the Gram-positive (S. epidermidis and S. aureus) to the Gram negative pathogens (P. aeruginosa, E. coli) and to different members of the Candida genus [35]. The understanding of microbial infections related to the biofilm development on tissues or indwelling devices was possible by using different qualitative and quantitative in vitro specific assays. In our study we have also used both assays for studying the susceptibility of microbial cells grown in suspension, as well as of those developed in biofilms, called planktonic cells.
The qualitative screening of the tested compounds at 10 mg/ml concentration showed that GIO-NPs were slightly active only on two of the tested strains, that is, E. faecalis 29212 and P. aeruginosa 1397. In order to investigate the dose-concentration effects of the tested GIO-NPs, we have further chosen these strains to be tested in the presence of a range of twofold serial dilutions of the tested compounds.
The last decades studies of microbial adherence to different substrata led to the conclusion that the survival of microorganisms in the natural habitats, including medical ecosystems, is dependent on their capacity to adhere to different surfaces/substrata and to form biofilms. A biofilm is a sessile microbial community composed of cells embedded in a matrix of extracellular polymeric substances attached to a substratum or interface. The matrix is primarily of microbial origin and the cells encased in this matrix present a modified phenotype, being metabolically more efficient and well protected, exhibiting resistance to different stress factors, including host defense mechanisms and antibiotics [36]. All tested concentrations of GIO-NPs strongly stimulated the microbial growth in suspension, the intensity of this stimulatory activity being proportional with the tested concentrations in the range 5 mg/mL–0.625 μg/mL, thereafter entering a plateau in the range 0.313–0.01 μg/mL (Figure 4).
The same general behavior was observed also in case of microbial biofilms, but, in case of P. aeruginosa 1397 biofilms, at lower concentrations, the GIO-NPs inhibited the ability of this strain to develop biofilms on the inert substratum (Figure 5).
The GIO-NPs exhibited a dose-dependent inhibitory effect on ability to develop biofilms of the Gram-negative P. aeruginosa 1397 and the Gram-positive E. faecalis ATCC 29212 strains. The GIO-NPs exhibited a strong inhibitory effect for different concentration on P. aeruginosa 1397 ability to develop bioflms on the inert substratum.
4. Conclusions
Glycerol iron oxide nanoparticles were synthesized by coprecipitation method. An average glycerol iron oxide size of 4.2 nm was found by X-ray diffraction and a 4.7 nm was found by transmission electron microscopy. The attachment of the glycerol on the iron oxide particles surface was revealed by Fourier transform infrared (FTIR) spectroscopy. In the case of P. aeruginosa 1397 biofilms, at low concentrations, the glycerol iron oxide inhibited the ability of this strain to develop biofilms on the inert substratum.
Acknowledgments
The authors would like to thank Professor Mariana Carmen Chifiriuc of Microbiology Immunology Department, Faculty of Biology, and University of Bucharest for assistance with antimicrobial tests and for the constructive discussions. This work was supported by the Ministry of Education of Romania, Project no. C2-06 under program CEA-IFA.