Fabricating High-Performance T2-Weighted Contrast Agents via

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Fabricating High Performance T2-Weighted Contrast Agent via Adjusting Composition and Size of Nanomagnetic Iron Oxide Jianmin Xiao, Guilong Zhang, Junchao Qian, Xiao Sun, Jie Tian, Kai Zhong, Dongqing Cai, and Zhengyan Wu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b00428 • Publication Date (Web): 02 Feb 2018 Downloaded from http://pubs.acs.org on February 2, 2018

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ACS Applied Materials & Interfaces

Fabricating High Performance T2-Weighted Contrast Agent via Adjusting Composition and Size of Nanomagnetic Iron Oxide

Jianmin Xiao,||,†,‡ Guilong Zhang,||,† Junchao Qian,||,┴ Xiao Sun,† Jie Tian,§ Kai Zhong,*,† Dongqing Cai,*,† and Zhengyan Wu*,†

†

Key Laboratory of High Magnetic Field and Ion Beam Physical Biology, Hefei

Institutes of Physical Science, Chinese Academy of Sciences, Hefei 230031, People’s Republic of China ‡

University of Science and Technology of China, Hefei 230026, People’s Republic of

China ┴

Hefei Cancer Hospital, Hefei Institutes of Physical Science, Chinese Academy of

Sciences, Hefei 230031, People’s Republic of China §

Material Test and Analysis Lab, Engineering and Materials Science Experiment

Center, University of Science and Technology of China, Hefei 230026, People’s Republic of China

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ABSTRACT: Magnetic relaxation switch (MRS) demonstrated that the aggregated nanomagnetic iron oxide (NMIO) nanocrystal possessed a lower T2 value and better relaxivity compared with monodispersed NMIO nanocrystal. However, we found that NMIO nanocluster (NMIONC) showed a different MRI property in comparison with NMIO nanocrystal. Herein, three types of NMIONC were used to explore the effects of size and compositions on the variations of magnetism and MR contrast ability. It was found that the transverse relaxation rate (r2) of NMIONC depended on the contact area between particles and water molecules. The smaller size and higher solubility could carry out higher contact area between NMIONC and water molecules. Therefore, the monodispersed NMIONC showed a better T2 contrast ability in comparison with the aggregated NMIONC. In addition, for NMIONC with the same composition, the magnetism and contrast ability gradually increased with the particle size decreasing. In vivo, NMIONC that possessed the best solubility and the smallest size showed the most effective MR contrast effect for the liver region of mice. As a result, the size and composition of NMIONC played important roles for enhancing contrast behavior. This study provides a new idea to develop high-performance T2 contrast agents (CAs) by modulating the size and composition of particles. KEYWORDS: Magnetic relaxation switch, Nanomagnetic iron oxide, T2 contrast agent, size, compositions, magnetic behavior

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INTRODUCTION Nanomagnetic iron oxide (NMIO) has been widely explored for biomedical applications thanks to the outstanding balance among magnetic property, easily functionalized surface, and good biocompatibility.1-6 The superparamagnetic state of NMIO ensures no remanent moment and coercive force in the absence of an external magnetic field, which avoids the risk of uncontrolled aggregation in vivo. Currently, NMIO has been utilized as magnetic resonance contrast agent (MRCA) because NMIO can shorten the transverse relaxation time (T2) of water protons in MR imaging process and help to obtain T2-weighted contrast enhancement and high-quality image.7-9 In addition, some studies found that the monodispersed NMIO nanocrystals exhibited higher T2 relaxation time and lower relaxation rate in comparison with the aggregated NMIO nanocrystals.10-14 Therefore, NMIO nanocrystals could often act as a magnetic relaxation switch (MRS) and be used to detect molecular interactions. However, there are few reports about NMIO nanocluster (NMIONC) as MRS and MRCA because the factors that affect T2 relaxation signal of NMIONC are still not clear. Therefore, it is necessary to make contrast behavior clear via studying physical parameters of NMIONC, and this will be beneficial to designing Fe-based CA and MRS with high quality. The

synthetic methods

of

NMIONC include co-precipitation, so-gel,

micro-emulsion, and solvothermal synthesis.15-19 Except for the solvothermal synthesis, NMIONC fabricated by the above mentioned methods shows some unexpected shortcomings: (1) uncontrolled particle size; (2) poor dispersion in the 3



solution; (3) few functional groups, which directly limits the development of NMIONC as T2-weighted CA.20 In addition, for better clinical applications, these NMIONCs need further surface modification such as coating with various polymers including polyacrylic acid (PAA), Poly(dopamine), citric acid (CA), poly(γ-glutamic acid) (PGA), poly(vinyl alcohol) (PVA), polyethyleneimine (PEI) to obtain the desired functions.21-26 However, most of the employed strategies usually involve multiple steps of chemical reaction, which are costly and time-consuming. To avoid this, one-pot synthesis of NMIONC with active groups can be realized via solvothermal reaction, and shows some advantages (i.e. adjustable particle size, numerous functional groups, high purity, and good aqueous solubility of the products).27 In this work, we synthesized three types of NMIONC (PVP@NMIONC, PEI@NMIONC, and PAA@NMIONC) with varying sizes by solvent-thermal reaction, and investigated their crystal structures, magnetic properties, and T2-weighted contrast abilities (Scheme 1). It could be seen that saturation magnetizations and relaxivities of NMIONC with the same composition gradually increased with the size decreasing. Moreover, the magnetic behavior of NMIONC began to transform from ferromagnetism to paramagnetism with the size decreasing, which would be beneficial for contrast enhancement. In addition, the compositions of NMIONC could significantly improve the solubility of particles (PAA@NMIONC > PEI@NMIONC > PVP@NMIONC), and then increase the contact area between NMIONC and water molecules, so that T2 contrast ability of NMIONC was 4


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dramatically enhanced. In vivo, NMIONC with the best solubility and the smallest size showed the best MR contrast effect for the liver tissue of mice than other CAs. Therefore, the monodispersed NMIONC possessed a better contrast ability (low T2 value and high relaxation rate) compared with the aggregated NMIONC, which meant the MRI properties of NMIONC were different from that of NMIO nanocrystals. EXPERIMENT SECTION Materials. All chemical reagents were used as received without further purification. Fe(acac)3 (98%) and polyethyleneimine (99%) were provided by Aladdin Chemical CO. Ltd. (Shanghai, China). FeCl3·6H2O (99%), ethylene glycol (EG, 99%), diethylene glycol (DEG, 99%), anhydrous ethanol (99.9%), trithanolamine (TEA, 99.9%), and anhydrous sodium acetate (99.7%) were purchased from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). Sodium acrylate (98%) was purchased from Nanjing Chemlin Chemical Industry Co. Ltd. In addition, polyvinylpyrrolidone (PVP-K30, 99.9%) was provided by Fluka-Solarbio Co. (Beijing, China). Synthesis

of

PVP@NMIONC,

PEI@NMIONC,

and

PAA@NMIONC.

PEI@NMIONC could be fabricated via the following method: Fe(acac)3 (0.5 g) was dissolved into the mixed solution (50 mL) of EG and DEG with different ratios for 30 min at 80oC. Next, PEI (1.5 g) was added into the resulting solution to form a brown turbid solution for 30 min. Afterward, TEA (3 mL) was added under stirring to form a transparent wine-red solution. The resulting solution was then transferred to an autoclave and kept at 200oC for 12 h. Finally, the black product was collected and washed at least three times with distilled water and alcohol, respectively. The size of 5



PEI@NMIONC could be adjusted via varying the ratios of EG to DEG, and then PEI@NMIONC fabricated by 5:0, 3:2, and 1:4 (EG to DEG) were designated as PEI@NMIONC-1, PEI@NMIONC-2, and PEI@NMIONC-3, respectively. PVP@NMIONC could also be prepared via the similar method as follows: FeCl3·6H2O (0.5 g) was dissolved into the mixed solution (50 mL) of EG and DEG with different ratios for 30 min at 80oC. Next, PVP (1.5 g) was added into the resulting solution for 30 min. Subsequently, sodium acetate (2 g) was added under stirring for 45 min. The resulting solution was then transferred to an autoclave and kept at 200oC for 24 h. Finally, the black product was collected and washed using the same method. Similarly, the size of PVP@NMIONC could be adjusted via varying the ratios of EG to DEG, and then PVP@NMIONCs fabricated by 5:0, 3:2, and 1:4 (EG to DEG) were designated as PVP@NMIONC-1, PVP@NMIONC-2, and PVP@NMIONC-3, respectively. Finally, PAA@NMIONC was synthesized via the similar method as follows: Fe(acac)3 (0.5 g) was dissolved into the mixed solution (50 mL) of EG and DEG with different ratios for 30 min at 80oC. Next, sodium acrylate (1.5 g) was added into the resulting solution for 30 min. Subsequently, sodium acetate (2 g) was added under stirring for 45 min. The resulting solution was then transferred to an autoclave and kept at 200oC for 24 h. Finally, the black product was collected and washed as the same method. PAA@NMIONCs fabricated by 5:0, 3:2, and 1:4 (EG to DEG) were designated as PAA@NMIONC-1, PAA@NMIONC-2, and PAA@NMIONC-3, respectively. 6


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MR Experiment. MR studies were performed on a 9.4 T/400 mm wide bore scanner (Agilent Technologies, Inc., Santa Clara, CA, USA), using a volume radiofrequency fields coil. In vitro MR experiments, the scanning procedure began with a localizer and then a series of spin echo images were acquired for transverse relaxation time (T2) measurement, identical in all aspects (repetition time (TR) 2500 ms, effective echo time (TE) 5.6 ms, band width (BW) 25 kHz, slice thickness 1 mm, matrix 96×96, 3 average) except for 20 echoes time (TI) which was varied linearly from 10 to 2500 ms. Signal intensity (SI) versus TI relationships were fit to the following exponential T2 decay model by nonlinear least squares regression: SI(TI) =A1*exp(-TI/T2)+SI(0). The T2 relaxivity (r2) was determined by a linear fit of the inverse relaxation time (1/T2) as a function of the iron concentration which was determined using an ICP-MS (7200plus, Thermo Fisher Scientific Co., USA). For in vivo MR studies, the mice used for the experiment were treated in accordance with the Ethics Committee Guidelines in University of Science and Technology of China. Firstly, the mice with the similar weight (20-22 g) were conducted on MR scanning to precisely evaluate the T2 contrast ability of synthetic NMIONC in vivo. Subsequently, three types of NMIONC (PEI@NMIONC, PVP@NMIONC, and PAA@NMIONC) were intravenously injected into mice with a dose of 2 mg(NMIONC)/kg(body weight). T2-weighted images of mice were obtained by 9.4 T MRI scanner at post-injection 15 min, 30 min, and 60 min. The imaging parameters were set as follows: fast spin echo, TR = 400.0 ms, TE = 10.0 ms, field of View (FOV) = 40 mm × 40 mm and matrix size = 128 × 128, slice thickness = 1 mm 7



(12 slices, gap = 0), 1 average, and BW = 50 kHz. Characterization. The morphologies and microstructure were observed using a transmission electron microscope (TEM) (JEM-ARM200F, JEOL Co., Japan). The crystal structure was observed via an X-ray diffractometer (XRD) (TTR-III, Rigaku Co., Japan). Particle size distribution measurements were conducted on a dynamic light scattering (DLS) detector (Nanotrac Wave II, Microtrac Co. USA). The structure and composition could be confirmed using an FT-IR spectrometer (iS10, Nicolet Co., USA). RESULTS AND DISCUSSION Three types of NMIONC could be synthesized via the solvent-thermal decomposition of ferric acetylacetonate or ferric chloride under the mixture of EG and DEG containing PEI, PVP, or PAA, respectively, and the size of NMIONC could be adjusted via varying the ratios of EG to DEG. Notably, PEI@NMIONCs were fabricated through a 12 h reaction, while PVP@NMIONCs and PAA@NMIONCs were prepared through a 24 h reaction. This was because PEI@NMIONCs fabricated through the 12 h reaction showed the similar physicochemical properties in comparison with the 24 h reaction according to XRD, TG, TEM, and magnetism analyses (Figure S1). However, PVP@NMIONCs and PAA@NMIONCs displayed plenty of tiny crystals and did not form exact spherical shape as shown in Figure S2, so it was hard to explore the size effect. Thereby, considering experimental efficiency, the reaction time of 12 h was selected for the preparation of PEI@NMIONC, and the reaction time of 24 h was used to synthesize PVP@NMIONC and PAA@NMIONC. 8


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The size and structure of NMIONC were analyzed as shown in Figure 1. Three types of NMIONC showed uniform spherical shape, and the size of NMIONC increased with the amount of EG in the solvent increasing. Compared to the same type of NMIONC, PVP@NMIONC-1, PEI@NMIONC-1, and PAA@NMIONC-1 showed the largest size, and PVP@NMIONC-3, PEI@NMIONC-3, and PAA@NMIONC-3 showed the smallest size. In addition, it could be seen that PVP@NMIONC-1 and PVP@NMIONC-2 displayed serious agglomeration, while PAA@NMIONC-3 and PEI@NMIONC-3 possessed good dispersion. Interestingly, for PAA@NMIONC, it could be seen that the spherical shape gradually became loose and even split with the size decreasing, implying that PAA@NMIONC-3 possessed a higher specific surface area than other NMIONC. Dynamic laser scattering (DLS) was employed to analyze the hydrodynamic diameter of NMIONC in order to illustrate the existence form of particles in aqueous solution (Figure 2a). It could be seen that the narrowed curves were shown in all of NMIONC samples, indicating that NMIONC could be dispersed in the solution. However, it could be found that the average hydrodynamic size of PVP@NMIONC was much higher than the size shown in TEM image (Figure 2b, insets of Figure 1), indicating that PVP@NMIONC was not stable and easily agglomerated in the solution. Subsequently, the crystal structures of NMIONC samples were analyzed by XRD, and the results exhibited the diffraction peaks assigned to the spinel structure of magnetite (Figure 2c and d), corresponding to Fe3O4 (JCPDS NO. 89-0691). In addition, it could be seen from samples 1 to 3 that the peak width of NMIONCs 9



gradually increased, while the strength of peak became weakened, which could be attributed to smaller size and lower crystalline degree. These results were consistent with those of TEM and DLS analysis. The compositions of NMIONC samples were investigated via FT-IR spectra. As shown in Figure 2d, in all of curves, the peak at 584 cm−1 was attributed to the typical Fe-O stretching vibration. In addition, PAA@NMIONC showed an apparent carboxyl (-COOH) peak at 1721 cm-1, indicating that particles contained abundant of PAA chains. For PEI@NMIONC, the peaks observed at 1075, 1631, 2920, and 3421 cm-1 were assigned to -C-N stretching vibration, -N-H bending vibration, -C-H and -N-H stretching vibration, respectively, suggesting that PEI chains were successfully decorated into NMIONC. For PVP@NMIONC, the peaks at 1048, 1645, and 2922 cm-1 could be respectively ascribed to -C-N stretching vibration, -C=O stretching vibration, -C-H stretching vibration, indicating that PVP was successfully introduced into NMIONC. According to above analysis, it could be seen that PAA@NMIONC and PEI@NMIONC possessed plenty of hydrophilic groups because of the existence of carboxyl and amino group, which would be beneficial to enhance the dispersion and stability of NMIONC in aqueous solution. These results implied that PAA@NMIONC and PEI@NMIONC might possess a higher contact area between NMIONC and water molecules than PVP@NMIONC. It was known that the solubility of NMIONC depended on the species and contents of hydrophilic organic substance. Therefore, it was necessary to determine the content of organic substance coated on the NMIONC. As shown in Figure S3, NMIONC with the same 10


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composition showed the similar organic content, indicating that NMIONC with the same composition possessed the similar solubility. Subsequently, single NMIONC was observed via high-magnification TEM images (Figure 3). Interestingly, it was found that three types of NMIONCs consisted of a cluster of numerous iron oxide tiny crystals, indicating that fabrication of NMIONC was a self-assembly process. Additionally, tiny crystals in PVP@NMIONC gradually became small with the decreasing size of PVP@NMIONC, but tiny crystals in PEI@NMIONC and PAA@NMIONC showed no obvious change in size. Moreover, the dominant size distribution of tiny crystals in PAA@NMIONC and PEI@NMIONC was about 4-7 nm, but the size of tiny crystals in PVP@NMIONC-1 to PVP@NMIONC-3 varied from 2-3 nm to 15-20 nm, respectively. Specifically, compared to PVP@NMIONC and PEI@NMIONC, the structure of PAA@NMIONC became loose with the size decreasing, which would be beneficial to enhance the contact area between NMIONCs and water molecules. The magnetic performance of three types of NMIONCs was investigated by a superconducting

quantum

interference

device

(SQUID).

Field-dependent

magnetization (M-H) curves indicated that NMIONCs showed different magnetic properties with the composition and size varying, and saturation magnetization moments (Ms) ranged from 52.0 to 96.8 emu/g at 300 K (Figure 4). Moreover, all of NMIONC samples had no coercive force and remanence at 300 K, indicating that the prepared NMIONCs were soft ferromagnetism or superparamagnetism. Ms values of PEI@NMIONC (samples 1 to 3) were 59.1, 62.5, and 77.7 emu/g, respectively. 11



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Meanwhile, Ms values of PVP@NMIONC (samples 1 to 3) and PAA@NMIONC (samples 1 to 3) were 67.5, 71.8, 73.6, 52.0, 85.3, and 96.8 emu/g, respectively. It was noted that Ms of NMIONC with the same composition gradually increased with the size decreasing, which was different from previous reports. This might be because the arranged direction of magnetic domain for tiny crystals in NMIONC became more ordered with the size decreasing under an external magnetic field. Meanwhile, zero-field cooling (ZFC) and field cooling (FC) measurements exhibited that all curves of NMIONC samples overlapped at high temperature, but split at low temperature (Figure 5). Moreover, the blocking temperatures of PEI@NMIONC and PAA@NMIONC gradually decreased with the size decreasing, indicating that magnetic behavior of PEI@NMIONC and PAA@NMIONC began to transform from ferromagnetism to superparamagnetism at room temperature. The blocking

temperatures

of

PEI@NMIONC-3,

PAA@NMIONC-2,

and

PAA@NMIONC-3 were respectively 302, 284, and 251 K, indicating that these particles possessed fine superparamagnetic behavior at physiological temperature. Superparamagnetic NMIONC would be beneficial for contrast enhancement because superparamagnetic nanocluster could enhance the local inhomogeneities of water proton and then lead to a reduction of the transverse relaxation time (T2).28 Within the measured temperature range (2-400 K), the curves of PVP@NMIONC samples still split, indicating that all of PVP@NMIONC samples were typical ferromagnetism. Based on preceding analysis, it could be speculated that NMIONC with superparamagnetic

behavior

(PEI@NMIONC-3, 12


PAA@NMIONC-2,

and

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PAA@NMIONC-3) possessed a better T2 contrast ability than ferromagnetic particles (PVP@NMIONC

samples,

PAA@NMIONC-1,

PEI@NMIONC-1,

and

PEI@NMIONC-2). The contrast ability using MR tubes with different concentrations of NMIONC was then investigated, providing directly visual evidence by distinguishing darkness (T2) of the images (Figure 6c). Both PAA@NMIONC and PEI@NMIONC had a significant contrast enhancement in T2-weighted imaging with the concentrations increasing. However, PVP@NMIONC showed a weak contrast effect, indicating that the contrast ability of PVP@NMIONC was poor. In addition, the corresponding transverse relaxivity (r2) values of PAA@NMIONC, PEI@NMIONC, and PVP@NMIONC were calculated via the ratio of proton relaxation (1/T2) to iron concentrations (Figure 6a). The r2 values of PEI@NMIONC (samples 1 to 3), PVP@NMIONC (samples 1 to 3), and PAA@NMIONC (samples 1 to 3) were 202.5, 343.3, 542.1, 132.6, 143.3, 155.6, 180.7, 409.3, and 663.6 mM-1s-1, respectively. The difference of relaxivities could be attributed to the fact that the hydrophilic groups on the surface of PEI@NMIONC (amino group) and PAA@NMIONC (carboxyl group) significantly improved the solubility of particles in aqueous solution and could enhance the contact area between NMIONCs and water molecules, so that PAA@NMIONC and PEI@NMIONC possessed higher relaxivities and better MR signal

enhancement

than

PVP@NMIONC.

Moreover,

NMIONC

with

superparamagnetism exhibited the higher relaxivities than the particles with ferromagnetism, indicating that magnetic behavior also played an important role to 13



enhance T2 contrast ability. In addition, it could be seen in Figure 6b that saturation magnetization of NMIONC with the same composition was proportional to T2 relaxivity. According to the reported theory,29 saturation magnetization played a key role for enhancing transverse relaxation, and a larger Ms led to a higher r2 value. Therefore, T2 contrast abilities of NMIONC showed a similar trend to their saturation magnetizations, which was consistent with the results obtained in this work. In addition, the opposite trend was observed for the size of NMIONC, a smaller size brought out a higher r2 value, and the relation among the relaxivity, single particle size, hydrodynamic diameter, and magnetism of NMIONC was listed in the Table S1. The reason might be because the same type of NMIONC with smaller size possessed better superparamagnetic property and higher contact area with water molecules. According to the Solomon-Bloembergen theory,30 the relaxation rate was related to the particle properties including the size, composition, and so on. Additionally, it could be found in Figure S4 and S5 that the relaxation rate of PVP@NMIONC dramatically decreased with standing time increasing, which could be attributed to the precipitation and aggregation of the particles. Therefore, the r2 values reported for PVP@NMIONC might be underestimated during T2 measurements. For example, determining T2 relaxation time cost more than 15 minutes per sample, but the stable time of PVP@NMIONCs in the solution was only 10 min, causing PVP@NMIONCs precipitated in the solution. In a word, it was found that T2-weighted contrast ability of NMIONC relied heavily on both contact area and magnetic forces acting on the NMIONC. In addition, the size and composition of NMIONC could effectively adjust 14


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magnetic behavior and the contact area between NMIONCs and water molecules, so that T2 contrast ability of NMIONC was optimized. Before in vivo experiment, the stability of three types of NMIONCs in 5% FBS was investigated (Figure S6 and S7). The results showed that NMIONC possessed excellent dispersibility, and no precipitation appeared. Subsequently, it could be observed in TEM images that NMIONCs still kept a spherical shape, and did not disintegrate into small single particles. These results suggested that these NMIONCs possessed a good stability in FBS. Meanwhile, the pharmacokinetic of NMIONCs was investigated via detecting the compositions in urine and feces. As shown in Figure

S8,

the

excrements

of

mice

injected

with

PVP@NMIONC-3,

PEI@NMIONC-3, and PAA@NMIONC-3 at 48 h possessed the high Fe content, and the excretion Fe amounts of mice were 459 µg for PVP@NMIONC-3, 422 µg for PEI@NMIONC-3, and 243 µg for PAA@NMIONC-3. The results suggested that these NMIONCs could be effectively excreted out from body. To estimate the contrast abilities of NMIONC samples in vivo, the mice injected by the different types of NMIONCs were observed by MRI. It has been demonstrated previously that accumulation of particles easily occurs in liver because of the uptake of mononuclear phagocyte systems (MPS) in liver.29 Therefore, the liver of mice was chosen as the region of interest and performed T2-weighted MR imaging. We investigated the T2 imaging of liver both pre- and post-injection of different types of NMIONCs at a dose of 2.0 mg(NMIONC)/kg(mouse body weight) through tail vein injection. As shown in Figure 7a, the significant signal attenuation could be observed 15



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for all groups at 15, 30, and 60 min post-injection of transverse plane in the liver region. The liver images of the mice injected by PVP@NMIONC-1 slightly darkened at 15 min post-injection, reached maximum darkness at 30 min, and then showed a brighter image at 60 min than that at 30 min, indicating that PVP@NMIONC-1 particle began to be excreted from the body at 60 min post-injection. Moreover, the same trend was observed in liver of mice treated with PVP@NMIONC-2 and PVP@NMIONC-3. In addition, the liver image of the mice treated with PEI@NMIONC-1 and PEI@NMIONC-2 displayed relatively strong dark signals at 15 min post-injection, and reached maximum signal intensity at 30 min. However, for PEI@NMIONC-3, the liver region image quickly darkened at 15 min post-injection, and then brightened after 30 min, indicating that PEI@NMIONC-3 could be quickly cleared. This might be because the particles with small size easily penetrated into various organs and could be rapidly excreted out of the body via the glomerular filtration. For PAA@NMIONC samples, obvious dark signals were obtained in liver region, indicating PAA@NMIONC possessed good T2 contrast ability. Especially, the strongest dark signal in liver of the mice treated with PAA@NMIONC-3 was observed at 15 min post-injection. However, after 30 min post-injection, the liver signal

began

to

slightly

brighten

compared

to

15

min,

implying

that

PAA@NMIONC-3 was also gradually cleared from body. These results demonstrated that PAA@NMIONC had a fast contrast-enhanced effect for the liver, which could provide more useful information for accurate diagnosis of liver disease. According to above analysis, it could be concluded that the size of NMIONCs played an important 16


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role for bio-distribution and blood circulation in vivo. Subsequently, we quantitatively analyzed the signal changes in liver region of mice treated with different types of NMIONCs. Signal to noise ratio changes (∆SNR) could be calculated by the equation (∆SNR = |SNRpost-SNRpre|/SNRpre). As shown in Figure 7b, the ∆SNR values (%) of PEI@NMIONC (samples 1 to 3), PVP@NMIONC (samples 1 to 3), and PAA@NMIONC (samples 1 to 3) at 15 min post-injection are 26.6±3.6, 36.1±4.2, 45.2±5.5, 22.3±2.3, 21.9±3.4, 32.1±4.1, 12.5±2.1, 49.5±5.7, and 56.2±6.1, respectively. PAA@NMIONC-3 particles presented the highest contrast enhancement effects, and the maximal ∆SNR was up to 56.2 ± 6.1% for transverse plane, which was ~2.1 times higher than that of PEI@NMIONC-1, ~2.5 times higher than that of PVP@NMIONC-1, and ~4.5 times higher

than

that

of

PAA@NMIONC-1.

These

results

suggested

that

PAA@NMIONC-3 possessed the best contrast ability in vivo and could have great potential as an excellent T2 contrast agent (CA) for sensitive MR imaging. Moreover, the high T2 contrast ability in vivo meant a low injection dose, indicating low cost, less side effects, and better clinical application. Besides, for PEI@NMIONC-3, PAA@NMIONC-2, and PAA@NMIONC-3, the ∆SNR started to decrease at 30 min post-injection, further confirming that the retention time of NMIONC in vivo could be adjusted via varying particle size. CONCLUSIONS In summary, by adjusting the size and compositions of NMIONC, we revealed the key roles for enhancing magnetic properties and T2-weighted contrast abilities. 17



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The saturation magnetizations and T2 relaxivities of NMIONC with the same composition gradually increased with the size decreasing. In addition, the magnetic behavior of NMIONCs began to gradually transform from a typical ferromagnetism to superparamagnetism with the size decreasing, which conferred them strong T2 contrast

ability.

Besides,

PEI@NMIONC-3,

PAA@NMIONC-2,

and

PAA@NMIONC-3 showed obvious superparamagnetic behavior at physiological temperature, and possessed excellent T2 contrast ability. In addition, the compositions of NMIONC could significantly affect the solubility of particles (PAA@NMIONC > PEI@NMIONC > PVP@NMIONC), and then improved their T2 contrast ability because of the varying contact area between NMIONC and water molecules. Systemic delivery of NMIONCs with different sizes and compositions significantly enhanced the T2-weighted contrast effect of liver, which would be beneficial for accurate imaging. Specially, PAA@NMIONC-3 showed the highest T2 relaxivity and the best signal sensitivity for liver imaging in vivo than other NMIONC CAs. Therefore, this work may provide a strategy for the development of high-performance T2 CA and MRS by adjusting the size and composition of particles.

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ASSOCIATED CONTENT Supporting Information: The Supporting Information is available free of charge on the ACS Publications website. TG curves data; hydrodynamic size distribution data; transverse relaxation rates data; particle photograph observation; TEM; biometabolism data; the list of particles size, magnetism, and T2 relaxation rate. AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected] (K.Z.), [email protected] (D.C.), [email protected] (Z.W.). Author Contributions ||

J.X., G.Z., and J.Q. are co-first authors.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS The authors acknowledge financial support from the National Natural Science Foundation of China (No. 21407151), the Youth Innovation Promotion Association of Chinese Academy of Sciences (No. 2015385), the Key Program of Chinese Academy of Sciences (No. KSZD-EW-Z-022-05), the Science and Technology Service Programs of Chinese Academy of Sciences (Nos. KFJ-STS-ZDTP-002 and KFJ-SW-STS-143), and the Science and Technology Major Project of Anhui Province 19



(No. 17030701051).

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REFERENCES: (1) Gupta, A. K.; Gupta, M. Synthesis and Surface Engineering of Iron Oxide Nanoparticles for Biomedical Applications. Biomaterials 2005, 26, 3995-4021. (2) Wu, W.; He, Q.; Jiang, C. Magnetic Iron Oxide Nanoparticles: Synthesis and Surface Functionalization Strategies. Nanoscale Res. Lett. 2008, 3, 397-415. (3) Figuerola, A.; Corato, R. D.; Manna, L.; Pellegrino, T. From Iron Oxide Nanoparticles Towards Advanced Iron-based Inorganic Materials Designed for Biomedical Applications, Pharmacol. Pharmacol. Res. 2010, 62, 126-143. (4) Beeran, A. E.; Nazeer, S. S.; Fernandez, F. B.; Muvvala, K. S.; Wunderlich, W.; Anil, S.; Vellappally, S.; Rao, M. S.; John, A.; Jayasree, R. S.; Varma, P. R. An Aqueous Method for The Controlled Manganese (Mn2+) Substitution in Superparamagnetic Iron Oxide Nanoparticles for Contrast Enhancement in MRI. Phys. Chem. Chem. Phys. 2015, 17, 4609-4619. (5) Zhang, G. L.; Du, R. H.; Zhang, L. L.; Cai, D. Q.; Sun, X.; Zhou, Y.; Zhou, J.; Qian, J. C.; Zhong, K.; Zheng, K.; Kaigler, D.; Liu, W. Q.; Zhang, X.; Zou, D. H.; Wu, Z. Y. Gadolinium-Doped Iron Oxide Nanoprobe as Multifunctional Bioimaging Agent and Drug Delivery System. Adv. Funct. Mater. 2015, 25, 6101-6111. (6) Cheng, Z. Y.; Dai, Y. L.; Kang, X. J.; Li, C. X.; Huang, S. S.; Lian, H. Z.; Ma, P. G.; Lin, J. Gelatin-Encapsulated Iron Oxide Nanoparticles for Platinum (IV) Prodrug Delivery, Enzyme-Stimulated Release and MRI. Biomaterials 2014, 35, 6359-6368. (7) Chertok, B.; Moffat, B. A.; David, A. E.; Yu, F.; Bergemann, C.; Ross, B. D.; Yang, V. C. Iron Oxide Nanoparticles as A Drug Delivery Vehicle for MRI Monitored 21



Magnetic Targeting of Brain Tumors. Biomaterials 2008, 29, 487-496. (8) Qiao, R. R.; Yang, C. H.; Gao, M. Y. Superparamagnetic Iron Oxide Nanoparticles: from Preparations to in Vivo MRI Applications. J. Mater. Chem. 2009, 19, 6274-6293. (9) Materia, M. E.; Guardia, P.; Sathya, A.; Leal, M. P.; Marotta, R.; Corato, R. D.; Pellegrino, T. Mesoscale Assemblies of Iron Oxide Nanocubes as Heat Mediators and Image Contrast Agents. Langmuir 2015, 31, 808-816. (10) Kim, G. Y.; Josephson, L.; Langer, R.; Cima, M. J. Magnetic Relaxation Switch Detection of Human Chorionic Gonadotrophin. Bioconjugate Chem. 2007, 18, 2024-2028. (11) Zhao, M.; Josephson, L.; Tang, Y.; Weissleder, R. Magnetic Sensors for Protease Assays. Angew. Chem. Int. Ed. 2003, 42, 1375-1378. (12) Perez, J. M.; Josephson, L.; O'Loughlin, T.; Högemann, D.; Weissleder, R. Magnetic Relaxation Switches Capable of Sensing Molecular Interactions. Nat. Biotechnol. 2002, 20, 816-820. (13) Perez, J. M.; O'Loughin, T.; Simeone, F. J.; Weissleder, R.; Josephson, L. DNA-based Magnetic Nanoparticle Assembly Acts as a Magnetic Relaxation Nanoswitch Allowing Screening of DNA-cleaving Agents. J. Am. Soc. Chem. 2002, 124, 2856-2857. (14) Taktak, S.; Sosnovik, D.; Cima, M. J.; Weissleder, R.; Josephson, L. Multiparameter Magnetic Relaxation Switch Assays. Anal. Chem. 2007, 79, 8863-8869. 22


Page 22 of 34

Page 23 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(15) Yoon, K. Y.; Kotsmar, C.; Ingram, D. R.; Huh, C.; Bryant, S. L.; Milner, T. E.; Johnston, K. P. Stabilization of Superparamagnetic Iron Oxide Nanoclusters in Concentrated Brine with Cross-linked Polymer Shells. Langmuir 2011, 27, 10962-10969. (16) Xu, H.; Cui, L.; Tong, N.; Gu, H. Development of High Magnetization Fe3O4/Polystyrene/Silica Nanospheres Via Combined Miniemulsion/Emulsion Polymerization. J. Am. Chem. Soc. 2006, 128, 15582-15583. (17) Zhang, X.; Xu, X.; Li, T.; Lin, M.; Lin, X.; Zhang, H.; Sun, H; Yang, B. Composite Photothermal

Platform

of

Polypyrrole-enveloped

Fe3O4

Nanoparticle

Self-assembled Superstructures. ACS Appl. Mater. Interfaces 2014, 6, 14552-14561. (18) Deng, H.; Li, X. L.; Peng, Q.; Wang, X.; Chen, J. P.; Li, Y. D. Monodisperse Magnetic Single-Crystal Ferrite Macro-spheres. Angew. Chem. Int. Ed. 2005, 44, 2782-2785. (19) Gao, J.; Ran, X.; Shi, C.; Cheng, H.; Cheng, T.; Su, Y. One-step Solvothermal Synthesis of Highly Water-soluble, Negatively Charged Superparamagnetic Fe3O4 Colloidal Nanocrystal Clusters. Nanoscale 2013, 5, 7026-7033. (20) Qiao, R.; Yang, C.; Gao, M. Superparamagnetic Iron Oxide Nanoparticles: From Preparations to in Vivo MRI Applications. J. Mater. Chem. 2009, 19, 6274-6293. (21) Ge, J. P.; Hu, Y. X.; Biasini, M.; Beyermann, W. P.; Yin, Y. D. Superparamagnetic Magnetite Colloidal Nanocrystal Cluster. Angew. Chem. Int. Ed. 2007, 46, 4342-4345. 23



(22) Liao, N.; Wu, M.; Pan, F.; Lin, J.; Li, Z.; Zhang, D.; Wang, Y.; Zheng, Y.; Peng, J.; Liu, X.; Liu, J. Poly(dopamine) Coated Superparamagnetic Iiron Oxide Nanocluster for Noninvasive Labeling, Tracking, and Targeted Delivery of Adipose Tissue-derived Stem Cells. Sci Rep 2016, 6, 1-5. (23) Sahoo, Y.; Goodarzi, A.; Swihart, M. T.; Ohulchanskyy, T. Y.; Kaur, N.; Furlani, E. P.; Prasad, P. N. Aqueous Ferrofluid of Magnetite Nanoparticles: Fluorescence Labeling and Magnetophoretic Control. J. Phys. Chem. B 2005, 109, 3879-3885. (24) Luo, B.; Xu, S.; Luo, A.; Wang, W. R.; Wang, S. L.; Guo, J.; Yao, L.; Zhao, D. Y.; Wang, C. C. Mesoporous Biocompatible and Acid-degradable Magnetic Colloidal Nanocrystal Clusters with Sustainable Stability and High Hydrophobic Drug Loading Capacity. ACS nano 2011, 5, 1428-1435. (25) Zhang, G. L.; Lu, S. Y.; Qian, J. C.; Zhong, K.; Yao, J. M.; Cai, D. Q.; Cheng, Z. L.; Wu, Z. Y. Magnetic Relaxation Switch Detecting Boric Acid or Borate Ester through One-Pot Synthesized Poly (vinyl alcohol) Functionalized Nanomagnetic Iron Oxide. ACS Appl. Mater. Interfaces 2015, 7, 16837-16841. (26) Wang, X.; Zhou, Z.; Wang, Z.; Xue, Y.; Zeng, Y.; Gao, J.; Chen, X. Gadolinium Embedded Iron Oxide Nanoclusters as T1–T2 Dual-modal MRI-visible Vectors for Safe and Efficient siRNA Delivery. Nanoscale 2013, 5, 8098-8104. (27) Tian, Y.; Yu, B.; Li, X.; Li, K. Facile Solvothermal Synthesis of Monodisperse Fe3O4 Nanocrystals with Precise Size Control of One Nanometre as Potential MRI Contrast Agents. J. Mater. Chem. 2011, 21, 2476-2481. 24


Page 24 of 34

Page 25 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(28) Hardy, P. A.; Henkelman, R. R. Transverse Relaxation Rate Enhancement Caused by Magnetic Particulates. Magn. Reson. Imaging 1989, 7, 265-275. (29) Yang, L.; Ma, L.; Xin, J.; Li, A.; Sun, C.; Wei, R.; Ren, B.; Chen, Z.; Lin, H.; Gao, J. Composition Tunable Manganese Ferrite Nanoparticles for Optimized T2 Contrast Ability Chem. Mat. 2017, 29, 3038-3047. (30) Thorek, D. L. J.; Tsourkas, A. Size, Charge and Concentration Dependent Uptake of Iron Oxide Particles by Non-Phagocytic Cells. Biomaterials 2008, 9, 3583-3590.

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Figures

Scheme 1. Schematic illustration of the factors enhancing T2-weighted contrast ability.

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Figure 1. TEM images and the size distribution (insets) of PVP@NMIONC, PEI@NMIONC, and PAA@NMIONC samples. When the ratios of EG to DEG in the solvent were 5/0, 3/2, and 1/4, respectively, different sizes of NMIONCs could be obtained. Scale bar: 200 nm, the unit of size in inset: nm.

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Figure 2. (a) Particle size distribution, (b) average particles size, (c) XRD patterns, and (d) FT-IR spectra of PVP@NMIONC, PEI@NMIONC, and PAA@NMIONC samples.

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Figure 3. High-magnification TEM images of PVP@NMIONC, PEI@NMIONC, and PAA@NMIONC samples.

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Figure 4. (a) Hysteresis loops and (b) the saturation magnetization values of PEI@NMIONC, PVP@NMIONC, and PAA@NMIONC samples at 300 K.

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Figure 5. Temperature dependence of ZFC and FC magnetization curves for PEI@NMIONC, PVP@NMIONC, and PAA@NMIONC samples.

31



Figure 6. (a) Transverse relaxation rates of PEI@NMIONC, PVP@NMIONC, and PAA@NMIONC samples calculated through the ratio of 1/T2 to Fe concentration; (b) the magnetism and relaxivity changes of PEI@NMIONC, PVP@NMIONC, and PAA@NMIONC with the size varying; (c) MR T2-weighted maps of PEI@NMIONC, PVP@NMIONC, and PAA@NMIONC samples.

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Figure 7. In vivo T2-weighted MR imaging of liver region at transverse plane and the related quantitative analysis of signal changes at 9.4 T. (a) T2-weighted MR images of mice treated with NMIONC (injection dosage: 2 mg/kg) pre-injection and post-injection 15, 30, 60 min; (b) Corresponding signal to noise ratio changes (∆SNR) in liver region after administration.

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TOC Graphic

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Fabricating High-Performance T2-Weighted Contrast Agents via

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