Highly stable silica-coated manganese ferrite nanoparticles as high-efficacy T 2 contrast agents for magnetic resonance imaging

Highly stable silica-coated manganese ferrite nanoparticles were fabricated for application as magnetic resonance imagining (MRI) contrast agents. The manganese ferrite nanoparticles were synthesized using a hydrothermal technique and coated with silica. The particle size was investigated using transmission electron microscopy and was found to be 40–60 nm. The presence of the silica coating on the particle surface was confirmed by Fourier transform infrared spectroscopy. The crystalline structure was investigated by X-ray diffraction, and the particles were revealed to have an inverse spinel structure. Superparamagnetism was confirmed by the magnetic hysteresis curves obtained using a vibrating sample magnetometer. The efficiency of the MRI contrast agents was investigated by using aqueous solutions of the particles in a 4.7 T MRI scanner. The T1 and T2 relaxivities of the particles were 1.42 and 60.65 s-1 mM-1, respectively, in water. The ratio r2/r1 was 48.91, confirming that the silica-coated manganese...


I. INTRODUCTION
2][3] MR images are generated by the excitation and energy release of hydrogen protons inside the body in a strong magnetic field.The nuclear spins (hydrogen protons) have different relaxation times depending on the water content and surroundings of the imaging.Two different relaxation times, T 1 and T 2 , are used in MRI. 4 T 1 mainly depends on the rate of energy transfer from the nuclear spins to the neighboring molecules, and T 2 depends on the dephasing process of the nuclear spins caused by the neighboring magnetic inhomogeneity.Highcontrast MRI is achieved by introducing contrast agents to decrease the relaxation times of the nuclear spins in tissues.In this process, the tissue-dependent uptake of the contrast agents is considered. 5,6wing to the lack of reticuloendothelial systems (for example, Kupffer cells in the liver) in cancer tissues, these tissues cannot capture the contrast agents, for example, magnetic nanoparticles, and recognize them as external invaders.Thus, a T 1 contrast agent accelerates T 1 relaxation at a normal tissue site, which strengthens the signal from that site, thus providing a brighter image than that of the cancer site.On the other hand, a T 2 contrast agent accelerates T 2 relaxation in a normal tissue, which weakens the MR signal at that site, whereas the MR signal at the cancer site remains unaltered.Therefore, a T 2 contrast agent can be used to distinguish between cancer and normal tissues.
Gadolinium-based contrast agents, such as Gd-diethylenetriaminepentacetate (DTPA), have been widely used since MRI was first applied as a clinical diagnostic technique.Gadolinium is a paramagnetic material that accelerates the relaxation of nuclear spins. 7,8However, it is toxic and therefore chelated with DTPA to eliminate the toxicity.Gadolinium-based contrast agents are widely used as T 1 contrast agents to increase the contrast of the bright images, indicating normal tissues, in MRI.Paramagnetic manganese in the form of Mn-dipyridoxyl diphosphate (DPDP) and Mn-ethylenediaminetetraacetic acid (EDTA)-PP is also used as a contrast agent. 9,10In Mn-DPDP, toxic manganese is chelated with DPDP, while manganese is inserted into liposomes in Mn-EDTA-PP.
Since the late 1990s, iron oxide-based nanoparticle contrast agents have been investigated and clinically used as T 2 contrasts agents.They compose of magnetic nanoparticle core and biocompatible coating material, which prevents aggregation and sedimentation, and thereby allows high biological tolerance. 11The magnetic particles are toxic and therefore must be coated with biocompatible materials.Various coating materials are used for commercial nanoparticle contrast agents, such as dextran (Feridex, Resovist, Sinerem, Feraheme), polyethylene glycol (PEG; Clariscan), and silica (GastroMARK).The coated particles have a diameter of a few tens of nanometers (see Table I below).2][23] These contrast agents contain a material that provides exchangeable protons.The saturation magnetization of the proton can be transferred to the neighboring water molecules if a radio frequency (RF) wave absorbable by the proton is applied.Due to this magnetization transfer, T 2 relaxation of the nuclear spins is accelerated.One such contrast agent, PARACEST, is composed of a paramagnetic element (lanthanide element) and a CEST (sugar or amino acid) material.In this combination, the paramagnetic element provides the chemical shift and the CEST material provides the exchangeable proton.The main advantages of this contrast agent are that it allows on-off function of the contrast enhancement because the proton exchange is initiated by RF waves, and that the RF wavelength can be controlled by choosing various paramagnetic and CEST materials.
Recently, researchers have focused on the use of manganese ferrite nanoparticles as MRI contrast agents due to their high magnetic susceptibility. 24,25Several groups have investigated manganesebased nanoparticles as an alternative to gadolinium for reducing the risk of toxicity. 26Manganese also possesses a high spin quantum number and proton exchange kinetics.As mentioned above, some manganese-based complexes such as Mn-DPDP have already been approved for clinical use. 27n the present study, manganese ferrite nanoparticles were synthesized by a hydrothermal route.Silica was employed as a coating material to ensure high stability and biocompatibility of the manganese ferrite nanoparticles.The morphological and crystalline structure and the magnetic properties of the nanoparticles were investigated using various analytical tools.The applicability of these particles as T 2 contrast agents was also confirmed.

Company or Contrast agent
Coating material Particle size

II. EXPERIMENTAL
and NaOH were purchased from Sigma Aldrich and were used as received without further modification.Deionized water was used throughout the reaction.The manganese ferrite nanoparticles were synthesized using a one-step hydrothermal technique in a 100-mL Teflon-lined stainless-steel reactor with a 60% filling degree.In brief, stoichiometric amounts of metal chloride precursors were dissolved with 20 mL deionized water in a 100-mL beaker using a magnetic stirrer, followed by the addition of 0.5 M NaOH solution until the pH reached 11, at which point the color of the mixture turned brown.The solution was continuously stirred for 1 h at room temperature under argon atmosphere to obtain a homogenous suspension.Then, this homogeneous mixture was transferred into a Teflon-lined stainless-steel reactor and kept in a vacuum oven (JEIO Tech OV-11-120) at 160 • C for 12 h.The solution was then naturally cooled to ambient temperature in a vacuum oven.The obtained sample was magnetically decanted using a permanent magnet to separate the unreacted precursors.The washing cycle was repeated several times by dispersing the nanoparticles in water and ethanol and using a magnet to separate the product from the solution and was continued until complete removal of the unreacted products.Finally, the nanoparticles were dispersed in 50 ml water for silica coating.
Silica coating was performed using a previously reported technique. 28In brief, 60 mg of the manganese ferrite nanoparticles was dispersed in water and placed in an ultrasonic bath for 20 min to obtain a good dispersion.Subsequently, the particles were dispersed in ice-cold 0.1 M nitric acid in an ultrasonic bath followed by separation of the nanoparticles by centrifugation.Then, the particles were re-dispersed in a 1 M citric acid solution.The particles were centrifuged again and alkalized by introducing a few drops of ammonia until pH reached 11, and then dispersed in water.The water suspension of the particles was added to a mixture of ethanol (150 mL), water (35 mL), and ammonia (7 mL) followed by addition of 0.6 mL of tetraethyl orthosilicate while stirring and continuously stirred at 40 • C for 6 h.A highly stable suspension of the silica-coated manganese ferrite nanoparticles was obtained and separated by high-speed (13500 rpm) centrifugation and then dispersed in water for further characterization.
The crystalline structure of the nanoparticles was investigated by X-ray diffraction (XRD; X'pert PRO, PANalytical).The shape and size of the nanoparticles were examined using transmission electron microscopy (TEM; HT 7700, Hitachi Ltd).The chemical composition analysis was performed using inductively coupled plasma (ICP) spectroscopy (IRISAP, Thermo Jarrell Ash).The concentration of the nanoparticles in the aqueous solution was also obtained by ICP.A vibrating sample magnetometer (VSM; MPMS, Quantum Design) was used to measure the magnetic properties of the nanoparticles.Fourier transform infrared (FTIR; Nicolet 380, Thermo Scientific USA) measurements were performed to evaluate the status of the coating.MRI contrast effects were observed using a 4.7 T MRI system (Bruker Biospec 47/40).

III. RESULTS AND DISCUSSION
The TEM images of the silica-coated manganese ferrite nanoparticles are shown in Fig. 1.We can see that cores consist of multiple manganese ferrite particles, and measure less than 15 nm in diameter.Average diameter of coated particles was about 40 nm as seen in the inset of Fig. 1, which is the histogram of 100 particles obtained from TEM images.
FTIR spectra of the silica-coated and bare manganese ferrite nanoparticles are shown in Fig. 3.The broad band at 3400 cm -1 corresponded to the O-H group stretching, while the band at 1620 cm -1 was attributed to the vibration of the O-H group present on the surface of the samples. 29,30n addition, the absorption band at 1100 cm −1 was the characteristic peak of the anti-symmetric stretching vibrational mode of Si-O-Si siloxane bridges.The absorption peak at 950 cm -1 was due to the contribution of the Si-O-H stretching vibration, 31 while the band at 800 cm -1 was due to the SiO 4 ring vibration. 32The band at about 600 cm -1 corresponded to the Fe-O stretching in the Fe-O-Si bond. 33In summary, the FTIR spectra confirmed the bonding of the silica to the surface of the manganese ferrite nanoparticles.
The magnetic hysteresis curves of the silica-coated and bare manganese ferrite nanoparticles at room temperature are shown in Fig. 4. The saturation magnetization values for the coated and bare particles were 10 and 7.5 emu/g, respectively, which were much lower than the corresponding bulk values of 80 emu/g. 34,35On the other hand, the coercive forces for the coated and bare particles were 20 and 35 Oe, respectively, showing the superparamagnetic behavior.The coercive force also showed a symmetric behavior, indicating no exchange bias.The small values of saturation magnetization for the nanoparticles could be explained by some important factors, such as cationic distribution, impurity phase, and surface spin structure, determining the magnetic properties. 34,36In manganese ferrite, the cations Mn and Fe were distributed at the tetrahedral A sites and octahedral B sites according to the inverse spinel crystalline structure.Some authors suggested that the redistribution of manganese and iron in the octahedral and tetrahedral sites upon annealing strengthened the exchange interaction, thus increasing the saturation magnetization. 379][40] They observed that particle size played an important role in the cationic distribution at the lattice sites of manganese ferrite. 41Low saturation magnetization similar to that observed in the present study was previously reported. 37,38,42he surface effects and the magnetic dead layer on the nanoparticle surface due to the large surface-tovolume ratio in the nanoparticle systems were found to be responsible for the saturation magnetization of the nanoparticles being lower than the bulk value.
In the silica-coated nanoparticles, the saturation magnetization was lower than that of the bare particles, whereas the coercive force was higher.This behavior was expected, since the silica-metal bond on the surface of the nanoparticles reduced their magnetic moment, leading to a decrease in the magnetization and an increase in the coercive force. 43or relaxation time measurements, six aqueous samples with different particle concentrations ranging from 0.13 to 0.92 mM were prepared.The amount of iron and manganese in the liquid was determined using ICP spectroscopy.The liquid sample retained its colloidal suspension over a few weeks before the relaxation measurements without any precipitates.T 1 and T 2 of the nuclear spins in the aqueous solutions of the magnetic particles were measured using an MR scanner.
For T 1 measurements, the inversion recovery pulse sequence was used.In this sequence, the signal intensity is expressed as a function of T 1 as follows: By comparing the MR intensities with Eq. 1, we could obtain the T 1 values for samples with three representative particle concentrations, as shown in Fig. 5.The Carr-Purcell-Meiboon-Gill multiple spin-echo pulse sequence was used for the T 2 measurements.Signal intensity as function of T 2 is expressed as follows: This relationship was used to determine the T 2 values.Fig. 6 shows the T 2 values for samples with three representative particle concentrations.
The dependence of the MR image intensities on the particle concentration for T 1 and T 2 relaxations is shown in Fig. 7.The particle concentration increased as we moved from left to right.For T 1 relaxation, the MR images became brighter with increasing particle concentration.On the other hand, for T 2 relaxation, the MR images become darker with increasing particle concentration.
Relaxivity is a measure of the ability of an MRI contrast agent to increase the relaxation of the surrounding nuclear spins and can be used to improve the contrast of MR images.Relaxivity is expressed in the units of s -1 per mM of magnetic components (iron and manganese) in the liquid.The relaxivities (1/T im ) of the nuclear spins in an aqueous solution of the magnetic nanoparticles can be expressed as 44 1 where i = 1 or 2, 1/T i represents the relaxivity of the nuclear spins without the nanoparticle contrast agent, r i is the relaxivity of the nuclear spins per mM of magnetic components in the liquid, and C represents the concentration of nanoparticles in the aqueous solution.
The plots of 1/T 1 versus particle concentration is shown in Fig. 8.In this figure, the slope gives the T 1 relaxivity, which is denoted as r 1 .The value of r 1 was determined to be 1.24 s -1 mM -1 .The plots of 1/T 2 versus particle concentration is shown in Fig. 9.The value of r 2 , the T 2 relaxivity, was 60.65 s -1 mM -1 .A contrast agent with a large relaxivity can give the same contrast effect with a lower dose than that with a small relaxivity.Also, the ratio r 2 /r 1 is an indicator of the efficiency of T 2 contrast agents.A higher value of this ratio represents better T 2 contrast efficacy.The r 2 /r 1 value calculated for our particles was 48.91, which was much larger than that of previously reported particles, as shown in Table I.
For comparison, the relaxivities of the manganese-based nanoparticles and commercial contrast agents are summarized in Table I.Sana et al. 45 synthesized manganese-ferritin nanocomposites and measured the relaxivities using a 3 T MRI scanner.The r 1 and r 2 values of their particles were 10 and 74 s -1 mM -1 , respectively.Thus, the ratio r 2 /r 1 was 7.4, indicating that these particles could be applicable as T 2 contrast agents.Li et al. 25 reported ultrasmall manganese ferrite nanoparticles with r 1 and r 2 values of 6.61 and 35.92 s -1 mM -1 , respectively, at 4.7 T. The particle size of these nanoparticles was 2.2 nm.Yang et al. 46 synthesized tetraethylene glycol (TEG)-coated manganese ferrite nanoparticles with an average diameter of 7 nm.The T 2 relaxivity of the particles dispersed in water at 0.5 T was 189.3 s -1 mM -1 , while the T 1 relaxivity of the particles in the cells was 36.8 s -1 mM -1 .The value of T 2 relaxivity of the particles in water was much larger than that of other reported particles.This was because only the amount of iron was considered while calculating the relaxivity.Leal et al. 47 studied the particle size and magnetic field dependence of the relaxivities.They synthesized PEG-coated manganese ferrite nanoparticle with various diameters of 6, 7.5, 9, 12, and 14 nm and observed the T 1 and T 2 relaxivities using 1.5 T and 9 T MRI scanners.They found that both r 1 and r 2 strongly depended on the particle size and magnetic field.The largest r 2 /r 1 value was found to be 152 for the 9-nm particles using the 9 T scanner.L. Yang et al. observed effect of manganese content on magnetization and relaxivity of manganese ferrite particles.Magnetization of the particles increased as manganese content increased up to 0.43; however, magnetization decreased upon further manganese doping.They also measured relaxivity at two different fields of 0.5 and 7 T.At 7 T, T 2 relaxivity increased sharply, demonstrating the effect of high magnetic moment of manganese on T 2 relaxivity. 18In Table I, the relaxivities of iron oxide-based commercial T 2 contrast agents are also provided.Their r 2 /r 1 values were lower than 10, but this value is much larger than that of Gd-DTPA and Mn-DPDP contrast agents.Thus, commercial iron oxide-based contrast agents are primarily used as T 2 contrast agents.Comparing the relaxivity data of our manganese particles with previous results, we concluded that our particles can be used as high-efficacy T 2 contrast agents.

IV. CONCLUSION
We prepared highly stable silica-coated manganese ferrite nanoparticles and revealed their MRI contrast abilities.The biocompatible silica coating resulted in highly stable aqueous solutions of manganese ferrite nanoparticles without any precipitation for long times.The particles showed superparamagnetic behavior suitable for MRI contrast agents.The particles in the aqueous solution revealed the strong effects for accelerating the T 2 relaxation of nuclear spins, indicating that these particles are suitable as T 2 contrast agents for MRI.Furthermore, the r 2 /r 1 value for the prepared particles was much higher (49.81) than that of other reported nanoparticles.These results confirmed that our silica-coated manganese ferrite nanoparticles could be used as high-efficacy T 2 contrast agents.

FIG. 4 .
FIG. 4. Magnetic moments of bare and silica-coated manganese ferrite nanoparticles as a function of the applied magnetic field.The particles exhibit superparamagnetic behavior with negligible coercive force.

FIG. 6 .
FIG.6.T 2 relaxation for three representative samples.It can be observed that T 2 relaxation is faster for the sample with a larger particle concentration.

FIG. 7 .
FIG.7.Concentration dependence of the MR image intensity at a fixed imaging time.It can be observed that both T 1 and T 2 relaxations are faster for the sample with a higher particle concentration.

FIG. 8 .
FIG.8.Plot of 1/T 1 as a function of particle concentration.The slope of the straight line gives T 1 relaxivity of the nanoparticles.

TABLE I .
Relaxivities of prepared nanoparticles and commercial MRI contrast agents.