Synthesis and Characterization of Water Dispersible Iron Oxide (γ-Fe2O3) Nanoparticles for Biomedical Applications

In this study, L-lysine coated γ-Fe2O3 nanoparticles were synthesized by a chemical approach in two steps. In the first step γ-Fe2O3 nanoparticles were synthesized by a polyol-reduction method. XRD analysis confirmed the presence of cubic maghemite phase with an average crystallite size of 9.2 nm. SEM analysis showed that the prepared γ-Fe2O3 nanoparticles have a spherical structural morphology with the tendency of agglomeration and with size in the range 8.3610.69 nm. The γ-Fe2O3 nanoparticles were coated with L-lysine in the second stage in an aqueous dispersion with ultrasonication followed by a gentle heating at 40oC. FT-IR spectroscopy confirmed the presence of L-lysine on the nanoparticles surface and the Zeta potential also supported the coating of nanoparticles with a hydrophilic layer of amino acid (L-lysine) and a good stability in aqueous medium. Hysteresis loop shows a ferromagnetic behavior at room temperature for both samples.


Introduction
In the last years, sensitive magnetic nanoparticles have found multiples applications in different areas of bioscience, biotechnology, and environmental technologies. In terms of applications in biomedicine, these are based on the use of selected properties such as magnetic separation, magnetic targeting, heat generation, increased MRI contrast [1][2][3]. For all these applications, magnetic nanoparticles must be stable to oxidation, biocompatible, not agglomerated, and exhibit high magnetization [4]. The most common used magnetic biomaterials are magnetite (Fe3O4) and maghemite (γ-Fe2O3). Spherical magnetic nanoparticles with a diameter of 5 -15 nm are ideal particles for most forms of diagnosis or therapy. Iron oxide nanoparticles may have a spherical shape when prepared in solution or in gas. For this reason, in the last period different methods and processes for the preparation of iron oxide nanoparticles have been intensively studied and improved. Many chemical synthesis methods are reported till present for iron oxide nanoparticles preparation: co-precipitation [5][6][7][8][9][10], thermal decomposition [11,12], hydrothermal method [13], the solvothermal method [14], the sonochemical method [15,16], the electrochemical method [17], the microemulsion method [18], and others [19][20][21][22]. In order to obtain a magnetically sensitive biocompatible material it is necessary to stabilize the iron oxide nanoparticles either by modifying the surfaces or by incorporating them in biocompatible matrices. The biopolymers used for the modification of magnetic oxide particles must be biocompatible, biodegradable, non-toxic, non-thrombogenic, non-immunogenic and inexpensive. The most used biopolymers for coating of magnetic oxide particles reported in the literature are: polyethylene glycol (PEG) [23], polyvinyl alcohol (PVA) [24], dextran [25], chitosan [26], polyvinylpyrrolidone (PVP) [27]. Another way to modify the surfaces of magnetic nanoparticles for biomedical applications is by bonding on their surface suitable chemicals that play an important role in the body. Amino acids are part of the desired chemical substances for modifying the surfaces of magnetic nanoparticles. Of the amino acids, L-lysine plays an important role in the body as a promoter for calcium absorption in the digestive tract, collagen production, is used by several hormones and enzymes and is required for tissue repair [28]. In this study, γ-Fe2O3 nanoparticles with surface coated with an amino acid (L-lysine) were prepared in two steps. The γ-Fe2O3 nanoparticles were prepared in a first step by a polyol-reduction method. In the second step, the γ-Fe2O3 nanoparticles were coated with an amino acid (L-lysine) by an ultrasonication technique. Modifying the surface of γ-Fe2O3 nanoparticles with L-lysine ensures the dispersibility, stability in water, and biocompatibility required for diagnosis and targeted drug delivery for cancer therapy.

Synthesis of iron oxide (γ-Fe2O3) nanoparticles (sample PIb)
An amount of 4.04g of Fe(NO3)3•9H2O was dissolved in 50 mL of PEG using magnetic stirring for 30 min. using a 250 mL tree-neck round bottomed flask equipped with condenser, magnetic stirrer, thermometer and heating system. After this stage, it was added 0.375g of PVP as stabilizing agent in the polyol-iron salt solution by magnetic stirring and heating at 100ºC for another 30 min. The obtained solution was subsequently heated up to 240ºC. At this stage it has been added an amount of 4.1015 g of CH3COONa as reducing agent and it was observed the formation of brown colour precipitate. The reaction solution was refluxed for 4h under these conditions of reaction. The mixture was then cooled down to the room temperature and it was centrifuged in order to remove the PEG and PVP excess traces. Then, the magnetically separated precipitate was washed three times successively with deionized water and absolute ethylic alcohol, and at the end it was dried in a vacuum oven at a temperature of 100°C overnight.

Synthesis of γ-Fe2O3 coated with L-lysine nanoparticles (sample PIIb)
0.22 g of the γ-Fe2O3 nanoparticles thus prepared were ultrasonically dispersed in 25 mL of H2O D.I (Deionized water) for 10 min. Then, 4 mL of 1% L-lysine solution was added to this suspension and has been dispersed ultrasonically for another 30 min. The reaction mixture thus obtained was then heated to 40ºC for 3 h under magnetic stirring. After cooling to room temperature, the nanoparticles were separated from the suspension by magnetic decantation and washed three times with H2O D.I and finally with absolute ethylic alcohol, then were dried under vacuum at room temperature overnight.

Characterization
The powders of γ-Fe2O3 nanoparticles and γ-Fe2O3 nanoparticles coated with L-lysine were analysed by X-ray diffraction (XRD) technique in order to establish the crystalline structure and to evaluate the crystallite size and the lattice parameters. X-ray diffractometry analysis was performed with the D8Discover (Bruker) diffractometer, configured on the primary optics with a primary radiation tube of Cu (λ = 1.540598Å), 0.6 mm Göebel mirror, and on the secondary optics with a 1D LynxEye detector. The diffractograms were recorded with an angular increment of 0.04°, at a scan speed of 1s/step. Qualitative analysis was performed using the ICDD Release 2015 database. Considering the main crystallographic phases, by the Rietveld analysis, the parameters of the elementary cell, respectively the average crystallite size were determined. Fourier Transform Infrared (FT-IR) spectroscope (Perkin Elmer Spectrum 100) was used to characterize the L-lysine coated γ-Fe2O3 nanoparticles. The powder samples were ground with KBr and compressed into a pellet. FT-IR spectra were recorded in range of 4000 -400 cm -1 with a resolution of 4 cm -1 . The morphology and particle size of the as prepared samples were investigated by FESEM-FIB (Workstation Auriga) scanning electron microscope equipped with a dispersive X-ray spectroscope (EDS) detector (Oxford Instruments). Elemental compositions were evaluated by energy dispersive X-ray spectroscopy (EDS) area scanning system. The magnetic properties of iron oxide samples were measured with a vibrating -sample magnetometer (VSM) Lake Shore 7300 model at room temperature. The prediction of the dispersion stability and particle size of the γ-Fe2O3 coated with L-lysine in aqueous solution was determined using NanoBrook ZetaPlus Zeta Potential Analyzer (Brookhaven, USA).

Results and discussions XRD analysis
The X-ray powder diffraction pattern of the sample (PIb) of γ-Fe2O3 nanoparticles processed by a polyol-reduction synthesis method is presented below in Figure 1. The peaks can be indexed by the (2 2 0), (3 1 1), (4 0 0), (5 1 1) and (4 0 0) crystalline planes. All the diffraction peaks in the patterns of sample (PIb) matched well with the standard γ-Fe2O3 reflection according to the ICDD PDF2 (01-089-5892) data sheet. As it is well known, both structures of magnetite (Fe3O4) and maghemite (γ-Fe2O3) nanoparticles have same XRD peaks. However, considering that black color is characteristic for magnetite and brown for maghemite, the sample in our experiment is of brown color which supports the successful formation of the maghemite phase. The crystallite size was obtained using Debye-Scherer equation [29] considering the main peak (311). The crystallite size and the main parameters of the as obtained samples determined are presented in Table 1.

FT-IR analysis
FT-IR spectra of sample (PIb) of iron oxide nanoparticles synthesized by polyol-reduction method and of sample (PIIb) of nanoparticles of iron oxide coated with L-lysine are given in Figure 2(a-b).  For samples PIb and PIIb, IR spectra exhibits two absorption bands at 441 cm -1 , 564 cm -1 and respectively at 401 cm -1 and 564 cm -1 , which are assigned the Fe-O stretching modes corresponding in the maghemite phase [28]. In the spectrum in Figure 2(a) the absorption bands at 3454 cm -1 and 1641 cm -1 are assigned to O-H stretching and deformation vibration which indicates the presence of hydroxyl groups on the surface of γ-Fe2O3 nanoparticles [27]. Also, in this spectrum the presence of two peaks is observed at 2925 cm -1 and 2854 cm -1 given by the symmetrical and asymmetrical stretching vibrations of the C-H bonds, due to the presence of the stabilizing agent (PVP) on the surface of the nanoparticles. In the spectrum in Figure 2 (b) two peaks are observed at 1636 cm -1 and 1391 cm -1 assigned to the vibrations of asymmetric and symmetric stretching of carboxyl anion from the structure of L-lysine. The splitting between the asymmetric and symmetric stretching of carboxyl group is higher than 200 cm -1 indicating a unidentate coordination between the carboxyl group and the hydroxyl group on the nanoparticles surface [8]. The peak at 2925 cm -1 is given by asymmetrical stretching vibration of the C-H bond and peak at 3442 cm -1 is assigned to the stretching vibration of N-H bond from the amino acid structure. The absorption peak at 2361 cm -1 may be due to the absorption of CO2 on the surface of nanoparticles. According to these results, FT-IR analysis confirms the coating of γ-Fe2O3 nanoparticles thus prepared with L-lysine.

EDS-analysis
By the EDS analysis of the chemical composition of the samples it was evaluated the EDS spectrum of the as synthesized γ-Fe2O3 nanoparticles (Figure 3(a)). It was noticed the existence of Fe, O and C elements, the C element being related to the presence of PVP capping agent on the nanoparticle surface, also in agreement with the FT-IR analysis [29]. Further, EDS pattern of the L-lysine coated γ-Fe2O3 nanoparticles (Figure 3(b)) evidenced the Fe, O, C elements and in addition the presence of the N element. The result is in agreement with the result of the FT-IR analysis and confirms the presence of the superficial amino acid layer (L-lysine) on the γ-Fe2O3 nanoparticles thus prepared.
(a) (b) Figure 3. The EDS pattern of γ-Fe2O3 nanoparticles obtained by polyol-reduction synthesis method: (a) nanoparticles not coated with L-lysine and (b) nanoparticles coated with L-lysine

SEM analysis
The morphological scanning electron image of the γ-Fe2O3 nanoparticles synthesized by polyolreduction method is presented in Figure 4. The as prepared γ-Fe2O3 nanoparticles morphology indicate an approximate spherical shape being noticed also a predisposition to form conglomerates. The size of particles, as evaluated by the SEM analysis, it was in range 8.36 -10.69 nm. Also the γ-Fe2O3 nanoparticles coated with L-lysine ( Figure 5) are of spherical shape with size in range 8.84 -11.62 nm. The increase in nanoparticle size in this case is explained by the presence of the L-lysine layer.

VSM analysis
The magnetic properties of the nanoparticles of γ-Fe2O3 and γ-Fe2O3 coated with L-lysine were measured at room temperature by vibrating sample magnetometry and hysteresis loops are presented in Figure 6.
The saturation magnetization (Ms), coercive field (Hc) and remanence magnetization (Mr) values are given in Table 2.  From the shape of the hysteresis loops it conclude that the nanoparticles have ferromagnetic behaviour at room temperature.
The saturation magnetization (Ms = 63.32 emu/g) value for the γ-Fe2O3 nanoparticles (sample PIb) is lower than that of the bulk γ-Fe2O3 (76 emu/g) due to the presence of PVP at surface the nanoparticles and the small size effect of the γ-Fe2O3 particles confirmed by the well-known direct dependence between magnetization and the diameter of small particles (diameters smaller than ca.15 nm). The Ms (MS = 54.43 emu/g) of γ-Fe2O3 nanoparticles coated with L-lysine (sample PIIb) decreases due to the diamagnetic amino acid layer on the surface of the nanoparticles and also due to the quantitative reduction of γ-Fe2O3 https://doi.org /10.37358/Rev.Chim.1949 Rev. Chim., 71 (10) in the sample. Therefore, the γ-Fe2O3 nanoparticles coated with L-lysine have magnetic properties characteristic for biomedical applications. Figure 7(a-b) shows dynamic light scattering measurement results for nanoparticles of γ-Fe2O3 and γ-Fe2O3 coated with L-lysine. Particles size analysis revealed bimodal distribution of nanoparticles with an effective diameter of 161.3 nm and 0.365 polydispersity index for γ-Fe2O3 nanoparticles, and an effective diameter of 270.5 nm with 0.327 polydispersity index for γ-Fe2O3 coated with L-lysine nanoparticles. The effective diameter of γ-Fe2O3 coated with L-lysine nanoparticles is larger than uncoated γ-Fe2O3 nanoparticles, which proves the presence L-lysine layer on their surface. The mean hydrodynamic diameters obtained by DLS method are also larger than those measured by SEM, because DLS measures the aggregated clusters of nanoparticles in aqueous solution. In order to determine the stability of the aqueous dispersion of γ-Fe2O3 coated with L-lysine nanoparticles, the Zeta potential was also performed. The values of Zeta potential recorded at different time intervals on an aqueous solution (0.01%) are shown in Figure 8(a-b).

Particle size and zeta potential analysis
The value of the Zeta potential decreases in time from -35.56 mV (initially) to the value of -33.90 mV (after 1 month). Maintaining the high value of the Zeta potential outside the range of +30 ÷ -30 mV shows a good stability in aqueous medium of γ-Fe2O3 coated with L-lysine nanoparticles thus prepared.

Conclusions
In this study we have developed a facile chemical approach for synthesis of water dispersible iron oxide (γ-Fe2O3) nanoparticles in two steps. In the first step were synthesized γ-Fe2O3 nanoparticles by a polyol-reduction method. XRD analysis confirmed the unique phase of maghemite with average crystallite of 9.2 nm. The surface coverage of the nanoparticles thus prepared with L-lysine in the second stage was confirmed by FT-IR, EDS and zeta potential analysis. The zeta potential values of -35.56 mV (at baseline) and of -33.90 mV (after time of 1 month) recorded on the aqueous dispersion of γ-Fe2O3 nanoparticles coated with L-lysine indicate a good dispersibility in water and stability over time. The https://doi.org/10.37358/RC.20.10.8353 VSM analysis evidenced a ferromagnetic behavior of the nanoparticles at room temperature. The good magnetic properties (Ms = 54.43 emu/g) and good dispersion and stability in the aqueous medium meet the requirements for biomedical applications.