Synthesis and characterization of PVP coated gadolinium oxide nanoparticles for imaging applications

. In this work, we present the synthesis and applications of Gd 2 O 3 @PVP nanoparticles as an efficient contrast agent for MRI and CT techniques. Gd 2 O 3 @PVP nanoparticles have been successfully synthesized by the polyol method using ethylene glycol and poly(vinylpyrrolidone) as solvent and surfactant, respectively. The structure, morphology and characteristic properties of the materials are thoroughly investigated by SEM, TEM, UV-Vis, XRD, FTIR and DLS measurements. As an important result, NPs synthetized under optimized conditions have a diameter in the range of 12 nm and exhibit a good contrast signal in magnetic resonance imaging and computed tomography at relatively low concentration ([NPs] = 0.1 mM for MRI and 1.25 mg.mL -1 for CT). In particular, the concentration of Gd 2 O 3 @PVP nanoparticles used in CT is 10 times lower than that of the commercial Iobitridol product (i.e., 12


INTRODUCTION
Among different medical imaging techniques, Magnetic Resonance Imaging (MRI) appears as one of the best candidates to visualize bio-target inside human body by means of magnetic relaxation of proton measurement.However, one of the drawbacks of this technique is the low kinetic of magnetic relaxation process that could lead to a burden on patients.Thus, an urgent need in accelerating the magnetic relaxation is mandatory which allows to shorten the diagnosis time and to increase the applicability of the MRI [1].Accordingly, contrast agents possessing large spin magnetic moments (e.g.7.9 µB for Gd 3+) have attracted widespread attention because their strong magnetic interaction with protons in the human body leads to the significant acceleration of the magnetic relaxation of these protons, thus defines and increases the magnetic contrast [2].Commonly used contrast agents are based on gadolinium (Gd) complexes with linear or cyclic structures such as gadopentetate (trade name Magnevist), gadobenate (Multihance), gadodiamide (Omniscan), gadoteridole (Prohance) or gadobutrol (Gadovist).These are paramagnetic substances with small sizes, so they have the effect of increasing the speed of R 1 vertical recovery.The biggest disadvantage of Gd 3+ complexes is the release of toxic Gd 3+ ions due to weak chelates binding between Gd 3+ centers and surrounding organic particles.In addition, the number of paramagnetic centers in each complex molecule is limited (usually 1 Gd 3+ ion/complex molecule), in order to increase contrast, the amount of sample used will have a rather high concentration [3 -5].
Recently, Gd 2 O 3 nanoparticles have been of particular interest, for example in MRI application research due to their ability to improve positive contrast and less toxicity compared to the complex state.Possibly, this is because the number of Gd 3+ paramagnetic centers (the positive contrast factor) per unit mass of the nanosized Gd 2 O 3 (Gd content is about 60 %) is much larger than in the complex form (Gd accounts for about 10 -16 %).Studies also show that nano-sized Gd 2 O 3 is more chemically stable than Gd 3+ complexes [5].
The research has initially been successfully implemented; the positive contrast material system for MRI imaging has a large R 1 vertical recovery rate.It is biocompatible, stable in physiological environment.The practical applications based on the combination of Gd 2 O 3 nanomaterials and biomedical molecules will be possibly potential.The obtained results are not only limited to basic research in the field of diagnostic imaging, such as MRI, computed tomorgraphy (CT), but also become a premise for realizing the application of nanotechnology in treatment (leading to drug delivery, thermotherapy) and diagnostic imaging [6][7][8].
Currently, there are many methods of synthesizing nanoparticles in general and Gd 2 O 3 in particular.They are co-precipitation, microemulsion, polyol and thermal decomposition methods in organic solvents.Each method has advantages and disadvantages [7 -9].Among them, the polyol method is a relatively effective approach to synthesize nanoparticles with sizes from 2 to 15 nm, in which the formation and size growth of nanoparticles is easily controlled by controlling the factors affecting the chemical reaction such as temperature and reaction time [10][11][12].Besides, the solvents commonly used in the polyol method are multifunctional alcohols such as ethylene glycol, diethylene glycol or polyethylene glycol which in some cases act as metal ion reducing agents.At the same time, they also act as the surface coating to protect the nanoparticles from agglomeration and prevent the formation of hydroxides.
In this study, polyol method is used to study the fabrication of Gd 2 O 3 nanoparticles for MRI and CT imaging applications.The obtained Gd 2 O 3 nanoparticle surfaces were then encapsulated with polyvinylpyrrolidone (PVP) polymer as a bio-responsive shell.This hydrophobic encapsulation controls surface charge and cytocompatibility of NPs in vivo.PVP is known as an inert, non-poisonous, heat-resistant, stable to pH, biocompatible and biodegradable polymer.It contains a hydrophilic component (pyrrolidone base) and a significantly hydrophobic group (alkyl group).There are highly polar amide groups in the pyrrolidone ring, apolar methylene and methane groups along its backbone.It is well known to be a stabilizer to prevent the aggregation of nanoparticles because of steric hindrance effect.The characterizations of the Gd 2 O 3 @PVP sample such as size, morphology and surface properties were investigated using scanning electron microscopy (SEM), transmission electron microscopy (TEM), X-ray diffraction (XRD), Fourier-transform infrared spectroscopy (FT-IR), dynamic light scattering (DLS) and Zeta potential measurements.The ability to increase contrast of nanofluids obtained in MRI and CT imaging techniques is investigated.

Preparation of Gd 2 O 3 @PVP nanoparticles
In a round flask, 8 mmol (3.25 g) of gadolinium acetate (Gd(CH 3 CO 2 ) 3 .4H 2 O) was poured into 50 mL of ethylene glycol (EG) solvent.The mixture was rigorously stirred until the precursor was completely dissolved.Then, 10 mL of an aqueous solution containing 0.8 g of NaOH and 2.0 g of PVP was added.The mixture was heated at 140 o C for 1 hour.Then the entire product was transferred to a Teflon lined stainless steel autoclave followed by a second heating step at a fixed temperature, ranging from 150 o C to 240 o C for 4 -8 hours.Afterwards, the mixture was cooled down to room temperature and Gd 2 O 3 nanoparticles were collected by centrifugation at 10,000 rpm for 8 minutes and washed 3 -4 times with distilled water.

Characterization
The Gd 2 O 3 nanoparticle morphology was studied by Transmission Electron microscopy (TEM, JEM1010-JEOL) and Scanning Electron Microscopy (SEM, Hitachi S-4800).X-ray diffraction method was used to investigate the crystal structure and phase composition of Gd 2 O 3 nanoparticles.The UV-Vis spectra of the samples were recorded on a Jasco V-670 spectrometer.Fourier transform infrared (FT-IR) spectra were recorded using a Nicolet 6700 spectrometer.Dynamic light scattering (DLS) and zeta potential measurement were used to investigate the distribution and stability of Gd 2 O 3 nanoparticles in aqueous medium.
The clinical 1.5T MRI scanner (Siemens Magnetom, Germany) and 128-Somatom Perspective CT scanner (Siemens, Germany) were used to measure longitudinal relaxation (r 1 ) and CT images of the Gd 2 O 3 @PVP sample, respectively.

Characterization of PVP coated Gd 2 O 3 nanoparticles
The reaction temperature is an important factor affecting the shape and the size development of nanoparticles.The reactions were conducted at 150 ºC, 180 ºC, 200 ºC, and 240 ºC. Figure 1 shows the SEM images of the obtained samples.From these SEM images, it can be seen that the nanoparticles obtained by the polyol method in ethylene glycol are spherical, at 150 ºC, the obtained particles are large in size and stick together (Figure 1a).The nanoparticles become smaller at 180 o C, but the size is still quite large (Figure 1b).When the reaction temperature is increased to 200 o C, the particles are much smaller with a fairly uniform size of about 10 nm.The decrease in particle size with reaction temperature can be explained that at a high temperature, the diffusion rate of Gd 3+ ions increases leading to the formation of many small nuclei in solution and reduces the Gd 3+ source to feed up for the growth stage of nanoparticles and thus smaller nanoparticles are obtained.However, when the temperature continues to increase to 240 o C, the size of Gd 2 O 3 nanoparticles is larger, but the obtained particles still have a spherical shape with a size of about 20 -30 nm.This can be explained that for temperatures higher than 200 o C, the decomposition of Gd(CH 3 COO) 3 precursors takes place faster, leading to the formation of a large number of Gd 3+ cations in solution, and as a result, this abundant source of Gd 3+ is available for further growth of nanoparticles.The result is the formation of larger sized particles.After choosing the suitable temperature of 200 o C for the synthesis of Gd 2 O 3 nanoparticles, the reaction time is the subject of further investigation.The reaction time is varied to be 4 hours, 6 hours and 8 hours.The SEM images of the Gd 2 O 3 samples are shown in Figure 2. From the results obtained, after a reaction time of 4 hours (Figure 2a), the particle size is still large, some large clusters exist, the morphology is uneven, and these clusters seem to be complex, not yet reacted.For a reaction time of 6 hours, the nanoparticles are smaller in size (about less than 10 nm) and have a uniform spherical shape (Figure 2b).Possibly, the short reaction time (4 hours) is not long enough for a complete reaction.It can be seen that the duration of 6 hours is the optimal reaction time for the formation, division, and crystallization of Gd 2 O 3 nanoparticles at the reaction temperature of 200 o C.However, as can be seen in Figure 2c, for longer reaction time (8 hours), the particles collide with each other, then stick together to form large particles.For application as a contrast agent in MRI and CT scanning techniques, the surface of Gd 2 O 3 nanoparticles is encapsulated with polyvinylpyrrolidone (PVP) polymer as a biocompatible shell.This shell not only prevents the release of harmful Gd +3 ions into the body and regulates the interaction of water molecules with Gd +3 ions on the particle surface, but also protects the nanoparticles from the agglomeration.Figure 3 shows the TEM images and size distribution diagram of the Gd 2 O 3 nanoparticles with and without using PVP under the same synthesis conditions.From Figure 3a, it can be observed that in the absence of PVP, the Gd 2 O 3 nanoparticles are clustered together to form large clusters with a size of approximately 100 nm.In contrast, in the presence of PVP, the shape of the obtained Gd 2 O 3 nanoparticles is almost spherical.They are relatively small and highly uniform.Besides, it can be found that each Gd 2 O 3 nanoparticle has an organic shell on its surface and the average size of each Gd 2 O 3 @PVP nanoparticle is about 12.5 nm (Figures 3b, 3c).  Figure 4a shows the X-ray diffraction pattern of the synthesized Gd 2 O 3 particles.The obtained results show peaks at diffraction angles of 28.5; 32.6; 35.3; 46.2; and 56.5, corresponding to the lattice planes of (222), (400), (411), (440), and (622), respectively, in the cubic structure of Gd 2 O 3 .In addition, Figure 4b shows the absorption spectrum of Gd 2 O 3 nanoparticles at wavelengths between 200 and 500 nm.The absorption edge can be observed in the UV region at 270 nm, which is the characteristic absorption wavelength of microscopic Gd 2 O 3 nanoparticles.Besides, in the sample, there is also an impressive small peak in the absorption spectrum at position 230 nm, which is an insignificant peak of the remaining solvent.
To determine the binding capacity between solvent molecules and PVP on the nanoparticle surface, we carried out an FT-IR analysis of the Gd 2 O 3 @PVP sample.The results are shown in Figure 5a.The peaks at 2922 cm -1 and 2854 cm -1 are the absorption site of the symmetric bond and the asymmetric bond of C-H.The peak position at 3457 cm -1 of O-H shows the waterabsorbed surface of the particles.At 1568 cm -1 and 1402 cm -1 on the FT-IR spectra, there is asymmetric and symmetric contraction.The peak corresponding to the contraction of functional group (COO-) locates at 1078 cm -1 .It shows the presence of C-O bonds on the grain surface.More specifically, the infrared spectrum of PVP-coated particles has an absorption peak at position 1648 cm -1 corresponding to the amide group of PVP formed by C=O and C-N bonds.This combination leads to the absorption peak in shift relating to the absorption peak of C=O (1750 cm -1 -1700 cm -1 ).Furthermore, there is a strong absorption band at the peak position of 613 cm -1 .This peak indicates the presence of Gd-O oscillation of Gd 2 O 3 .The above analysis results are consistent with previous studies other authors on using PVP coating agents [13] on nanoparticle systems.From the position of the characteristic absorption peaks of the bonds (C-H), (C-O), (N-C=O), functional groups (COO-) appearing on the corresponding coated sample, it proves that the Gd 2 O 3 nanoparticle surface was successfully functionalized by PVP coating.Dynamic light scattering is used to analyze and evaluate the hydrodynamic size of Gd 2 O 3 @PVP nanoparticles dispersed in water, as shown in Figure 5b.The water-dispersed Gd 2 O 3 @PVP nanoparticle has an average diameter of 91.2 nm with a sharp peak and high concentration.Besides, the Zeta potential value of the Gd 2 O 3 @PVP sample is +19.5 mV with 100 % area, indicating good dispersion and stability in their aqueous environment.Figure 6 shows the results of the contrast agent for the MRI technique test applying the Gd 2 O 3 @PVP nanoparticle sample.The sample with a concentration of 0.025 mM affects the contrast signal.There is an excellent contrast even at a concentration of 0.1 mM when TR = 300 ms and at 0.05 mM when TR = 600 ms.The increased concentrations change almost negligibly because the contrast of the sample is maximum.Thus, it can be said that the Gd 2 O 3 @PVP nanoparticle used in the MRI technique could make an increase in the potential R 1 recovery.Moreover, nanoparticles from Gd 2 O 3 coated with PVP biocompatible substance are introduced into the body in a smaller amount, so it is safer.Figure 7 shows the CT images of a commercial Iobitridol sample and a Gd 2 O 3 @PVP sample.The obtained result indicates that the Gd 2 O 3 @PVP sample has better potential CT scanning applications than the commercial sample.Specifically, a concentration of 1.25 mg/mL gives a good CT signal with an intensity equal to the signal of a commercial Iobitridol sample at a concentration of 12.5 mg/mL.The signal of 5 mg/mL -Gd 2 O 3 @PVP sample is as good as that of 25 mg/mL -commercial sample.It can be seen that the small-sized spherical Gd 2 O 3 @PVP nanoparticle is superior with good X-ray dispersion and absorption.Thus, the liquid sample from Gd 2 O 3 @PVP provides a potential good contrast for CT techniques.

CONCLUSIONS
Gd 2 O 3 nanoparticles are successfully synthesized by the polyol method in the presence of EG.The obtained Gd 2 O 3 nanoparticles with a size of 12 nm are introduced into the aqueous medium by the ligand exchange method using PVP phase transition agent.Thanks to their small Gd 2 O 3 core size and hydrophilic PVP coating, the Gd 2 O 3 @PVP nanoparticles show a significant improvement in increasing the contrast in MRI and CT scans.The results of MRI and CT scans show excellent image quality, much better than the commercial complexes in use even at high concentrations.This result has implications for reducing the dose of contrast agent in vivo in both MRI and CT imaging modalities.This is also a prerequisite result for the study of dual contrast agents for multimodal imaging applications (combining MRI and CT at the same time).

Figure 2 .
Figure 2. SEM images of Gd 2 O 3 nanoparticles synthesized at a temperature of 200 o C with reaction times of 4 hours (a), 6 hours (b), and 8 hours (c).

Figure 3 .
Figure 3. TEM images of samples Gd 2 O 3 (a), Gd 2 O 3 @PVP (b) and size distribution histogram of Gd 2 O 3 @PVP at 200 o C for 6 hours.

Figure 5 .
Figure 5. FT-IR spectra (a) and DLS spectrum (b) of Gd 2 O 3 @PVP nanoparticles formed at a temperature of 200 o C and reaction time of 6 hours.