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Article

The Influence of Milling Conditions on the Structure and Properties of Fe3O4 Nanoparticles for Biomedical Applications

by
Vladislav A. Mikheev
1,
Timur R. Nizamov
1,
Polina I. Nikolenko
1,
Anna V. Ivanova
1,
Alexander I. Novikov
1,
Irina V. Dorofievich
1,
Alexey S. Lileev
1,
Maxim A. Abakumov
1,2 and
Igor V. Shchetinin
1,*
1
Department of Physical Materials Science, University of Science and Technology «MISIS», Moscow 119049, Russia
2
N.I. Pirogov Russian National Research Medical University, Moscow 117997, Russia
*
Author to whom correspondence should be addressed.
Crystals 2024, 14(12), 1028; https://doi.org/10.3390/cryst14121028
Submission received: 15 November 2024 / Revised: 23 November 2024 / Accepted: 25 November 2024 / Published: 27 November 2024
(This article belongs to the Section Polycrystalline Ceramics)
Browse Figures
Versions Notes

Abstract

:
In this work, a new two-stage scalable method for the synthesis of magnetite nanoparticles for biomedical applications is proposed. The influence of the milling time, medium, and surfactants on the formation of the structure, magnetic, and functional properties of magnetite nanoparticles has been studied. Comprehensive investigation of the formation of the structure and properties of magnetite nanoparticles has been carried out using X-ray diffraction analysis, scanning and transmission electron microscopy, Mössbauer spectroscopy, measurements of magnetic properties, specific loss power (SLP), and cytotoxicity. It was shown that the milling medium of water with the addition of trisodium citrate is a harsher milling condition compared to octadecene-1 with the addition of oleic acid. Continuous milling for 50 h allowed to obtain a fraction of colloidally stable nanoparticles at the level of 80–90%. Harsher milling conditions led to the formation of a larger fraction of superparamagnetic particles, which reduced the coercivity and SLP. The maximum SLP value of 1140 W/g was reached by large particles, while nanoparticles had decreased SLP values of 100–190 W/g, which was completely determined by the coercivity dependence. Different synthesis conditions allowed obtaining particles with different cytotoxicity against PC-3 cells.

1. Introduction

Magnetic nanoparticles have great potential for application in nanobiotechnology, biochemistry, nanomedicine, and biomaterials [1,2,3]. Magnetite is among the very promising materials for that, due to its unique combination of magnetic properties and excellent biocompatibility. The magnetic properties of magnetite can be controlled in a wide range by doping. Therefore, magnetite nanoparticles have become the basis for the development of materials for diagnostics [4,5,6,7,8], therapy [9,10,11,12,13,14,15], and theranostics [16,17]. For example, the fabrication of chlorella-based magnetic microrobots that contain Fe3O4 nanoparticles has been reported recently [18]. Also, the efficiency of magnetite as a component of theranostic platforms that are responsive to pH has been discussed [19].
Superparamagnetic nanoparticles are promising for use as contrast agents for MRI and targeted delivery since they are not prone to aggregation and can be easily transported by the bloodstream through the body. For the purposes of magnetic hyperthermia, in which nanoparticles injected into the bloodstream or directly into the tumor are heated by a high-frequency field, the use of superparamagnetic particles is not very advisable. This is due to the fact that several mechanisms of heating nanoparticles in an external field are distinguished: specific loss [20], eddy currents [21], Brownian and Néel relaxation [20,21,22], and friction loss [20,23]. From the point of view of increasing the specific loss, it is advantageous for the material to have high values of saturation magnetization, remanence, and coercivity. However, for biomedical applications, the limits of the parameters of the external magnetic field are justified as Ha∙f < 5 × 109 A/(m·s) [24,25], where Ha and f represent the amplitude and the frequency of the external magnetic field, respectively. Exceeding this limit can cause overheating of healthy tissues by eddy currents. Thus, the use of magnetic field generators with a frequency of 200 kHz limits the coercivity of the material at Hc < 25 kA/m, while the area of the magnetic hysteresis loop should be maximum.
It is also worth considering that during the transition of magnetite from bulk to nanoparticle state, due to structural transformation, the properties of the material change as well. So, when the particle size decreases at the nanometer scale, the saturation magnetization decreases due to a larger fraction of surface area relative to the volume of the material, which induces the formation of a non-collinear spin structure of the atoms near the surface [26,27]. Coercivity also depends on the particle size [28]—it increases gradually as the particle size decreases, peaking at a ‘single-domain’ size. The most common values of the single-domain size of magnetite in the literature are ≈70–80 nm for cubic particles [29,30] and ≈120 nm for spherical-shaped ones [31]. However, there are papers where larger values of 160 nm [32], and smaller values of 30–50 nm are reported [33,34,35,36]. A further decrease in the particle size leads to a decrease in the coercivity and a transition to a superparamagnetic state characterized by zero coercivity. The superparamagnetic threshold size of magnetite is reported to be less than 20 nm [37,38,39,40], but values of about 10 nm appear in the literature more often [41]. Thus, the synthesis of Fe3O4 nanoparticles with a set of properties required for biomedical applications is a complex task.
Currently, a variety of chemical methods for the synthesis of magnetite nanoparticles have been developed: co-deposition, aging, decomposition, hydrothermal, etc. [1,2,3]. They allow you to control the size and shape of particles, as well as to modify the surface for solving applied problems. At the same time, the main disadvantages of chemical methods for the synthesis of magnetite nanoparticles are low yield (at the level of grams), the lack of scalability of the synthesis technology, and low to moderate reproducibility. Therefore, the exclusive usage of chemical synthesis methods restrains the application of magnetite nanoparticles in biomedicine. So, there is a need for alternative synthesis methods that would make it possible to produce magnetite nanoparticles in greater quantities. One of the promising scalable methods may be the method of mechanochemical synthesis. In this paper, a new two-stage method for the synthesis of magnetite nanoparticles is proposed. We investigated the effect of the milling time, medium, and surfactants (surface stabilizers) on the formation of the structure and magnetic and functional properties for application in biomedicine.

2. Materials and Methods

All the reagents used in this work were supplied by Sigma-Aldrich (Burlington, VA, USA), except for distilled water, which was prepared in the Laboratory of Biomedical Nanomaterials at NUST MISIS, Moscow, Russia.
To produce magnetite in this work, we used iron powder (99.99%) and aqueous hydrogen peroxide solution (37%), which were mixed and loaded into milling jars. The mechanochemical synthesis was carried out using the ‘Activator 2S’ planetary type high-energy ball mill (Activator, Novosibirsk, Russia); steel jars and balls, ball-to-powder mass ratio 200:20, at jar rotation speed of 800 rpm. This synthesis was conducted for 25 h in an air atmosphere.
In the next stage, the obtained magnetite was wet-milled using the PM400 planetary ball mill (Retsch, Haan, Germany), steel jars, and 3–15 mm balls; the jar rotation speed was 400 rpm; jars filled with air. The milling was carried out in two different media: water-based and octadecene-based. The maximum duration of milling was 50 h. For the water-based medium, trisodium citrate (97%) was used as a surfactant; the mass ratio between balls, powder, water, and trisodium citrate was 200:2:20:1. For the medium based on octadecene-1 (95%), oleic acid (90%) was used as a surfactant; the mass ratio between balls, powder, water, and oleic acid was 200:2:30:2. The samples milled with oleic acid were diluted 5-fold with butanol-1 (≥99.4%) and centrifuged at 3000 rpm for 10 min. The resulting precipitate was washed with ethanol (96%) and then dispersed in a measured amount of chloroform (99%) to prepare a solution with a Fe3O4 concentration of 10 mg/ml. Hydrophilization was performed with slight improvements following the protocol outlined in [42]. The chloroform solution was diluted 5-fold with toluene (≥99.5%) and mixed with an equal volume of 2 mM Pluronic F-127 solution in water. The mixture was sonicated intensively until a gray emulsion formed. The emulsion was centrifuged at 14,000 rpm for 30 min, after which the organic phase was replaced with an equal amount of water. The mixture was sonicated vigorously again, followed by centrifugation under the same conditions. The resulting precipitate was dispersed in water and used for subsequent experiments.
X-ray diffraction (XRD) analysis was carried out using the MiniFlex600 diffractometer (Rigaku; Tokyo, Japan), CoKα radiation (λavg = 0.179012 nm), Kβ-filter, linear D/teX detector (Rigaku; Tokyo, Japan). Phase analysis and calculation of both lattice spacings and substructure parameters of phases were carried out by the Rietveld method using the PDXL-2 software (Rigaku; Tokyo, Japan) together with the PDF-2 powder diffraction pattern database (ICDD; Newtown Square, PA, USA). The instrumental broadening was excluded using the LaB6 standard, which does not contain physical broadening.
Mössbauer spectra were obtained using the MS-1104Em spectrometer (Analytical Instrumentation Department of Research Institute of Physics, SFU; Rostov-on-Don, Russia), with 57Co isotope into the Rh matrix, at room temperature, transmission mode with constant acceleration. The spectra were calibrated with the standard α-Fe sample and analyzed using the Univem MS software (v 2.1.6.73, Analytical Instrumentation Department of Research Institute of Physics, SFU; Rostov-on-Don, Russia).
Magnetic properties of the samples were measured by vibrating sample magnetometry (VSM) using the VSM-250 magnetometer (Dexing Magnet Tech Co.; Xiamen, China) in the external magnetic field of ±1592 kA/m at room temperature.
Morphology of the synthesized materials was investigated by both scanning electron microscopy (SEM) using the JSM-IT500 microscope (JEOL; Tokyo, Japan) and transmission electron microscopy (TEM) using the JEM-1400 microscope (JEOL; Tokyo, Japan) at an acceleration voltage of 120 kV in both bright-field and dark-field modes and selected area diffraction (SAED). First, the investigated nanoparticles were dispersed in an ethanol solution using an ultrasonic bath. Then, the obtained suspension was deposited on a carbon-coated copper specimen holder. Particle size measurements were carried out using the dark-field mode, which is more advantageous as compared to the bright-field mode since single particles that overlay each other in agglomerates become visually distinct. The diameter of a particle was measured in a horizontal direction using the iTEM software (Olympus; Tokyo, Japan). The number of measurements per sample was above 350. Lognormal function was used to fit the size distribution histogram and to calculate an average particle size.
Magnetic hyperthermia was investigated in a water medium with the concentration of Fe3O4 nanoparticles of 10 mg/ml under external field amplitude Ha ≈ 20 kA/m at a frequency f = 261 kHz. The heating of the samples was registered with an infrared camera while applying the alternating magnetic field into the chamber. The obtained data were used for the calculation of specific loss power (SLP) and intrinsic loss power (ILP) by the method described in the article.
The concentration of Fe in solutions was determined using the ferrozine test by measuring the absorption at λ = 560 nm on the Thermo Scientific Multiskan GO spectrophotometer (Thermo Fisher Scientific Corporation; Waltham, MA, USA) in photometric mode. The iron concentration was determined from the calibration curve.
The hydrodynamic size and Zeta potential of the nanoparticles were determined by dynamic light scattering (DLS) using the Zetasizer Nano ZS instrument (Malvern Instruments; Malvern, UK) in plastic cuvettes at 25 °C.
PC-3 human prostate cancer cells were cultured in a 5% CO2 atmosphere at 37 °C in DMEM/F12 medium containing 10 % fetal bovine serum (Sigma-Aldrich; Burlington, VT, USA), 1% L-glutamine (Gibco; Waltham, MA, USA), and 1% antibiotics (penicillin and streptomycin). Tests for cytotoxicity were performed 3 times in triplicate. The data presented in the histograms contain the mean value of cell viability ± SD (standard deviation). The statistical significance of differences between the groups was determined using a parametric paired one-tailed t-test. The differences were considered statistically significant at * p < 0.05, ** p < 0.01, *** p < 0.001.

3. Results and Discussion

3.1. Synthesis and Characterization of Magnetite

According to XRD analysis (Figure 1a, Table 1), the milling of Fe powder together with hydrogen peroxide for 25 h led to mechanochemical synthesis of spinel-type structure Fe3O4 (H11, Fd-3m). A similar technique has been used in works that have confirmed the formation of magnetite [43,44].
Mössbauer spectroscopy confirmed magnetite formation as well, since the specter was represented by two sextets that correspond to the presence of Fe ions in two non-equivalent sites. The iron ions in these sites are tetrahedrally and octahedrally surrounded by oxygen ions. The ST sextet corresponded to tetrahedral surrounding and had an isomer shift value Is of 0.28 mm/s and the hyperfine magnetic field µ0H of 48.9 T. The other SO sextet corresponded to octahedral surrounding of Fe ions and had an isomer shift value Is of 0.66 mm/s and the hyperfine magnetic field µ0H of 45.7 T. The quadrupole splitting Q was zero for both sextets, which is common for magnetite. The ratio between ST and SO sextet areas (S) was close to 0.5, which corresponds to the stoichiometric magnetite since the spinel structure has twice as many octahedral sites.
According to SEM (Figure 1c), the powder obtained after milling was presented in the form of agglomerates of about 10 μm in size that consisted of smaller particles of <1 μm.
The synthesized samples demonstrated a specific saturation magnetization value of 75.8 A·m2/kg (Figure 1d). The value was lower than that of a stoichiometric magnetite in a massive state of 92 A·m2/kg [45,46], which can be a sign of other phases’ presence (especially hematite). However, the presence of the other phases was not detected either by XRD phase analysis (Figure 1a, Table 1) or by Mössbauer spectroscopy (Figure 1b). The decrease in the specific saturation magnetization could also be caused by the formation of nanoparticles. The anisotropy of a particle is known to decrease along with its size due to an increase in surface contribution. As a result, the saturation magnetization of the particles decreases. This effect is common for nanoparticles, and it has been observed multiple times for the nanoparticles of magnetite [43,47], and other systems [48,49]. According to this fact, the particles synthesized at that stage might have had a very wide size distribution, and if so, they could not be used for biomedical applications. Thus, the obtained sample was further milled separately in two different media: (a) water with an addition of trisodium citrate as a surfactant and (b) octadecene-1 with an addition of oleic acid as a surfactant.

3.2. The Influence of the {Water + Trisodium Citrate} Medium on the Milling Process

The magnetite produced at the first stage of work was milled in a water medium with an addition of trisodium citrate for 50 h in total. The jars were unloaded completely after 5, 10, 20, 30, and 50 h of milling (samples cit-5 h, cit-10 h, cit-20 h, cit-30 h, and cit-50 h, respectively) to conduct a systematic and comprehensive study of the material.
According to XRD analysis, the milling did not change the phase composition of the sample (Figure 2, Table 1). After 50 h of milling, the diffractogram was still represented by the magnetite phase exclusively (mass fraction = 100%). The lattice spacing of the magnetite phase was changing insignificantly with the milling time (the values were within statistical error). Meanwhile, the analysis of diffraction lines’ broadening revealed that the crystallite size decreased monotonically from 23.7 ± 0.5 to 11.6 ± 0.3 nm during milling.
The results of magnetic properties measurements (Figure 3a, Table 1) showed that the specific saturation magnetization consistently decreased during milling, which might correlate with the growth of nanoparticles’ relative share. With the reduction in particle size, the surface contribution increases, reducing the anisotropy of the particle. The continuous decrease in the coercivity during milling might be explained by the formation of superparamagnetic particles since the superparamagnetic limit for magnetite is reported to be at the level of 10 nm [41].
However, the XRD analysis and magnetic properties measurements are both integral methods that do not provide information about the size distribution of particles. Therefore, defining the quantity of nanoparticles in the synthesized samples is a fairly important issue. To estimate the quantity of nanoparticles with respect to the mass of the whole sample for different milling times, the following method is proposed. The milled samples were dispersed in an aqueous solution in an ultrasonic bath, and then the concentration of iron was measured (marked as the ‘milled state’). After that, the solution was centrifuged at 2000 rpm for 10 min and the iron concentration was re-measured (marked as the ‘supernatant’). While larger particles were precipitated during the centrifugation process, the remaining liquid lying above the precipitate (called the supernatant) contained nanoparticles that were stable in the solution. The average hydrodynamic size of the particles was measured by the DLS method for both the supernatant and the ‘milled state’. The fraction of nanoparticles that were stable in the aqueous solution was estimated using the following equation:
α s t a b = C s u p e r n a t a n t / C m i l l e d × 100 %
where αstab is the fraction of nanoparticles stable in the aqueous solution, Csupernatant is the concentration of iron in the supernatant (after the centrifugation), and Cmilled is the concentration of iron before the centrifugation (= ‘milled state’).
The DLS analysis (Figure 3b) showed that for a short milling time (less than 20 h), an average hydrodynamic particle size remained at the level of 250–300 nm for the ‘milled state’, whereas for the supernatant, it decreased drastically after 10 h of milling, and then it remained at the level of 100–150 nm. After 50 h of milling, an average hydrodynamic particle size of the ‘milled state’ has almost approached that of the supernatant.
The analysis of the fraction of the nanoparticles stable in the aqueous solution (Figure 3c) revealed that the fraction was continuously growing during milling and reached 90% after 50 h of milling. However, both the XRD phase analysis and magnetic properties indicated the presence of superparamagnetic particles in the sample after 50 h of milling, which was not well consistent with the DLS data. Apparently, the superparamagnetic particles might have agglomerated during the centrifuging process, forming stable clusters of 100–150 nm that were detected by DLS.
The hyperthermia measurement (Figure 3d, Table 1) showed weak dependence of the SLP parameter for the supernatant from milling time, which remained at 100–140 W/g. That was well consistent with nanoparticles’ size dependence (Figure 3b), since smaller particles have the smaller coercivity, meaning less specific loss. On the contrary, the ‘milled state’ samples had a very high specific loss (up to 1140 W/g) at a short milling time (<20 h) due to their high coercivity values caused by a high particle size of 250–300 nm [50]. Increasing the milling time resulted in a reduction in both the particle size and the coercivity for the ‘milled state’. But at 50 h of milling, SLP for the supernatant and for the ‘milled state’ reached comparable values due to comparable particle sizes for both samples.
TEM imaging of the Fe3O4 sample milled in the water medium with the addition of trisodium citrate for 50 h (Figure 4a) has confirmed the XRD data. Bright-field images demonstrated small equiaxial particles. The selected area diffraction image was represented by a magnetite pattern (Figure 4b). In the dark-field images, there were distinctly observed very small particles in the Bragg condition (Figure 4c). Using those images, a particle size distribution histogram was constructed (Figure 4d). The obtained average size value of 8.5 ± 2.4 nm was well consistent with the XRD data and therefore proved that DLS measured cluster size instead of single particle due to the tendency of the investigated particles to agglomerate during centrifugation. Moreover, according to TEM, the fraction of particles with sizes less than 10 nm was more than 70%.

3.3. The Influence of the {Octadecene-1 + Oleic Acid} Medium on the Milling Process

An identical study of milling time influence on magnetite was made in the medium of octadecene-1 with the addition of oleic acid. After 5, 10, 20, 30, and 50 h of milling (samples ol-5 h, ol-10 h, ol-20 h, ol-30 h, and ol-50 h, respectively), the jars were unloaded completely; the samples were washed off from octadecene-1 with butanol-1 and ethanol and then dispersed in chloroform.
According to XRD phase analysis, the milling in octadecene-1 + oleic acid for up to 50 h did not change the phase composition of the powder (Figure 5, Table 2). The samples were single-phase and represented by magnetite exclusively (mass fraction = 100%), similar to those milled in water + trisodium citrate. Lattice spacings of the magnetite phase were changing insignificantly with the milling time (the values were within statistical error). However, the analysis of the diffraction lines’ broadening revealed that the crystallite size decreased monotonically from 23.7 ± 0.5 to 14.1 ± 0.3 nm during milling (Table 2). Comparison of crystallite size of the samples milled in different media (Table 1 and Table 2) showed that in the medium of water + trisodium citrate, smaller average crystallite sizes of 11.6 ± 0.3 nm were achieved.
The analysis of magnetic properties of the samples milled in octadecene-1 + oleic acid showed a monotonic decrease in the saturation magnetization from 75.8 ± 0.7 to 61.7 ± 0.5 nm (Table 2, Figure 6a) due to the reasons mentioned above. Comparison of saturation magnetization of the samples milled in different media (Table 1 and Table 2) showed that the product of milling in octadecene-1 + oleic acid had higher saturation magnetization, explained by higher particle size. The analysis of saturation magnetization dependence was supported by XRD data as well: crystallite size values were monotonously decreasing during milling along with the saturation magnetization. The coercivity of the samples milled in octadecene-1 + oleic acid decreased slightly from 11.5 ± 0.6 to 10.1 ± 0.3 kA/m (Table 2), whereas that of samples milled in water + trisodium citrate reduced more significantly from 11.5 ± 0.6 to 5.9 ± 0.3 kA/m (Table 1). Such a noticeable difference can be explained by the formation of a larger number of superparamagnetic nanoparticles, which were less than 10 nm.
Thus, both XRD analysis results and magnetic properties measurements indicate octadecene-1 + oleic acid to be a softer milling medium that allows for obtaining larger particles.
Hydrophilization was achieved using Pluronic F-127, a surfactant composed of two terminal hydrophilic polyethylene glycol (PEG) blocks and a central hydrophobic polypropylene glycol (PPG) block. The polymer adsorbs onto the hydrophobic surface of oleic acid-capped nanoparticles via its hydrophobic PPG segments, while the hydrophilic PEG segments extend into the aqueous medium. This mechanism allows Pluronic F-127 to act as a phase transfer agent, facilitating the transition of nanoparticles from a hydrophobic (toluene) phase to a hydrophilic (water) phase.
The results of particle hydrodynamic size measurements by DLS (Figure 6b) showed that average particle size monotonously decreased during milling from 370 to 150 nm for the ‘milled state’ samples, although for supernatant it was at the level of 100 nm and practically independent of the milling time. The obtained values of hydrodynamic size (Figure 6b) differed from those for milled water + trisodium citrate (Figure 3b). These differences may be related to the better properties of oleic acid as a surfactant compared with trisodium citrate. The results of the study of the fraction of stable nanoparticles (Figure 6c) showed that the fraction was continually increasing during the milling process and reached 80% after 50 h of milling. Analysis of the change in the fraction of stable nanoparticles showed the following: In the case of the water + trisodium citrate medium, the alpha parameter increased very quickly up to 60–75% at the initial stages of milling (up to 20 h), though it did not grow so intensely when milling in octadecene-1 + oleic acid. The obtained dependences can be explained by more harsh milling conditions in the case of the water + trisodium citrate medium, leading to rapid, intense formation of very small superparamagnetic particles along with large particles. That results in a very wide size distribution of the particles produced. In contrast, milling in the octadecene-1 + oleic acid medium is a softer process, and the resulting particles have a narrower size distribution.
This assumption was confirmed by measurements of the specific loss power (Figure 3d and Figure 6d, Table 1 and Table 2). The samples milled in water + trisodium citrate had significantly higher values (compared to the other medium) of the SLP parameter for the ‘milled state’. This was caused by the presence of a considerable quantity of large single-domain particles with high coercivity, which provided higher specific loss. SLP values for the supernatant derived from the two milling media corresponded to each other roughly.
TEM imaging of magnetite milled in octadecene-1 + oleic acid for 50 h was in good agreement with the XRD data. Bright-field images (Figure 7a) demonstrated small equiaxial particles of about 10 nm in size. The selected area diffraction image was represented by a magnetite pattern (Figure 7b). In the dark-field images, there were distinctly observed very small particles in the Bragg condition (Figure 7c). Using those images, a particle size distribution histogram was constructed (Figure 7d). The obtained average size value of 12.0 ± 2.9 nm was well consistent with the XRD data. The fraction of particles with sizes less than 10 nm was about 20%, and it was several times lower than that of the sample milled in water + trisodium citrate. A comparison of the constructed distribution histograms (Figure 4d and Figure 7d) suggested that milling in water + trisodium citrate allows obtaining of smaller particles.

3.4. Cytotoxicity

The results of cytotoxicity investigations of Fe3O4 nanoparticles produced by high-energy ball milling in the two different media for 50 h are presented in Figure 8a. While the particles milled in water + trisodium citrate demonstrated toxicity, the ones milled in octadecene-1 + oleic acid and then modified with Pluronic did not show a strong toxic effect. The difference in toxicity can be related to particle size, since the fraction of particles less than 10 nm prevailed (was more than 70%) in the sample milled in water + trisodium citrate. This effect might as well be related to different states of the particle surface when using different surfactants.
The significant difference in cytotoxicity might be related to the different surface structures of the tested nanoparticles. The citrate-modified sample exhibited relatively high cytotoxicity (Figure 8a) and a significant negative charge on the surface (Figure 8b). Such a surface could lead to the slow dissolution of the iron oxide core when nanoparticles were internalized by cells into vesicles—digestive organelles with an acidic pH that can drop to as low as 4.7 over time. This acidic environment promoted the release of Fe3+ ions, which possess high oxidative activity, leading to the generation of reactive oxygen species and triggering a programmed cell death pathway called ferroptosis [51]. In the case of the Pluronic-modified sample, the iron oxide core was encased within a double shell of organic compounds—oleic acid and Pluronic—and had a slight positive charge (Figure 8b). This structure has a hydrophobic interlayer between the core and the surrounding medium, which may inhibit core dissolution and reduce the leakage of Fe3+ ions [52].

4. Conclusions

  • For the first time, a method for the synthesis of magnetite by mechanochemical synthesis of iron with hydrogen peroxide is proposed. According to XRD analysis and Mössbauer spectroscopy, stoichiometric magnetite was synthesized. SEM imaging of the obtained Fe3O4 powder demonstrated agglomerates of 10 µm approximately that consisted of smaller particles of <1 µm, which could not be used for biomedical applications. In this regard, the obtained materials were subjected to wet high-energy ball milling in 2 different media.
  • Wet milling of the synthesized Fe3O4 in water with trisodium citrate additives led to a decrease in particle size and an increase in the fraction of colloidally stable nanoparticles to 90% after milling for 50 h. The TEM results correlated well with the XRD data and confirmed the formation of nanoparticles of 8.5 ± 2.4 nm. The hydrodynamic particle size decreased during the milling process, which was followed by a decrease in the coercivity of the samples due to the formation of superparamagnetic particles. The specific loss power (SLP) was completely determined by the dependence of the coercivity and was primarily formed by the specific loss.
  • Wet milling of synthesized Fe3O4 in an octadecene-1 medium with the addition of oleic acid turned out to be a softer method of milling. It also led to the formation of nanoparticles with an average size of 12.0 ± 2.9 nm according to TEM, which was well consistent with the XRD analysis. The fraction of colloidally stable nanoparticles increased to 80% after milling for 50 h. The supernatant SLP values obtained for nanoparticles milled in octadecene-1 with the addition of oleic acid were higher than that in the other medium, due to larger particle size and higher coercivity, which contributed to the increase in specific loss.
  • The synthesized Fe3O4 nanoparticles in water + trisodium citrate showed a clear toxic effect on PC-3 cells compared to the particles milled in octadecene-1 with oleic acid, which did not show a strong toxic effect. This result is explained by the difference in the sizes of the synthesized particles and the surface condition. The proposed two-stage method with post-milling in octadecene-1 + oleic acid can be used for the industrial production of magnetite nanoparticles for biomedical applications.

Author Contributions

Conceptualization, T.R.N. and I.V.S.; methodology, T.R.N.; validation, A.I.N. and I.V.D.; formal analysis, P.I.N. and A.V.I.; investigation, V.A.M., T.R.N. and A.V.I.; resources, M.A.A.; data curation, P.I.N.; writing—original draft preparation, V.A.M. and I.V.S.; writing—review and editing, V.A.M.; visualization, A.I.N. and I.V.D.; supervision, A.S.L.; project administration, A.S.L.; funding acquisition, M.A.A. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Russian Science Foundation (RSF), grant number 23–73-00114.

Data Availability Statement

The data presented in this study are available in the article.

Conflicts of Interest

The authors declare no conflicts of interest.

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Crystals 14 01028 g001
Figure 1. (a) XRD pattern, (b) Mössbauer spectra, (c) microstructure, and (d) magnetic properties of the sample obtained by the mechanochemical synthesis method.
Figure 1. (a) XRD pattern, (b) Mössbauer spectra, (c) microstructure, and (d) magnetic properties of the sample obtained by the mechanochemical synthesis method.
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Crystals 14 01028 g002
Figure 2. Diffractograms of the samples milled in water + trisodium citrate for (a) 0 h, (b) 10 h, (c) 20 h, and (d) 50 h.
Figure 2. Diffractograms of the samples milled in water + trisodium citrate for (a) 0 h, (b) 10 h, (c) 20 h, and (d) 50 h.
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Crystals 14 01028 g003
Figure 3. The influence of milling time on the Fe3O4 nanoparticles’ properties after milling in water +trisodium citrate: (a) magnetic hysteresis loops, (b) DLS results for the ‘milled state’ and supernatant, (c) the fraction of the nanoparticles stable in the aqueous solution, and (d) SLP for the ‘milled state’ and supernatant, and coercivity dependence.
Figure 3. The influence of milling time on the Fe3O4 nanoparticles’ properties after milling in water +trisodium citrate: (a) magnetic hysteresis loops, (b) DLS results for the ‘milled state’ and supernatant, (c) the fraction of the nanoparticles stable in the aqueous solution, and (d) SLP for the ‘milled state’ and supernatant, and coercivity dependence.
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Crystals 14 01028 g004
Figure 4. TEM imaging of the Fe3O4 sample milled in water + trisodium citrate for 50 h: (a) bright-field, (b) SAED, (c) size distribution, and (d) dark-field.
Figure 4. TEM imaging of the Fe3O4 sample milled in water + trisodium citrate for 50 h: (a) bright-field, (b) SAED, (c) size distribution, and (d) dark-field.
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Crystals 14 01028 g005
Figure 5. Diffractograms of the samples milled in octadecene-1 + oleic acid for (a) 0 h, (b) 10 h, (c) 20 h, and (d) 50 h.
Figure 5. Diffractograms of the samples milled in octadecene-1 + oleic acid for (a) 0 h, (b) 10 h, (c) 20 h, and (d) 50 h.
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Crystals 14 01028 g006
Figure 6. The influence of milling time on the Fe3O4 nanoparticles’ properties after milling in octadecene-1 + oleic acid: (a) magnetic hysteresis loops, (b) DLS results for the ‘milled state’ and supernatant, (c) the fraction of the nanoparticles stable in the aqueous solution, and (d) SLP for the ‘milled state’ and supernatant, and coercivity dependence.
Figure 6. The influence of milling time on the Fe3O4 nanoparticles’ properties after milling in octadecene-1 + oleic acid: (a) magnetic hysteresis loops, (b) DLS results for the ‘milled state’ and supernatant, (c) the fraction of the nanoparticles stable in the aqueous solution, and (d) SLP for the ‘milled state’ and supernatant, and coercivity dependence.
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Crystals 14 01028 g007
Figure 7. TEM imaging of the Fe3O4 sample milled in octadecene-1 + oleic acid for 50 h: (a) bright-field, (b) SAED, (c) size distribution, and (d) dark-field.
Figure 7. TEM imaging of the Fe3O4 sample milled in octadecene-1 + oleic acid for 50 h: (a) bright-field, (b) SAED, (c) size distribution, and (d) dark-field.
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Crystals 14 01028 g008
Figure 8. (a) Cytotoxicity effect on PC-3 cells and (b) Zeta potential measurements of magnetite nanoparticles milled for 50 h in water + trisodium citrate and octadecene-1 + oleic acid.
Figure 8. (a) Cytotoxicity effect on PC-3 cells and (b) Zeta potential measurements of magnetite nanoparticles milled for 50 h in water + trisodium citrate and octadecene-1 + oleic acid.
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Table 1. Phase analysis, magnetic properties, and hyperthermia of the samples milled in water + trisodium citrate.
Table 1. Phase analysis, magnetic properties, and hyperthermia of the samples milled in water + trisodium citrate.
Milling Time, hParameters of the Fe3O4 PhaseMagnetic PropertiesSLP Milled State, W/gSLP
Supernatant, W/g
Lattice
Spacing, nm		d, nmε, %Hc, kA/mσr, A·m2/kgσs, A·m2/kg
00.8377 ± 0.000323.7 ± 0.50.23 ± 0.0211.5 ± 0.611.9 ± 0.275.8 ± 0.7--
50.8379 ± 0.000320.6 ± 0.50.19 ± 0.0211.4 ± 0.610.8 ± 0.270.9 ± 0.71147 ± 57139 ± 21
100.8383 ± 0.000317.5 ± 0.40.14 ± 0.0211.4 ± 0.610.4 ± 0.267.0 ± 0.61116 ± 56124 ± 20
200.8379 ± 0.000314.9 ± 0.30.16 ± 0.028.9 ± 0.46.7 ± 0.158.3 ± 0.6727 ± 43100 ± 19
300.8382 ± 0.000312.5 ± 0.30.17 ± 0.027.2 ± 0.45.6 ± 0.158.2 ± 0.6473 ± 38139 ± 23
500.8377 ± 0.000311.6 ± 0.30.16 ± 0.025.9 ± 0.35.0 ± 0.157.1 ± 0.5218 ± 23119 ± 17
d: crystallite size; ε: microstrain; Hc: coercivity; σr: specific remanence; and σs: specific saturation magnetization.
Table 2. Phase analysis, magnetic properties, and hyperthermia of the samples milled in octadecene-1 + oleic acid.
Table 2. Phase analysis, magnetic properties, and hyperthermia of the samples milled in octadecene-1 + oleic acid.
Milling Time, hParameters of the Fe3O4 PhaseMagnetic PropertiesSLP Milled State, W/gSLP
Supernatant, W/g
Lattice
Spacings, nm		d, nmε, %Hc, kA/mσr, A·m2/kgσs, A·m2/kg
00.8377 ± 0.000323.7 ± 0.50.23 ± 0.0211.5 ± 0.611.9 ± 0.275.8 ± 0.7--
50.8376 ± 0.000321.9 ± 0.50.21 ± 0.0211.2 ± 0.611.5 ± 0.269.5 ± 0.7405 ± 16193 ± 14
100.8385 ± 0.000319.4 ± 0.40.23 ± 0.0211.4 ± 0.611.0 ± 0.266.9 ± 0.6386 ± 15211 ± 15
200.8374 ± 0.000316.2 ± 0.30.20 ± 0.0211.4 ± 0.49.6 ± 0.162.2 ± 0.638 6± 15206 ± 14
300.8388 ± 0.000315.2 ± 0.30.22 ± 0.0210.6 ± 0.49.0 ± 0.162.4 ± 0.6290 ± 12183 ± 14
500.8379 ± 0.000314.1 ± 0.30.21 ± 0.0210.1 ± 0.38.7 ± 0.161.7 ± 0.5270 ± 14198 ± 14
d: crystallite size; ε: microstrain; Hc: coercivity; σr: specific remanence; and σs: specific saturation magnetization.
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Mikheev, V.A.; Nizamov, T.R.; Nikolenko, P.I.; Ivanova, A.V.; Novikov, A.I.; Dorofievich, I.V.; Lileev, A.S.; Abakumov, M.A.; Shchetinin, I.V. The Influence of Milling Conditions on the Structure and Properties of Fe3O4 Nanoparticles for Biomedical Applications. Crystals 2024, 14, 1028. https://doi.org/10.3390/cryst14121028

AMA Style

Mikheev VA, Nizamov TR, Nikolenko PI, Ivanova AV, Novikov AI, Dorofievich IV, Lileev AS, Abakumov MA, Shchetinin IV. The Influence of Milling Conditions on the Structure and Properties of Fe3O4 Nanoparticles for Biomedical Applications. Crystals. 2024; 14(12):1028. https://doi.org/10.3390/cryst14121028

Chicago/Turabian Style

Mikheev, Vladislav A., Timur R. Nizamov, Polina I. Nikolenko, Anna V. Ivanova, Alexander I. Novikov, Irina V. Dorofievich, Alexey S. Lileev, Maxim A. Abakumov, and Igor V. Shchetinin. 2024. "The Influence of Milling Conditions on the Structure and Properties of Fe3O4 Nanoparticles for Biomedical Applications" Crystals 14, no. 12: 1028. https://doi.org/10.3390/cryst14121028

APA Style

Mikheev, V. A., Nizamov, T. R., Nikolenko, P. I., Ivanova, A. V., Novikov, A. I., Dorofievich, I. V., Lileev, A. S., Abakumov, M. A., & Shchetinin, I. V. (2024). The Influence of Milling Conditions on the Structure and Properties of Fe3O4 Nanoparticles for Biomedical Applications. Crystals, 14(12), 1028. https://doi.org/10.3390/cryst14121028

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