Synthesis and Evaluation of Magnetic Nanoparticles for Biomedical Applications

In this study, iron oxide (IO) nanoparticles from various precursors have been synthesized using sonochemical method and characterized for their structural variability and toxicity. e iron oxide (IO) precursor solutions were prepared from iron acetate (IA), iron pentacarbonyl (IP), decalin, PEG (poly(ethylene glycol)), EG (ethylene glycol), PVA (poly(vinyl alcohol)), ββ-cyclodextrin (CD), and distilled water. ese precursor solutions were irradiated with high power ultrasound for 3 hours and heat treated as needed. ese as-prepared iron oxide nanoparticles were characterized using X-ray diffraction (XRD), Mössbauer spectroscopy, transmission electron microscopy (TEM), and magnetization measurements. XRD results show that all the particles are highly crystalline in nature and the particles sizes measured from TEM are approximately 5–20 nm. e maximum magnetization was observed for IO-IP at approximately 60.17 emu/g and the minimum was approximately 30.56 emu/g for IO-IA. ese results con�rm that the particles are superparamagnetic (SPM) in nature. Mössbauer spectroscopy veri�ed the magnetic nanoparticles are purely Fe3O4 and particles sizes varied by the nature of the precursor and coatings.


Introduction
Iron based nanomaterials have found applications in many areas of science and technology due to their unique magnetic properties [1][2][3][4][5].Recently, they are also used in biomedical applications such as MRI contrast agents for imaging [6], magnetic hyperthermia [7], and targeted drug delivery [8] because of their size compatibility [9] to cells, genes, and viruses.A size reduction of these materials from bulk to the nanoscale permits them to display various size related properties.Among other changes, a signi�cant decrease in size can alter reactivity, increase surface area, and change the magnetic properties [10][11][12].Iron oxide, for example, exhibits ferromagnetic behavior in bulk where it can reach full magnetization and the effect persists even aer removal of the magnetic �eld.ese same particles below a certain size (<20 nm) may exhibit a behavior similar to paramagnetism even when at temperatures below the Curie point.is is a nanoscale phenomenon, where the energy required to change the direction of the magnetic moment of a particle is comparable to the ambient thermal energy.e energy required to change the direction of magnetization of a crystallite is called the crystalline anisotropy energy and depends both on the material properties and the crystallite size.As the crystallite size decreases, so does the crystalline anisotropy energy, resulting in a decrease in the temperature at which the material becomes superparamagnetic.ese materials require the presence of a �eld to align their magnetic moments and magnetize.ey lose most or all of their magnetization once the �eld is removed.
SPM materials are potential candidates for targeted drug delivery applications because of their size compatibility and ability to become highly magnetized only in the presence of a magnetic �eld.Magnetic particles performing as drug carriers [13] involve systems that can be directed through the body to a speci�c location by an external magnet.e signi�cance of these drug systems is perhaps most imperative in the area of chemotherapy where severe side effects such as morphological alterations and functional impairment of the heart can occur [14].Cancer drugs perform by killing cancer cells, but are not restricted and consequently affect healthy cells as well.e side effects of these drugs may decrease while the effectiveness is increased if a majority of the drug is able to reach its targeted site.With the aid of an external magnet placed near the desired location, more of these drugs should be able to reach its destination and harm fewer healthy cells.
Due to the nature of these applications, which involves interactions with a magnetic �eld outside the body, it is necessary to use materials that can reach the highest possible magnetization.Currently, iron oxide, particularly magnetite (Fe 3 O 4 ), is commonly studied for these applications over other iron oxide structures such as maghemite (-Fe 2 O 3 ) due to a higher magnetic saturation [15].Similar synthesis routes can be used to produce either structure and require detailed analysis such as XRD and Mössbauer spectroscopy to determine the speci�c phase.Peaks for both Fe 3 O 4 and -Fe 2 O 3 occur at nearly the same 2 in XRD, making it difficult to distinguish.Fe 3 O 4 can be identi�ed by Mössbauer spectra taken at room temperature by the display of hyper�ne magnetic splitting that is not seen in Fe 2 O 3 [16,17].
Iron oxide nanoparticles for drug delivery applications can be synthesized through various methods including solution precipitation, decomposition, ultrasound chemistry, and microwave heating [18][19][20].Sonochemical techniques, the method used in this study proceeds by the creation, formation, and bursting of cavitations created from ultrasound waves.When the bubble collapses, it generates pressures and temperatures high enough to cause bond breakage.Because the cooling rates are also high, the particles' size remains low, and only nanosized materials are produced.is method has been proven by Suslick [21] and others [22][23][24][25] as a reliable, reproducible, and relatively easy way to synthesize both uncoated and in situ coated materials.All particles created for biorelated applications must be biocompatible, and therefore may require additional coatings.Coatings can also provide water dispersion stability and protection from degradation as well as increasing circulation time [26].Coatings may be added to the surface of the oxides through chemical or physical bonding [27] using techniques such as solvent evaporation and coupling agents [28] and can take place either in situ or aer-synthesis.Commonly used coatings include poly(lactic-co-glycolic acid) (PLGA) [29], Polyethylene glycol (PEG) [30], Poly vinyl alcohol (PVA), [31] and pluronic-127 [32].e efficiency of these coatings can be determined through cell viability studies at various concentrations of nanoparticles [33].In this study a uniform superparamagnetic Fe 3 O 4 nanoparticles were synthesized and characterized for their morphology, structural, magnetic and biocompatibility.

Synthesis of Fe 3 O 4 (IO-IP)
. IO-IP magnetic nanoparticles were synthesized using ultrasonic irradiation of iron pentacarbonyl (2 mL), decalin (40 mL), and PEG (20 mL) for 3 hrs at 50% power using a high intensity ultrasonic horn (Tihorn, 20 kHz, 100 W/cm 2 ).e reaction was carried out in open air within a stainless steel vessel at 30 ∘ C maintained by circulating water using a thermostat.e solution was diluted in ethanol and centrifuged (Allegra 64R centrifuge) at 15,000 rpm at 5 ∘ C. e precipitate was washed several times with ethanol and dried overnight under vacuum.e asprepared sample was analyzed using XRD, TEM, Mossbauer spectroscopy, and magnetic measurements.

Synthesis of PVA-Coated 𝐹𝐹𝐹𝐹 3 𝑂𝑂 4 (PVA-IO).
Following the same procedure as shown above, except the reaction was carried out in the presence of PVA (0.5 g); the as-prepared sample was heated at 300 ∘ C for 3 hrs to produce crystalline particles.

Synthesis of 𝐹𝐹𝐹𝐹 3 𝑂𝑂 4 (IO-IA).
Additional Fe 3 O 4 nanoparticles were synthesized through ultrasonic irradiation of iron acetate (1.0 g), ethylene glycol (40 mL), and distilled H 2 O solutions (100 mL).e reaction was carried out in open air in a glass beaker placed in a water bath at room temperature to prevent overheating and followed the same procedure as described earlier to obtain �nal product for characterization.

Testing Method. XRD analysis was carried out on a
Rigaku Diffractometer (D-MAX2000) from 3-80 ∘ of 2 at a scan speed of 0.200 ∘ /min.Samples were prepared by spreading the powdered specimen on a glass holder using ethanol; the powder dried before testing.TEM studies were conducted using a JEOL-2010 transmission electron microscope.Samples were prepared by dropping a colloidal solution of ethanol nanoparticles onto a carbon-coated (200 mesh) copper grid.Magnetization curves were obtained at room temperature in an induced magnetic �eld of 2-20 kOe using a Princeton Measurement Micromag.ese samples were prepared by placing a 200 mg of solid sample at the end of the �exible �ber and mounted perpendicular to magnetic �eld.e mass of the sample was calculated excluding the coating of PVA or CD.
e Mössbauer measurements were carried out using a conventional constant acceleration spectrometer operated in multichannel scaling mode.e gamma ray source used for the sample determinations and for iron-57 velocity calibrations consisted of 12 mCi of Co 57 in a rhodium metal matrix that was maintained at ambient temperature throughout spectral determinations.Using the latter source allowed for realization of reasonably high S/N velocity spectra in collection times approximately 24 to 48 hours.e spectrometer was calibrated using a 6-micron thick natural abundance iron foil.e center of the magnetic hyper�ne pattern of the latter foil (at ambient temperature) is taken as zero velocity.e line widths of the inner-most pair of Δ  = ±1 transitions of the latter Zeeman pattern were reproducibly determined to be 0.214 mm/s.e as-prepared samples were placed in nylon sample holders of 1 cm 2 cross-sectional area for transmission Mössbauer spectroscopy experiments.

Cell Cytotoxicity of Nanoparticles.
HEp-2 cells were purchased from the American Type Culture collection (ATCC, Manassas, VA; CCL-23) and were propagated by standard methods using minimum essential medium (MEM) supplemented with 10% fetal bovine serum (FBS), 2 mM L-Glutamine, 75 U/mL penicillin, 100 g/mL kanamycin, and 75 g/mL streptomycin.e effect of nanoparticles on cytotoxicity was measured by MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) dye reduction.HEp-2 cells were seeded in a 96-well plate at a density of 20,000 cells per well in MEM containing 5% FBS and grown overnight.Aer 24, 48, and 72 h aer-incubation of nanoparticles complexes, media were removed and cells were washed twice with sterile PBS.20 L of MTS (5 mg/mL) in sterile �ltered PBS was added to each well and then incubated for 2 h to allow formation of formazan crystals at 37 ∘ C. Aer 2 h, the absorbance of formazan products was measured at 490 nm using a microplate reader.

Results and Discussion
XRD analysis was carried out to determine the amorphous/ crystalline nature and impurities in the as-prepared nanoparticles.Figure 1 shows the XRD patterns of (a) as-prepared IO-IP, (b) PVA-IP heated at 300 ∘ C, (c) IO-IP heated at 300 ∘ C, (d) as-prepared CD-IA, and (e) as-prepared IO-IA.e powder X-ray pattern of Figure 1(a) clearly shows the asprepared uncoated, unheated sample synthesized from iron pentacarbonyl is highly amorphous and no crystalline peaks were observed.To further analyze the amorphous material, XRD was carried out for IO-IP (Figure 1(c)) heated to 300 ∘ C in an argon atmosphere.Figure 1(c) shows that the particles are highly crystalline and all the diffraction peaks match very well with magnetite JCPDS �le number 19-0629.No impurities were observed in this sample.Figure 1(b) shows PVA-IO heated at 300 ∘ C in argon atmosphere is also highly crystalline in nature and all peaks match with magnetite JCPDS �le number 19-0629.
e XRD diffraction patterns of as-prepared particles synthesized from iron acetate (IO-IA) are shown in Figure 1(e).ese diffraction peaks illustrate the highly crystalline nature of the particles.No XRD detectable impurities were observed in this sample as well.Unlike the iron oxide particles produced from the iron pentacarbonyl precursor, these iron oxides were highly crystalline and did not need any additional heat treatments.e peaks were also matched  with the JCPDS �le number 19-0629 for magnetite.Figure 1(d) depicts the XRD of as-prepared CD-IA; the diffraction peaks clearly suggest that the as-prepared particles are also highly crystalline and all peaks match very well with the JCPDS number 19-0629 for magnetite.ese XRD results visibly suggest that all the particles synthesized from various precursors and solvents produced only magnetite Fe 3 O 4 nanoparticles with various coating.TEM analysis was conducted to understand the morphology, surface coatings and crystal nature of the particles.Figure 2 represents the TEM micrograph of (a) IO-IP heated ese particles are also spherical in shape, and uniformly dispersed, and the sizes measured are approximately 10 nm with minimum agglomeration.is micrograph also clearly shows the lattice structure of iron oxide nanoparticles.It is noteworthy to mention that all the iron oxide nanoparticles prepared are mostly spherical.Most importantly, the particle sizes observed in all of the samples fall within the critical limits for drug delivery applications and are small enough to allow for size increase due to additional coatings or drug loading without reaching above the limits.e PVA-coated particles seem to be completely embedded in the PVA, thereby providing an environment for drug loading, protection from potential oxidation encountered in vivo, and yielding a much more biocompatible material.Magnetic properties were studied to determine the magnetic saturation and coercive �eld of (a) IO-IP heated at 300 ∘ C, (b) PVA-IO heated at 300 ∘ C, (c) as-prepared IO-IA, and (d) as-prepared CD-IA nanoparticles at room temperature are shown in Figure 3 and the results were summarized in Table 1.e magnetization/coercive �eld measured from Figure 3  e uncoated oxides (IO-IP) heated to 300 ∘ C were found to reach the highest magnetic saturation of approximately 60 emu/g as expected.With the addition of PVA, there is a signi�cant drop in   , which is attributed to the polymer.It was anticipated that a polymer coating would decrease the magnetization; here; it is decreased by half.e iron oxides synthesized from iron acetate were found to have magnetic saturations much less than those uncoated and produced by iron pentacarbonyl precursors.e CD-IA oxides unexpectedly reached a higher degree of magnetization of approximately 36.92 emu/g, compared to that of the uncoated oxides (IO-IA) which reached approximately 31.32 emu/g.e increase in magnetization of CD-IA is most likely due to the presence of the high crystalline nature of particles, which are ultimately more magnetic.
For comparison, we have noted that the saturation magnetization and coercive �eld for commercial [34] Fe 3 O 4 are 96.3 emu/g and 293 Oe, respectively.However, in our case, the magnetite particles are much smaller than the commercial magnetite; the magnetization of these magnetite nanoparticles at 1.6 T does not fully reach saturation.is huge di�erence in magnetization and coercive �eld is a clear indication of the nanostructure of the samples.e total magnetization of a specimen consists of small particles and decreases with decreasing particle size due to the increased dispersion in the exchange integral [35,36] and �nally reaches superparamagnetic (SPM) state when each particle acts as a single domain dipole moment with suppressed exchange interaction between particles.
Mössbauer spectroscopy was used to determine the �ne structure of iron oxide nanoparticles and their relative sizes.e ambient temperature Mössbauer spectra of (a) as-prepared IO-IP, (b) PVA-IO heated at 300 ∘ C, (c) as-prepared IO-IA, and (d) as-prepared CD-IA are shown in Figure 4 wherein one can see a progression from rapidly relaxing (super) paramagnetism (a) to slow paramagnetic relaxation (d) but not a fully resolved magnetic hyper�ne splitting.Stoichiometric magnetite can be written as [Fe 3+ ] tet [Fe 2+ Fe 3+ ] oct O 4 [16] and displays two sets of superimposed hyper�ne resonance lines representing the iron at the octahedral and tetrahedral sites where only one set of lines is present in Fe 2 O 3 .Also, magnetite displays hyper�ne splitting at room temperature, that is absent in maghemite.Spectra of IO-IP (Figure 4(a)) show evidence of the characteristic magnetite splitting.
Figure 4(b) which is of the PVA-coated particles illustrate widening peaks and a developing sextet, indicating an increase in particle size which is expected due to the additional polymer coating.e sextet becomes increasingly evident in the oxides synthesized from iron acetate, signifying a greater increase in particle size.As expected, the CD-IA has the most de�ned sextet, indicative of the largest size particles.is is a typical, systematic trend with increasing particle (actually magnetic domain) sizes for a �xed temperature and is also observed by other researchers [37,38] for a speci�c case of hematite (Fe 2 O 3 ) for a number of speci�c particle sizes.at is, with increasing particle size, the blocking temperature for the transition from rapidly relaxing superparamagnetism (for which the long time average molecular �eld is zero) to long range ordered bulk magnetic behavior increases.us the hierarchy for the particle size of the present samples implied by the ambient temperature results is particle size (a) < (b) < (c) < (d).On the other hand, for a �xed (small) particle size, one sees effects similar to those of increasing particle size (at �xed temperature) by decreasing the temperature [39].e spectrum of the largest particle size is for (d), of Figure 4 begins to exhibit the low velocity doublets (indicated by vertical arrows in the �gure) as seen in the larger particle size bulk Fe 3 O 4 .is con�rms that the syntheses procedures used for the preparation of samples (a) through (d) maintain the intrinsic "Fe 3 O 4 " identity of these materials.
Toxicity studies were carried out to determine the effect of the as-prepared coated and uncoated iron oxide nanoparticles on human cell viability at increasing concentrations and exposure time.e cytotoxicity of the nanoparticles was evaluated in HEp-2 cell line by the MTS assay.e nanoparticle concentrations ranged from 5 to 400 g/mL which is well above the conventional drug dosage concentration of 2 g/mL.e cell viability was observed at 24, 48, and 72 hours.5 g/mL of IO-IP and PVA-IO were added to the cells, 90% of the cell viability were observed.Cell viability decreased to 80% when IO-IP and PVA-IO concentrations were increased to 200 g/mL aer 24 h aer infection.ere was also a 80% of the cell viability when 100 g/mL of CD-IA and IO-IA were added to the cells.Cell viability continued decreasing about 60% when CD-IA and IO-IA were increased to 200 g/mL aer 24 h aer infection (Figure 5(a)).At the exposure time (48 h) and concentration (100 g/mL), the cells sustained at least 85% cell viability (Figure 5(b)).But, cell death was more prominent when cells were analyzed aer 72 h as shown in Figure 5(c).Overall, all the particles were determined to have minimal toxicity.As the exposure time increased, the coatings on the nanoparticles became less effective.

Conclusions
Uncoated and coated Fe 3 O 4 nanoparticles were prepared using simple one-step synthesis followed by heat treatments as needed.e use of ultrasonic chemistry provides an easy method to produce both coated and uncoated iron oxide nanoparticles that are of similar size, shape, and crystallinity by either the addition or omission of a polymer.In some instances, the polymer presence does decrease the potential magnetic susceptibility; however it provides an environment for drug loading.From toxicity studies, both the coated and uncoated particles were determined to have low toxicity.

F 2 :
TEM micrograph of (a) IO-IP heated at 300 ∘ C, (b) PVA-IO heated at 300 ∘ C, (c) as-prepared IO-IA, and (d) as-prepared CD-IA.at 300 ∘ C, (b) PVA-IO heated at 300 ∘ C, (c) as-prepared IO-IA, and (d) as-prepared CD-IA.Figure 2(a) shows that the iron oxide nanoparticles prepared from iron pentacarbonyl precursor and heated at 300 ∘ C are highly crystalline in nature and spherical in shape.e particles sizes measured from the micrograph are about approximately 5-10 nm.e nanoparticles prepared from PVA and iron pentacarbonyl are presented in Figure 2(b).e TEM micrograph shows that the particles are spherical in shape and are relatively less agglomerated when compared to the IO-IP heated sample with similar sizes.Figure 2(c) depicts the TEM micrograph of as-prepared iron oxide nanoparticles from IA. ese particles are also spherical in shape and sizes measured are approximately 15-20 nm with a little agglomeration.e iron oxide nanoparticles prepared from IA in the presence of CD are presented in Figure 2(d).

F 5 :
Toxicity studies of as-prepared oxide nanoparticles evaluated in HEp-2 cell lines by MTS assay at (a) 24 h, (b) 48 h, and (c) 72 h.