Polyaspartic Acid-Coated Paramagnetic Gadolinium Oxide Nanoparticles as a Dual-Modal T 1 and T 2 Magnetic Resonance Imaging Contrast Agent

Dual-Modal Abstract: Surface-coating polymers contribute to nanoparticle-based magnetic resonance imaging (MRI) contrast agents because they can affect the relaxometric properties of the nanoparticles. In this study, polyaspartic acid (PASA)-coated ultrasmall Gd 2 O 3 nanoparticles with an average particle diameter of 2.0 nm were synthesized using the one-pot polyol method. The synthesized nanoparticles exhibited r 1 and r 2 of 19.1 and = 53.7 s − 1 mM − 1 , respectively, (r 1 and r 2 are longitudinal and transverse water–proton spin relaxivities, respectively) at 3.0 T MR ﬁeld, approximately 5 and 10 times higher than those of commercial Gd-chelate contrast agents, respectively. The T 1 and T 2 MR images could be obtained due to an appreciable r 2 /r 1 ratio of 2.80, indicating their potential as a dual-modal T 1 and T 2 MRI contrast agent. images in mice. This result indicates that dual-modal T 1 and T 2 MRI is possible through a proper choice of hydrophilic polymers as surface-coating ligands of Gd 2 O 3 nanoparticles which provide appreciable r 2 /r 1 ratios. The in vivo animal imaging experiments were conducted


Synthesis
PASA-coated Gd 2 O 3 nanoparticles were synthesized through a polyol method ( Figure 1). An amount of 0.5 mmol of GdCl 3 ·6H 2 O and 0.1 mmol of PASA were added to 10 mL TEG in a three-necked round-bottom flask. The mixture was magnetically stirred at room temperature under atmospheric conditions until the precursor and PASA dissolved in TEG. In a separate beaker, NaOH solution was prepared by dissolving 2.0 mmol of NaOH in 10 mL TEG at room temperature using a magnetic stirrer. The NaOH solution was slowly added to the precursor solution using a pipette until the pH of the solution reached~10. At constant pH, the reaction temperature increased from room temperature to 110 • C and was maintained at that temperature for 15 h with magnetic stirring. After the reaction completion, the product solution was cooled to room temperature and transferred to a 500 mL beaker. The product nanoparticles were washed thrice with 400 mL ethanol. For this step, the product solution was magnetically stirred for 10 min and then, stored at a cool place (4 • C) until the nanoparticles settled down. The supernatant was decanted and the remaining product solution was washed again using ethanol. To remove ethanol and concentrate the nanoparticle solution sample, 400 mL triple-distilled water was added to the product solution and rotary evaporation was used to reduce the solution volume to approximately 50 mL. This process was repeated thrice. The obtained concentrated nanoparticle solution sample was split into two equal volumes: one part was subject to air-drying to obtain a powder sample for various characterizations and the other part was diluted using triple-distilled water to obtain a nanoparticle solution sample (30-50 mM Gd) for cytotoxicity and MRI analyses.
Appl. Sci. 2021, 11, x FOR PEER REVIEW 3 of 1 other part was diluted using triple-distilled water to obtain a nanoparticle solution sam ple (30-50 mM Gd) for cytotoxicity and MRI analyses.

Characterizations
The particle diameter (d) of the nanoparticles was measured using a high-resolution transmission electron microscope (HRTEM) (Titan G2 Chemi STEM CS Probe, FEI Hillsboro, OR, USA) operated at 200 kV acceleration voltage. The copper grid (PELCO No.160, TED PELLA, Inc., Redding, CA, USA) covered with carbon film was used to support nanoparticles for imaging. The hydrodynamic diameter (a) and zeta potential (ξ were measured using a dynamic light scattering (DLS) particle size and zeta potentia analyzer (Zetasizer Nano ZS, Malvern, Malvern, UK) using a 0.01 mM Gd solution sam ple. The crystal structural analyses of the nanoparticles before and after thermogravi metric analysis (TGA) were conducted using a multi-purpose powder X-ray diffraction (XRD) spectrometer (X'PERT PRO MRD, Philips, The Netherlands) with unfiltered CuKα radiation (λ = 1.54184 Å ). The Gd-concentration of the aqueous nanoparticle solution sample was measured using an inductively coupled plasma-atomic emission spectrom eter (ICP-AES) (IRIS/AP, Thermo Jarrell Ash Co., Waltham, MA, USA). A Fourier trans form-infrared (FT-IR) absorption spectrometer (Galaxy 7020A, Mattson Instrument, Inc. Madison, WI, USA) was used to study the surface coating of the nanoparticles. To record FT-IR absorption spectra, pellets of the powder samples in KBr were prepared and the spectra were recorded between 400 and 4000 cm −1 . A TGA instrument (SDT-Q600, TA Instrument, New Castle, DE, USA) was used to record the TGA curve of the powde sample between room temperature and 900 ℃ while air flowed over the sample. The average amount of PASA polymers coating a nanoparticle was quantified from the mass drop in the TGA curve after considering the initial mass drop from room temperature to ~110 °C due to water desorption. A vibrating sample magnetometer (VSM) (7407-S, Lake Shore Cryotronics Inc., Westerville, OH, USA) was used to obtain the magnetic proper ties of the powder sample by recording a magnetization (M) versus applied field (H) (or M−H) curve (−2.0 T ≤ H ≤2.0 T) at 300 K. The net M value of the sample (i.e., only the Gd2O3 nanoparticles without PASA coating) was estimated using the net mass of Gd2O nanoparticles extracted from the TGA curve.

In Vitro Cytotoxicity Tests
Cellular toxicity of an aqueous nanoparticle solution sample was assessed using a CellTiter-Glo Luminescent Cell Viability Assay (Promega, Madison, WI, USA). In this assay, a luminometer (Synergy HT, BioTek, Winooski, VT, USA) was used to quantify intracellular adenosine triphosphate. Human prostate cancer (DU145) and normal mouse hepatocyte (NCTC 1469) cell lines were used as test cells. Dulbecco's Modified Eagle

Characterizations
The particle diameter (d) of the nanoparticles was measured using a high-resolution transmission electron microscope (HRTEM) (Titan G2 Chemi STEM CS Probe, FEI, Hillsboro, OR, USA) operated at 200 kV acceleration voltage. The copper grid (PELCO No.160, TED PELLA, Inc., Redding, CA, USA) covered with carbon film was used to support nanoparticles for imaging. The hydrodynamic diameter (a) and zeta potential (ξ) were measured using a dynamic light scattering (DLS) particle size and zeta potential analyzer (Zetasizer Nano ZS, Malvern, Malvern, UK) using a 0.01 mM Gd solution sample. The crystal structural analyses of the nanoparticles before and after thermogravimetric analysis (TGA) were conducted using a multi-purpose powder X-ray diffraction (XRD) spectrometer (X'PERT PRO MRD, Philips, The Netherlands) with unfiltered CuKα radiation (λ = 1.54184 Å). The Gd-concentration of the aqueous nanoparticle solution sample was measured using an inductively coupled plasma-atomic emission spectrometer (ICP-AES) (IRIS/AP, Thermo Jarrell Ash Co., Waltham, MA, USA). A Fourier transform-infrared (FT-IR) absorption spectrometer (Galaxy 7020A, Mattson Instrument, Inc., Madison, WI, USA) was used to study the surface coating of the nanoparticles. To record FT-IR absorption spectra, pellets of the powder samples in KBr were prepared and the spectra were recorded between 400 and 4000 cm −1 . A TGA instrument (SDT-Q600, TA Instrument, New Castle, DE, USA) was used to record the TGA curve of the powder sample between room temperature and 900 • C while air flowed over the sample. The average amount of PASA polymers coating a nanoparticle was quantified from the mass drop in the TGA curve after considering the initial mass drop from room temperature to~110 • C due to water desorption. A vibrating sample magnetometer (VSM) (7407-S, Lake Shore Cryotronics Inc., Westerville, OH, USA) was used to obtain the magnetic properties of the powder sample by recording a magnetization (M) versus applied field (H) (or M−H) curve (−2.0 T ≤ H ≤ 2.0 T) at 300 K. The net M value of the sample (i.e., only the Gd 2 O 3 nanoparticles without PASA coating) was estimated using the net mass of Gd 2 O 3 nanoparticles extracted from the TGA curve.

In Vitro Cytotoxicity Tests
Cellular toxicity of an aqueous nanoparticle solution sample was assessed using a CellTiter-Glo Luminescent Cell Viability Assay (Promega, Madison, WI, USA). In this assay, a luminometer (Synergy HT, BioTek, Winooski, VT, USA) was used to quantify intracellular adenosine triphosphate. Human prostate cancer (DU145) and normal mouse hepatocyte (NCTC 1469) cell lines were used as test cells. Dulbecco's Modified Eagle medium and Rosewell Park Memorial Institute-1640 culture media were used for NCTC1469 and DU145 cells, respectively. Both cells were seeded on a 24-well cell culture plate and incubated for 24 h (5 × 10 4 cell densities, 500 µL cells per well, 5% CO 2 , and 37 • C). Five test nanoparticle solution samples were prepared by diluting an original nanoparticle solution sample with a sterile phosphate buffer saline solution. Two microliters of each test solution sample were added to the cells to obtain 10, 50, 100, 200, and 500 µM Gd-concentration, and the treated cells were then incubated for 48 h. The viability of the treated cells was measured and normalized with respect to the control cells. The measurement was repeated thrice for all treated cells to obtain average cell viabilities.

Relaxivity Measurements
Both longitudinal (T 1 ) and transverse (T 2 ) relaxation times as well as longitudinal (R 1 ) and transverse (R 2 ) map images were measured using a 3.0 T MRI scanner (MAGNETOM Trio Tim, Siemens, Munchen, Bayern, Germany) at 20 • C. Various concentrations of aqueous nanoparticle solution samples (0.1, 0.05, 0.025, 0.0125, 0.00625, and 0.0 mM Gd) were prepared in a series by diluting an original aqueous nanoparticle solution sample using triple-distilled water. T 1 relaxation times were measured using an inversion recovery method. T 2 relaxation times were obtained using a multiple spin-echo method with a Carr-Purcell-Meiboom-Gill pulse sequence. Then, r 1 and r 2 values were estimated from the slopes in the plots of 1/T 1 and 1/T 2 versus Gd-concentration, respectively.

In Vivo T 1 and T 2 MR Image Measurements
In vivo animal imaging was conducted based on the rules and regulations and permission of the animal research committee of the Korea Institute of Radiological and Medical Sciences (KIRAMS). The same 3.0 T MRI scanner used for relaxivity measurements was used to obtain T 1 and T 2 MR images. Two Institute of Cancer Research mice weighing 30 g (N mice = 2) were used for each modal imaging. The mice were anesthetized using 1.5% isoflurane in oxygen. Measurements were made before and after injecting an aqueous nanoparticle solution sample into mice tail veins. The injection doses were approximately 0.013 and 0.1 mmol Gd/kg for T 1 and T 2 MR image measurements, respectively. During measurements, the body temperature of the mice was maintained at 37 • C using a warm water blanket. After measurements, the mice were revived from anesthesia and placed in a cage with free access to food and water. The spin-echo sequence was used for MR image measurements at 3.0 T. The typical measurement parameters were as follows: echo time = 10 (or 37) ms, repetition time = 385 (or 1620) ms, pixel bandwidth = 299 (or 197) Hz, number of acquisitions = 8 (or 4), echo train length = 3 (or 13), flip angle = 120 o (or 120 o ), slice thickness = 1.0 (or 1.0) mm, and slice gap = 1.1 (or 1.1) mm for T 1 (or T 2 ) MR image measurements.

Nanoparticle Size and Structure
The particle diameters were ultrasmall (Figure 2a) and the average particle diameter (d avg ) was estimated to be 2.0 nm by fitting an observed particle diameter distribution to a log-normal function ( Figure 2b and Table 1). The average hydrodynamic diameter (a avg ) of the nanoparticles dispersed in triple-distilled water (0.01 mM Gd) was estimated to be 12.7 nm by fitting an observed DLS pattern to a log-normal function ( Figure 2c and Table 1). An aqueous nanoparticle solution sample is presented in Figure 3a. To confirm the colloidal dispersion of the nanoparticles in aqueous media, laser scattering (i.e., Tyndall effect) was conducted ( Figure 3b). Laser scattering was observed only in the nanoparticle solution sample whereas no such scattering was observed in reference triple-distilled water, confirming the colloidal dispersion. In addition, the average zeta potential (ξ avg ) was estimated to be −28.0 mV in aqueous media ( Figure 3c). This high value supported the observed good colloidal stability.
(wt.%) (1/nm 2 ) NPAA Magnetism 300 K (emu/g) r1 (s mM ) r2 (s mM ) 2.0 ± 0.1 12.7 ± 0.2 63.2 ± 0.2 0.29 ± 0.05 4 ± 1 −28.0 ± 0.2 Paramagnetic 1.95 ± 0.05 19.1 ± 0.1 53.7 ± 0.1 1 Average surface-coating amount in wt.% of the PASA polymers per nanoparticle. 2 Average grafting density (i.e., average number of polymers coating a nanoparticle unit surface area). 3 Average number of PASA polymers coating a nanoparticle. XRD patterns of the synthesized nanoparticles before and after TGA were recorded ( Figure 4). The XRD pattern of the as-synthesized nanoparticles was broad and structureless, indicating that they were poorly crystallized (i.e., amorphous) due to their ultrasmall particle size [20]. However, sharp peaks were obtained after TGA, indicating crystallization and particle size growth of the nanoparticles after TGA, as observed previously [21]. The observed XRD pattern after TGA was in good agreement with that of cubic Gd2O3 (JCPDS card no: 43-1014) and the estimated lattice constant (10.81 Å ) was comparable to the reported value (10.818 Å ) [22].  Table 1. Average particle diameter (d avg ), average hydrodynamic diameter (a avg ), surface-coating amount (P, σ, N PAA ), magnetic properties, and water-proton spin relaxivities (r 1 , r 2 ) of PASA-coated Gd 2 O 3 nanoparticles.   XRD patterns of the synthesized nanoparticles before and after TGA were reco ( Figure 4). The XRD pattern of the as-synthesized nanoparticles was broad and tureless, indicating that they were poorly crystallized (i.e., amorphous) due to the trasmall particle size [20]. However, sharp peaks were obtained after TGA, indic crystallization and particle size growth of the nanoparticles after TGA, as observed viously [21]. The observed XRD pattern after TGA was in good agreement with th cubic Gd2O3 (JCPDS card no: 43-1014) and the estimated lattice constant (10.81 Å comparable to the reported value (10.818 Å ) [22]. XRD patterns of the synthesized nanoparticles before and after TGA were recorded ( Figure 4). The XRD pattern of the as-synthesized nanoparticles was broad and structureless, indicating that they were poorly crystallized (i.e., amorphous) due to their ultrasmall particle size [20]. However, sharp peaks were obtained after TGA, indicating crystallization and particle size growth of the nanoparticles after TGA, as observed previously [21]. The observed XRD pattern after TGA was in good agreement with that of cubic

Surface Coating
The coating of PASA polymers on the nanoparticle surface recording an FT-IR absorption spectrum (bottom spectrum labele addition, FT-IR absorption spectra of bare Gd2O3 nanoparticles a (top and middle spectra labeled I and II in Figure 5a, respectively served absorption frequencies are provided in Table 2. Characterist PASA [23,24] were also observed in the FT-IR absorption spec Gd2O3 nanoparticles, confirming the surface coating. The frequen cm −1 between symmetric and asymmetric COO − stretches indicat were electrostatically bonded to Gd 3+ on a nanoparticle surface th [25], as drawn in Figure 5b. Considering numerous COO − groups p numerous bridge bonds are expected per PASA with a nanoparti bond is presented in Figure 5b.

Surface Coating
The coating of PASA polymers on the nanoparticle surface was demonstrated by recording an FT-IR absorption spectrum (bottom spectrum labeled III in Figure 5a). In addition, FT-IR absorption spectra of bare Gd 2 O 3 nanoparticles and PASA were taken (top and middle spectra labeled I and II in Figure 5a, respectively) as reference. The observed absorption frequencies are provided in Table 2. Characteristic absorption peaks of PASA [23,24] were also observed in the FT-IR absorption spectrum of PASA-coated Gd 2 O 3 nanoparticles, confirming the surface coating. The frequency difference of~196 cm −1 between symmetric and asymmetric COO − stretches indicated that COO − groups were electrostatically bonded to Gd 3+ on a nanoparticle surface through a bridge bond [25], as drawn in Figure 5b. Considering numerous COO − groups per PASA (i.e., n =~72), numerous bridge bonds are expected per PASA with a nanoparticle although only one bond is presented in Figure 5b. The coating amount of PASA polymers per nanoparticle was estimated in weight percentage (%) from a TGA curve (Figure 5c). The initial mass drop of 5.5% between room temperature and ~110 °C was due to water desorption, and the next mass drop of 63.2% was due to PASA burning through its reaction with flowing hot air. The final mass drop of 31.3% was due to the remaining Gd2O3 nanoparticles. To estimate the average  The coating amount of PASA polymers per nanoparticle was estimated in weight percentage (%) from a TGA curve (Figure 5c). The initial mass drop of 5.5% between room temperature and~110 • C was due to water desorption, and the next mass drop of 63.2% was due to PASA burning through its reaction with flowing hot air. The final mass drop of 31.3% was due to the remaining Gd 2 O 3 nanoparticles. To estimate the average number of PASA polymers coating a nanoparticle per unit surface area, a grafting density (σ) [26] was calculated to be 0.29 nm −2 using a molecular mass of PASA (i.e., 9900 amu), the average particle diameter estimated from HRTEM (i.e., 2.0 nm), and the bulk density of 7.407 g/cm 3 [27] for Gd 2 O 3 . By multiplying σ with the average nanoparticle surface area (= πd avg 2 ), the average number of PASA polymers coating a nanoparticle was estimated to be~4. The results are provided in Table 1.

In Vitro Cell Viability
As free Gd 3+ ions are toxic in vivo [28,29], in vitro cytotoxicity of PASA-coated Gd 2 O 3 nanoparticles was measured in DU145 and NCTC1469 cells. As shown in Figure 6, cell viabilities were approximately 100 and 77% in DU145 and NCTC1469 cells at 500 µM Gd, respectively, indicating good biocompatibility of the nanoparticles. The coating amount of PASA polymers per nanoparticle wa percentage (%) from a TGA curve (Figure 5c). The initial mass room temperature and ~110 °C was due to water desorption, and 63.2% was due to PASA burning through its reaction with flowing drop of 31.3% was due to the remaining Gd2O3 nanoparticles. To number of PASA polymers coating a nanoparticle per unit surfac sity () [26] was calculated to be 0.29 nm −2 using a molecular ma amu), the average particle diameter estimated from HRTEM (i.e., density of 7.407 g/cm 3 [27] for Gd2O3. By multiplying  with the surface area (= πdavg 2 ), the average number of PASA polymers coat estimated to be ~4. The results are provided in Table 1.

In Vitro Cell Viability
As free Gd 3+ ions are toxic in vivo [28,29], in vitro cytotoxicity nanoparticles was measured in DU145 and NCTC1469 cells. As s viabilities were approximately 100 and 77% in DU145 and NCTC14 respectively, indicating good biocompatibility of the nanoparticles

Magnetic Properties
Magnetic properties of PASA-coated Gd2O3 nanoparticles w obtaining an M−H curve at T = 300 K. As shown in Figure 7, the M hysteresis (i.e., zero value in coercivity and remanence), indicating

Magnetic Properties
Magnetic properties of PASA-coated Gd 2 O 3 nanoparticles were characterized by obtaining an M−H curve at T = 300 K. As shown in Figure 7, the M−H curve showed no hysteresis (i.e., zero value in coercivity and remanence), indicating paramagnetism of the nanoparticles, similar to bulk Gd 2 O 3 [30,31]. In the plot, the net M value of the nanoparticles (i.e., only Gd 2 O 3 nanoparticles without PASA) was used, which was estimated using the net mass of Gd 2 O 3 nanoparticles extracted from a TGA curve. The net M at H = 2.0 T was estimated to be 1.95 emu/g (Table 1). This appreciable M value is due to a high-spin magnetic moment (S = 7/2) of 4f-electrons of Gd 3+ .

2021, 11, x FOR PEER REVIEW
ing the net mass of Gd2O3 nanoparticles extracted from a TGA curve T was estimated to be 1.95 emu/g (Table 1). This appreciable M value magnetic moment (S = 7/2) of 4f-electrons of Gd 3+ .

R1 and R2 Values and R1 and R2 Map Images
As shown in Figure 8a, r1 and r2 values were estimated to be 1 (r2/r1 = 2.80) (Table 1), respectively, from the slopes in the plots Gd-concentration using an equation 1/Ti = riC + I (i = 1, 2) where C is and I is the intercept. The obtained r1 and r2 values were approxim higher than those of commercial Gd-chelates [5,6], respectively. Dos enhancements were observed in both R1 and R2 map images (Figur the ability of the nanoparticles to enhance both T1 and T2 MR contras

R 1 and R 2 Values and R 1 and R 2 Map Images
As shown in Figure 8a, r 1 and r 2 values were estimated to be 19.1 and 53.7 s −1 mM −1 (r 2 /r 1 = 2.80) (Table 1), respectively, from the slopes in the plots 1/T 1 and 1/T 2 versus Gd-concentration using an equation 1/T i = r i C + I (i = 1, 2) where C is the Gd-concentration and I is the intercept. The obtained r 1 and r 2 values were approximately 5 and 10 times higher than those of commercial Gd-chelates [5,6], respectively. Dose-dependent contrast enhancements were observed in both R 1 and R 2 map images (Figure 8b), demonstrating the ability of the nanoparticles to enhance both T 1 and T 2 MR contrasts in vitro.
Appl. Sci. 2021, 11, x FOR PEER REVIEW ing the net mass of Gd2O3 nanoparticles extracted from a TGA curve. The net T was estimated to be 1.95 emu/g (Table 1). This appreciable M value is due t magnetic moment (S = 7/2) of 4f-electrons of Gd 3+ .

R1 and R2 Values and R1 and R2 Map Images
As shown in Figure 8a, r1 and r2 values were estimated to be 19.1 and (r2/r1 = 2.80) (Table 1), respectively, from the slopes in the plots 1/T1 and Gd-concentration using an equation 1/Ti = riC + I (i = 1, 2) where C is the Gd-c and I is the intercept. The obtained r1 and r2 values were approximately 5 higher than those of commercial Gd-chelates [5,6], respectively. Dose-depend enhancements were observed in both R1 and R2 map images (Figure 8b), de the ability of the nanoparticles to enhance both T1 and T2 MR contrasts in vit

In Vivo T1 and T2 MR Images: Dual-Modal Imaging
As shown in Figure 9a, positive (or brightened) and negative (or darken were observed in T1 and T2 MR images of mice livers, respectively, after int jection of the nanoparticle solution sample into mice tails. Time-course cont of signal-to-noise ratios (SNRs) of a region-of-interest (ROI) (labeled as dotte

In Vivo T 1 and T 2 MR Images: Dual-Modal Imaging
As shown in Figure 9a, positive (or brightened) and negative (or darkened) contrasts were observed in T 1 and T 2 MR images of mice livers, respectively, after intravenous injection of the nanoparticle solution sample into mice tails. Time-course contrast changes of signal-to-noise ratios (SNRs) of a region-of-interest (ROI) (labeled as dotted circles) are plotted as a function of time before and after injection in Figure 9b. The contrast increased initially and then, decreased with time in T 1 MR images whereas the contrast decreased initially and then, slowly increased with time in T 2 MR images. These results indicated that PASA-coated Gd 2 O 3 nanoparticles should act as a dual-modal T 1 and T 2 MRI contrast agent. Ultrasmall nanoparticles (d <3.0 nm) can be excreted via the renal system [32,33]. Therefore, most of the nanoparticles were likely to be excreted via the renal system.

Discussion
In this study, PASA-coated Gd2O3 nanoparticles were synthesized v polyol method and characterized with various techniques. Their relaxom imaging properties were studied. Surface-coating ligands can affect r1 a Gd2O3 nanoparticles [16,17]. The PASA has not been used as a surface-coa thus, was investigated here. The r1 and r2 values of PASA-coated Gd2O were 19.1 and 53.7 s −1 mM −1 , respectively, which are much higher than commercial Gd-chelates. Interestingly, PASA-coated ultrasmall Gd2O3 na hibited an appreciable r2/r1 ratio of 2.80 and provided both T1 and T2 MR plain this, we used the relationship between hydrophilicity of polymers a polymer-coated Gd2O3 nanoparticles using various polymer-coated Gd2O studied here and previously [34][35][36]. As provided in Table 3, the PASA-coated Gd2O3 nanoparticles was higher than those [34][35][36] of the oth polymer-coated Gd2O3 nanoparticles. To understand this, the r2/r1 ratio o mer-coated Gd2O3 nanoparticles [34][35][36] was plotted as a function of COO percentage of polymers in Figure 10, showing that the r2/r1 ratio generally increasing COO − group weight percentage of the polymers. In this plot, i that among hydrophilic groups of the polymers, the COO − group could attract water molecules around the Gd2O3 nanoparticles through its negat electronegative oxygens. Thus, other hydrophilic groups such as C−O, were excluded from the hydrophilic group weight percentage of the poly indicates that a more water-attracting (or more hydrophilic) polymer can p ratio which is closer to one. This is because T1 water-proton spin relaxati strongly induced if more water molecules are attracted closer to the Gd2O according to the inner-sphere model [5]. Under such conditions, the r2/ close to one; thus, contrast agents can efficiently act as T1 MRI contrast ag

Discussion
In this study, PASA-coated Gd 2 O 3 nanoparticles were synthesized via the one-pot polyol method and characterized with various techniques. Their relaxometric and MRI imaging properties were studied. Surface-coating ligands can affect r 1 and r 2 values of Gd 2 O 3 nanoparticles [16,17]. The PASA has not been used as a surface-coating ligand and thus, was investigated here. The r 1 and r 2 values of PASA-coated Gd 2 O 3 nanoparticles were 19.1 and 53.7 s −1 mM −1 , respectively, which are much higher than those [5][6][7] of commercial Gd-chelates. Interestingly, PASA-coated ultrasmall Gd 2 O 3 nanoparticles exhibited an appreciable r 2 /r 1 ratio of 2.80 and provided both T 1 and T 2 MR images. To explain this, we used the relationship between hydrophilicity of polymers and r 2 /r 1 ratio of polymercoated Gd 2 O 3 nanoparticles using various polymer-coated Gd 2 O 3 nanoparticles studied here and previously [34][35][36]. As provided in Table 3, the r 2 /r 1 ratio of PASA-coated Gd 2 O 3 nanoparticles was higher than those [34][35][36] of the other hydrophilic polymer-coated Gd 2 O 3 nanoparticles. To understand this, the r 2 /r 1 ratio of various polymer-coated Gd 2 O 3 nanoparticles [34][35][36] was plotted as a function of COO − group weight percentage of polymers in Figure 10, showing that the r 2 /r 1 ratio generally decreased with increasing COO − group weight percentage of the polymers. In this plot, it was assumed that among hydrophilic groups of the polymers, the COO − group could most strongly attract water molecules around the Gd 2 O 3 nanoparticles through its negative charge and electronegative oxygens. Thus, other hydrophilic groups such as C−O, C=O, and NH were excluded from the hydrophilic group weight percentage of the polymers. This plot indicates that a more water-attracting (or more hydrophilic) polymer can provide the r 2 /r 1 ratio which is closer to one. This is because T 1 water-proton spin relaxation can be more strongly induced if more water molecules are attracted closer to the Gd 2 O 3 nanoparticles according to the inner-sphere model [5]. Under such conditions, the r 2 /r 1 ratio will be close to one; thus, contrast agents can efficiently act as T 1 MRI contrast agents. However, PASA has the lowest COO − group weight percentage among the polymers (Table 3 and Figure 10) and thus, the lowest water-attracting power around the Gd 2 O 3 nanoparticles. Therefore, PASA-coated Gd 2 O 3 nanoparticles will most weakly induce T 1 water-proton spin relaxation among polymer-coated Gd 2 O 3 nanoparticles in Table 3. This explains the observed appreciable r 2 /r 1 ratio of 2.80 in this study, which is higher than those of the other hydrophilic polymercoated Gd 2 O 3 nanoparticles in Table 3. Due to this, PASA-coated Gd 2 O 3 nanoparticles showed T 2 MR images as well as T 1 MR images, thus acting as a dual-modal T 1 and T 2 MRI contrast agent. This result indicates that dual-modal T 1 and T 2 MRI is possible through a proper choice of hydrophilic polymers as surface-coating ligands of Gd 2 O 3 nanoparticles with which the r 2 /r 1 ratio is appreciable.

Conclusions
In summary, surface-coating ligand plays an important role in relax properties of Gd2O3 nanoparticles. The PASA has not been used as ligand of Gd2O3 nanoparticles and thus, was investigated here. PASA-Gd2O3 nanoparticles were synthesized using the one-pot polyol meth laxometric and imaging properties in MRI were investigated. The PA nanoparticles were ultrasmall with a davg of 2.0 ± 0.1 nm and exhibited g bility up to 500 μM Gd as determined from cellular cytotoxicity tests. T Gd2O3 nanoparticles were paramagnetic and exhibited r1 = 19.1 ± 0.1 s −1 m ± 0.1 s −1 mM −1 , which are higher than those of commercial Gd-chelates.

Conclusions
In summary, surface-coating ligand plays an important role in relaxometric and MRI properties of Gd 2 O 3 nanoparticles. The PASA has not been used as a surface-coating ligand of Gd 2 O 3 nanoparticles and thus, was investigated here. PASA-coated ultrasmall Gd 2 O 3 nanoparticles were synthesized using the one-pot polyol method and their relaxometric and imaging properties in MRI were investigated. The PASA-coated Gd 2 O 3 nanoparticles were ultrasmall with a d avg of 2.0 ± 0.1 nm and exhibited good biocompatibility up to 500 µM Gd as determined from cellular cytotoxicity tests. The PASA-coated Gd 2 O 3 nanoparticles were paramagnetic and exhibited r 1 = 19.1 ± 0.1 s −1 mM −1 and r 2 = 53.7 ± 0.1 s −1 mM −1 , which are higher than those of commercial Gd-chelates. Due to an appreciable r 2 /r 1 ratio of 2.80, the PASA-coated ultrasmall Gd 2 O 3 nanoparticles exhibited both in vivo T 1 and T 2 MR images in mice. This result indicates that dual-modal T 1 and T 2 MRI is possible through a proper choice of hydrophilic polymers as surface-coating ligands of Gd 2 O 3 nanoparticles which provide appreciable r 2 /r 1 ratios.

Data Availability Statement:
The data presented in this study are available on request from the corresponding authors.