Functionalized Lanthanide Oxide Nanoparticles for Tumor Targeting, Medical Imaging, and Therapy

Recent progress in functionalized lanthanide oxide (Ln2O3) nanoparticles for tumor targeting, medical imaging, and therapy is reviewed. Among the medical imaging techniques, magnetic resonance imaging (MRI) is an important noninvasive imaging tool for tumor diagnosis due to its high spatial resolution and excellent imaging contrast, especially when contrast agents are used. However, commercially available low-molecular-weight MRI contrast agents exhibit several shortcomings, such as nonspecificity for the tissue of interest and rapid excretion in vivo. Recently, nanoparticle-based MRI contrast agents have become a hot research topic in biomedical imaging due to their high performance, easy surface functionalization, and low toxicity. Among them, functionalized Ln2O3 nanoparticles are applicable as MRI contrast agents for tumor-targeting and nontumor-targeting imaging and image-guided tumor therapy. Primarily, Gd2O3 nanoparticles have been intensively investigated as tumor-targeting T1 MRI contrast agents. T2 MRI is also possible due to the appreciable paramagnetic moments of Ln2O3 nanoparticles (Ln = Dy, Ho, and Tb) at room temperature arising from the nonzero orbital motion of 4f electrons. In addition, Ln2O3 nanoparticles are eligible as X-ray computed tomography contrast agents because of their high X-ray attenuation power. Since nanoparticle toxicity is of great concern, recent toxicity studies on Ln2O3 nanoparticles are also discussed.


Introduction
Medical imaging plays an important role in the pre-detection, diagnosis, and treatment of tumors [1]. Among the currently available medical imaging techniques, magnetic resonance imaging (MRI) allows whole-body imaging and outstanding microsoft tissue contrast and image resolution to reveal morphological and anatomical details of tissues [2,3]. As imaging agents, various nanoparticles have been developed due to their remarkable physical and chemical properties, which are superior to those of small molecules [4][5][6][7]. Moreover, nanoparticles can be easily surface-functionalized for advanced imaging and tumor targeting [8,9]. They can also provide longer blood circulation times than small molecules, which is conducive to tumor targeting and drug delivery to specific tumor cells [10,11].
Lanthanide oxide (Ln 2 O 3 ) nanoparticles (Ln = Gd, Tb, Dy, and Ho) are of special interest because they have appreciable magnetic moments at room temperature, which is useful for MRI [12][13][14][15], and high X-ray attenuation power, which is useful for X-ray computed tomography (CT) [16][17][18]. In addition, surface-modified Ln 2 O 3 nanoparticles exhibit improved properties, such as high-water proton spin relaxivities, high colloidal stabilities, and low toxicities [12][13][14]. For in vivo applications, nanoparticles should have an ultrasmall particle diameter (<3 nm) to allow their excretion from the body via the urinary system after   Nanoparticle contrast agents possess an additional advantage over molecular agents because cancer-targeting ligands and drugs can be easily attached to the nanoparticle surfaces. Small molecular ligands are less efficient in providing good colloidal stability to nanoparticles compared with polymer ligands, which is due to the presence of many hydrophilic binding groups in the polymers for attachment to the nanoparticles [39][40][41][42][43]. In addition, hydrophilic polymers can provide higher r 1 values than small molecular ligands [39][40][41] because they can attract more water molecules around the nanoparticles. Examples of these polymers are polyacrylic acid (PAA), polymethyl vinyl ether-alt-maleic acid (PMVEMA), and polyacrylic acid-co-maleic acid (PAAMA) having numerous COOH groups ( Figure 1) [39][40][41], which can serve as anchor groups of functional molecules, such as cancer-targeting ligands and drugs. For example, RGD-conjugated PAA-coated Gd 2 O 3 nanoparticles were reported by Ho et al. [44] (Figure 2). Ln 2 O 3 nanoparticles can also be synthesized in organic solvents via the thermal decomposition method. The synthesized nanoparticles can be further coated with hydrophilic ligands and then conjugated with cancer-targeting ligands. For example, CTX-poly(ethylene glycol)-N-(trimethoxysilylpropyl) ethylenediamine triacetic acid silane-coated Gd 2 O 3 nanoparticles (CTX-PEG-TETT-Gd 2 O 3 ) were reported by Gu et al. [45] as tumor-targeting contrast agents ( Figure 3). Here, the biocompatible poly(ethylene glycol) (PEG) layer is known to stabilize the Gd 2 O 3 nanoparticles, enhance blood circulation blood times, and improve colloidal stability [11].  hydroxysuccinimide, and UGNP = ultrasmall gadolinium oxide nanoparticle. Adapted with permission from [44], The Royal Society of Chemistry, 2020.
Ln2O3 nanoparticles can also be synthesized in organic solvents via the thermal decomposition method. The synthesized nanoparticles can be further coated with hydrophilic ligands and then conjugated with cancer-targeting ligands. For example, CTXpoly(ethylene glycol)-N-(trimethoxysilylpropyl) ethylenediamine triacetic acid silanecoated Gd2O3 nanoparticles (CTX-PEG-TETT-Gd2O3) were reported by Gu et al. [45] as tumor-targeting contrast agents ( Figure 3). Here, the biocompatible poly(ethylene glycol) (PEG) layer is known to stabilize the Gd2O3 nanoparticles, enhance blood circulation blood times, and improve colloidal stability [11].

Physicochemical Characterization
Various experimental techniques can be used to characterize the synthesized Ln2O3 nanoparticles [17,31], as summarized in Table 1. The particle diameters, the hydrodynamic diameters of nanoparticles dispersed in water, and the crystal structure of the nanoparticles can be measured using a high-resolution transmission electron microscope (HRTEM) operating at high acceleration voltages (>200 kV), a dynamic light scattering (DLS) particle size analyzer, and a powder X-ray diffraction (XRD) spectrometer, respectively. The attachment of ligands on the nanoparticle surfaces can be investigated by recording Fourier transform-infrared (FT-IR) absorption spectra using powder samples; powder samples may be pelletized in KBr for easy handling and acquisition of good signal-to-noise ratios using a small number of powder samples. The amount of surface-coat-

Physicochemical Characterization
Various experimental techniques can be used to characterize the synthesized Ln 2 O 3 nanoparticles [17,31], as summarized in Table 1. The particle diameters, the hydrodynamic diameters of nanoparticles dispersed in water, and the crystal structure of the nanoparticles can be measured using a high-resolution transmission electron microscope (HRTEM) operating at high acceleration voltages (>200 kV), a dynamic light scattering (DLS) particle size analyzer, and a powder X-ray diffraction (XRD) spectrometer, respectively. The attachment of ligands on the nanoparticle surfaces can be investigated by recording Fourier transform-infrared (FT-IR) absorption spectra using powder samples; powder samples may be pelletized in KBr for easy handling and acquisition of good signal-to-noise ratios using a small number of powder samples. The amount of surface-coating ligand can be estimated according to thermogravimetric analysis (TGA) curves. The elemental analysis (EA) can also be used to investigate the surface-coating amount and ligand composition (C/H/O/N/S) in metal oxide nanoparticles because metal oxide nanoparticles hardly decompose during EA. The Ln-concentration in an aqueous nanoparticle suspension can be determined using an inductively coupled plasma atomic emission spectrometer (ICPAES). The in vitro cellular cytotoxicity of the ligand-coated Ln 2 O 3 nanoparticles can be measured using various cell viability assay techniques, such as trypan blue dye exclusion staining, methylthiazoly tetrazolium (MTT) reduction, and water-soluble tetrazolium salt (WST) assay [46]. Cell viability measurements are generally performed using more than two cell lines to obtain consistent and reliable results because the cell viabilities generally depend on the cell lines. Magnetization (M) values of the nanoparticles can be estimated by recording M versus applied field (H) (M−H) curves at room temperature using a vibrating sample magnetometer (VSM). Net M values of the nanoparticles without ligand can be obtained using the net mass of the nanoparticles determined via TGA. For MRI contrast agents, r 1 and the transverse relaxivity (r 2 ) represent their ability to act as T 1 or T 2 MRI contrast agents. To measure the r 1 and r 2 values, longitudinal (T 1 ) and transverse (T 2 ) relaxation times are measured using an MRI scanner. The T 1 relaxation time measurements can be conducted using an inversion recovery method. For the T 2 relaxation times, the Carr-Purcell Meiboom-Gill pulse sequence can be used for multiple spin-echo measurements. The r 1 and r 2 values are then estimated from the slopes of the plots of 1/T 1 and 1/T 2 versus the Ln-concentration, respectively. In vivo T 1 or T 2 MR images can be obtained using an MRI scanner after intravenous injection of an aqueous nanoparticle suspension sample. In addition, the X-ray attenuation power of Ln 2 O 3 nanoparticles, which is higher than those of commercial iodine CT contrast agents, can be measured by recording phantom images using a CT scanner, and in vivo CT images can be obtained using a CT scanner after intravenous injection of an aqueous nanoparticle suspension sample. MRI MR image MRI X-ray attenuation power CT CT image CT 1 HRTEM = high-resolution transmission electron microscope; DLS = dynamic light scattering; XRD = X-ray diffraction; FT-IR = Fourier transform-infrared; EA = elemental analysis; TGA = thermogravimetric analysis; ICPAES = inductively coupled plasma atomic emission spectrometry; MTT = methylthiazoly tetrazolium; WST = water-soluble tetrazolium salt; VSM = vibrating sample magnetometer; MRI = magnetic resonance imaging; CT = computed tomography.

Particle Diameters and Surface Coatings
The imaging properties of nanoparticles depend on their particle and hydrodynamic diameters. Ln 2 O 3 nanoparticles synthesized via the polyol method generally have ultrasmall diameters (average particle diameter = 2.0 nm) [39][40][41][42][43][44]. Figure 4a-c shows various HRTEM images of Gd 2 O 3 nanoparticles [44,47,48]. Both particle and hydrodynamic diameters follow a log-normal function distribution; therefore, average particle and hydrodynamic diameters can be obtained by fitting the log-normal distribution function to the observed particle and hydrodynamic diameter distributions, respectively. The insets in Figure 4c show a nanoparticle suspension sample exhibiting laser light scattering (i.e., Tyndall effect) due to the collision of the nanoparticle colloids with the passing laser light, whereas triple-distilled water shows no light scattering [44]. This experiment shows the good colloidal dispersion of the nanoparticles in aqueous media. Due to the ultrasmall particle diameters, the XRD patterns of nanoparticle powder samples are generally broad and amorphous [49,50]. Nevertheless, upon performing a TGA from room temperature to high temperatures (>500 °C), sharp peaks corresponding to the (222), (400), (440), and (622) planes of a highly crystallized form of Ln2O3 having cubic structure are observed [50]. Examples of XRD patterns of ligand-coated Gd2O3 nanoparticles recorded before (i.e., as-prepared) and after TGA [44,47] are provided in Figure 5. The peak positions and the estimated cell constants of the TGA-analyzed samples are well consistent with those given in the literature [51]. The sharp peaks observed in the XRD patterns after TGA are due to the particle size growth and subsequent crystallization of the nanoparticles (Figure 5a,b), which was confirmed by Kattel Figure 5. XRD patterns of (a) cRGD-Gd2O3 nanoparticles [47] and (b) RGD-PAA-Gd2O3 nanoparticles [44] before (i.e., asprepared; bottom figures) and after TGA (top figures). Adapted from [44,47]. Copyright 2020 The Royal Society of Chemistry [44]; Copyright 2018 Wiley [47].
Surface coating of Ln2O3 nanoparticles with hydrophilic and biocompatible ligands is essential for their biomedical applications. The surface coating can be investigated by recording FT-IR absorption spectra. An FT-IR absorption spectrum of PAA-coated Gd2O3 nanoparticles is provided as an example at the bottom of Figure 6a [44,47,48]. Arrows and circles indicate nanoparticles. Insets in (c) show a magnified image of a nanoparticle (top right) and laser light scattering (i.e., Tyndall effect) due to the collision of the nanoparticle colloids with a passing laser light, whereas triple-distilled water shows no light scattering, confirming the good colloidal dispersion of nanoparticles in aqueous media (bottom left). Reproduced from [44,47,48]. Copyright 2020 The Royal Society of Chemistry [44]; Copyrights 2018 Wiley [47,48]. Due to the ultrasmall particle diameters, the XRD patterns of nanoparticle powder samples are generally broad and amorphous [49,50]. Nevertheless, upon performing a TGA from room temperature to high temperatures (>500 • C), sharp peaks corresponding to the (222), (400), (440), and (622) planes of a highly crystallized form of Ln 2 O 3 having cubic structure are observed [50]. Examples of XRD patterns of ligand-coated Gd 2 O 3 nanoparticles recorded before (i.e., as-prepared) and after TGA [44,47] are provided in Figure 5. The peak positions and the estimated cell constants of the TGA-analyzed samples are well consistent with those given in the literature [51]. The sharp peaks observed in the XRD patterns after TGA are due to the particle size growth and subsequent crystallization of the nanoparticles (Figure 5a,b), which was confirmed by Kattel et al. on the basis of HRTEM images [50,52].  [44,47,48]. Arrows and circles indicate nanoparticles. Insets show a magnified image of a nanoparticle (top right) and laser light scattering (i.e., Tyndall e due to the collision of the nanoparticle colloids with a passing laser light, whereas triple-dis water shows no light scattering, confirming the good colloidal dispersion of nanoparticles in a ous media (bottom left). Reproduced from [44,47,48]. Copyright 2020 The Royal Society of Che try [44]; Copyrights 2018 Wiley [47,48].
Due to the ultrasmall particle diameters, the XRD patterns of nanoparticle pow samples are generally broad and amorphous [49,50]. Nevertheless, upon performi TGA from room temperature to high temperatures (>500 °C), sharp peaks correspon to the (222), (400), (440), and (622) planes of a highly crystallized form of Ln2O3 ha cubic structure are observed [50]. Examples of XRD patterns of ligand-coated Gd2O noparticles recorded before (i.e., as-prepared) and after TGA [44,47] are provided in ure 5. The peak positions and the estimated cell constants of the TGA-analyzed sam are well consistent with those given in the literature [51]. The sharp peaks observed in XRD patterns after TGA are due to the particle size growth and subsequent crystalliza of the nanoparticles (Figure 5a  . XRD patterns of (a) cRGD-Gd2O3 nanoparticles [47] and (b) RGD-PAA-Gd2O3 nanoparticles [44] before (i.e., asprepared; bottom figures) and after TGA (top figures). Adapted from [44,47]. Copyright 2020 The Royal Society of Chemistry [44]; Copyright 2018 Wiley [47].
Surface coating of Ln2O3 nanoparticles with hydrophilic and biocompatible lig is essential for their biomedical applications. The surface coating can be investigate recording FT-IR absorption spectra. An FT-IR absorption spectrum of PAA-coated G nanoparticles is provided as an example at the bottom of Figure 6a [44] before (i.e., as-prepared; bottom figures) and after TGA (top figures). Adapted from [44,47]. Copyright 2020 The Royal Society of Chemistry [44]; Copyright 2018 Wiley [47].
Surface coating of Ln 2 O 3 nanoparticles with hydrophilic and biocompatible ligands is essential for their biomedical applications. The surface coating can be investigated by recording FT-IR absorption spectra. An FT-IR absorption spectrum of PAA-coated Gd 2 O 3 nanoparticles is provided as an example at the bottom of Figure 6a  stretching (1550 cm −1 ), and COO − symmetric stretching vibrations (1400 cm −1 ). The PAA and the nanoparticle were bonded through a coordination bonding between Gd 3+ on the nanoparticle surface as a hard acid and the COO − group of PAA as a hard base [53][54][55]. In this case, a stable nanoparticle colloid was formed in aqueous media because of the multiple bonding between many COO − groups of PAA (each PAA has~54 COO − groups) and a nanoparticle. On this basis, the bonding between PAA and the Gd 2 O 3 nanoparticle surface was proposed, as displayed in Figure 6b. Another example of surface coating on Gd 2 O 3 nanoparticles is provided in Figure 6c, which displayed the FT-IR absorption spectra of RGD, PAA (M w = 1800 Da), PAA-Gd 2 O 3 , and RGD-PAA-Gd 2 O 3 powder samples [44]. The presence of a C=O stretching band (1553 cm −1 ) of PAA in the FT-IR absorption spectrum of PAA-Gd 2 O 3 nanoparticles confirmed the PAA surface coating on the Gd 2 O 3 nanoparticles. This band was red-shifted by 144 cm −1 compared with free PAA (1697 cm −1 ), which can be attributed to the coordination bonding between the COO − groups of PAA and the Gd 3+ of the Gd 2 O 3 nanoparticles. As mentioned above, each PAA can form many coordination bonds with each Gd 2 O 3 nanoparticle via a hard acid (Gd 3+ )-hard base (COO − ) interaction [53][54][55]. The N−H bending vibration at 1544 cm −1 and the C-N stretching vibration at 1390 cm −1 of RGD [56] appeared in the FT-IR absorption spectrum of the RGD-PAA-Gd 2 O 3 nanoparticles, confirming the successful conjugation of RGD to PAA in the PAA-Gd 2 O 3 nanoparticles via amide bond formation. nanoparticle were bonded through a coordination bonding between Gd 3+ on the nanoparticle surface as a hard acid and the COO − group of PAA as a hard base [53−55]. In this case, a stable nanoparticle colloid was formed in aqueous media because of the multiple bonding between many COO − groups of PAA (each PAA has ~54 COO − groups) and a nanoparticle. On this basis, the bonding between PAA and the Gd2O3 nanoparticle surface was proposed, as displayed in Figure 6b. Another example of surface coating on Gd2O3 nanoparticles is provided in Figure 6c, which displayed the FT-IR absorption spectra of RGD, PAA (Mw = 1800 Da), PAA-Gd2O3, and RGD-PAA-Gd2O3 powder samples [44]. The presence of a C=O stretching band (1553 cm −1 ) of PAA in the FT-IR absorption spectrum of PAA-Gd2O3 nanoparticles confirmed the PAA surface coating on the Gd2O3 nanoparticles. This band was red-shifted by 144 cm −1 compared with free PAA (1697 cm −1 ), which can be attributed to the coordination bonding between the COO − groups of PAA and the Gd 3+ of the Gd2O3 nanoparticles. As mentioned above, each PAA can form many coordination bonds with each Gd2O3 nanoparticle via a hard acid (Gd 3+ )-hard base (COO − ) interaction [53][54][55]. The N−H bending vibration at 1544 cm −1 and the C-N stretching vibration at 1390 cm −1 of RGD [56] appeared in the FT-IR absorption spectrum of the RGD-PAA-Gd2O3 nanoparticles, confirming the successful conjugation of RGD to PAA in the PAA-Gd2O3 nanoparticles via amide bond formation.  [44]. The lowercase letters "s", "ss", "as", and "b" indicate stretch, symmetric stretch, antisymmetric stretch, and bend, respectively. Adapted from [39,44]. Copyrights 2018 and 2020 The Royal Society of Chemistry, respectively.
The EA can also be used to investigate the surface coating of nanoparticles. For example, in carbon-coated Gd2O3 nanoparticles (C@Gd2O3), the C/H/O content, in which H mostly stems from hydrocarbons and -OH groups and O mostly stems from -OH groups on the carbon-coating layer, was determined to be 28.1/3.2/26.5 in wt% [57]. The sum of  [44]. The lowercase letters "s", "ss", "as", and "b" indicate stretch, symmetric stretch, antisymmetric stretch, and bend, respectively. Adapted from [39,44]. Copyrights 2018 and 2020 The Royal Society of Chemistry, respectively.
The EA can also be used to investigate the surface coating of nanoparticles. For example, in carbon-coated Gd 2 O 3 nanoparticles (C@Gd 2 O 3 ), the C/H/O content, in which H mostly stems from hydrocarbons and -OH groups and O mostly stems from -OH groups on the carbon-coating layer, was determined to be 28.1/3.2/26.5 in wt% [57]. The sum of these wt% is 57.8%, which is fairly consistent with the 56.5% estimated from the TGA curve ( Figure 7). As can be seen in Figure 7, the remaining 37.0% corresponds to the mass wt% of Gd 2 O 3 in the C@Gd 2 O 3 nanoparticles. By performing a grafting density analysis [58], the wt% can be converted into the number of ligands grafted onto each nanoparticle surface [47,48]. these wt% is 57.8%, which is fairly consistent with the 56.5% estimated from the TGA curve ( Figure 7). As can be seen in Figure 7, the remaining 37.0% corresponds to the mass wt% of Gd2O3 in the C@Gd2O3 nanoparticles. By performing a grafting density analysis [58], the wt% can be converted into the number of ligands grafted onto each nanoparticle surface [47,48].

Magnetic Properties
To obtain high r1 and r2 values for enhanced MR contrast images, the nanoparticles must exhibit large M values at room temperature [59,60]. Figure 8a,b shows the M-H curves of Ln2O3 nanoparticles (Ln = Gd, Tb, Ho) [41,43]. Similar to the corresponding bulk materials [61][62][63], all of the nanoparticle samples are paramagnetic (i.e., no hysteresis, zero coercivity, zero remanence, and low M values) and exhibit appreciable unsaturated M values up to the measured H values at 300 K. From the mass-corrected M-H curve, the net M value of the Gd2O3 nanoparticles, which corresponds to the nanoparticles without ligands, at 2.0 T was estimated to be 1.71 emu/g. This appreciable value is due to the high 4f-electron spin magnetic moment (s = 7/2) of Gd 3+ having seven unpaired 4f electrons. Meanwhile, other Ln2O3 nanoparticles (Ln = Tb and Ho) exhibit net M values of 3.8 and 4.1 emu/g at 1.8 T (Figure 8b), respectively, which result from high 4f-electron spin-orbital magnetic moments, i.e., J = 6 for Tb 3+ and J = 8 for Ho 3+ . The slightly higher M value of the Ho2O3 nanoparticles compared with that of the Tb2O3 nanoparticles is due to the higher atomic magnetic moment (µ = 10.60 µB) of Ho 3+ compared with that of Tb 3+ (= 9.72 µB) [64], where µB is the Bohr magneton.

Magnetic Properties
To obtain high r 1 and r 2 values for enhanced MR contrast images, the nanoparticles must exhibit large M values at room temperature [59,60]. Figure 8a,b shows the M-H curves of Ln 2 O 3 nanoparticles (Ln = Gd, Tb, Ho) [41,43]. Similar to the corresponding bulk materials [61][62][63], all of the nanoparticle samples are paramagnetic (i.e., no hysteresis, zero coercivity, zero remanence, and low M values) and exhibit appreciable unsaturated M values up to the measured H values at 300 K. From the mass-corrected M-H curve, the net M value of the Gd 2 O 3 nanoparticles, which corresponds to the nanoparticles without ligands, at 2.0 T was estimated to be 1.71 emu/g. This appreciable value is due to the high 4f-electron spin magnetic moment (s = 7/2) of Gd 3+ having seven unpaired 4f electrons. Meanwhile, other Ln 2 O 3 nanoparticles (Ln = Tb and Ho) exhibit net M values of 3.8 and 4.1 emu/g at 1.8 T (Figure 8b), respectively, which result from high 4f-electron spin-orbital magnetic moments, i.e., J = 6 for Tb 3+ and J = 8 for Ho 3+ . The slightly higher M value of the Ho 2 O 3 nanoparticles compared with that of the Tb 2 O 3 nanoparticles is due to the higher atomic magnetic moment (µ = 10.60 µ B ) of Ho 3+ compared with that of Tb 3+ (= 9.72 µ B ) [64], where µ B is the Bohr magneton.
Pharmaceutics 2021, 13, x FOR PEER REVIEW these wt% is 57.8%, which is fairly consistent with the 56.5% estimated from th curve ( Figure 7). As can be seen in Figure 7, the remaining 37.0% corresponds to th wt% of Gd2O3 in the C@Gd2O3 nanoparticles. By performing a grafting density an [58], the wt% can be converted into the number of ligands grafted onto each nanop surface [47,48].

Magnetic Properties
To obtain high r1 and r2 values for enhanced MR contrast images, the nanopa must exhibit large M values at room temperature [59,60]. Figure 8a,b shows the curves of Ln2O3 nanoparticles (Ln = Gd, Tb, Ho) [41,43]. Similar to the correspondin materials [61][62][63], all of the nanoparticle samples are paramagnetic (i.e., no hysteresi coercivity, zero remanence, and low M values) and exhibit appreciable unsatura values up to the measured H values at 300 K. From the mass-corrected M-H cur net M value of the Gd2O3 nanoparticles, which corresponds to the nanoparticles w ligands, at 2.0 T was estimated to be 1.71 emu/g. This appreciable value is due to th 4f-electron spin magnetic moment (s = 7/2) of Gd 3+ having seven unpaired 4f ele Meanwhile, other Ln2O3 nanoparticles (Ln = Tb and Ho) exhibit net M values of 3 4.1 emu/g at 1.8 T (Figure 8b), respectively, which result from high 4f-electron spinmagnetic moments, i.e., J = 6 for Tb 3+ and J = 8 for Ho 3+ . The slightly higher M value Ho2O3 nanoparticles compared with that of the Tb2O3 nanoparticles is due to the atomic magnetic moment (µ = 10.60 µB) of Ho 3+ compared with that of Tb 3+ (= 9.72 µ where µB is the Bohr magneton.   [43]. Net M values of Ln 2 O 3 nanoparticles without ligands were used in the plots. Adapted from [41,43]. Copyrights 2020 and 2021 MDPI.

MR Imaging Properties: r 1 and r 2 Values
The r 1 and r 2 values are affected by the number of water molecules around the nanoparticles and the distance between them and, therefore, depend on the water-attracting ability of the ligands around the nanoparticles [59,60,65]. Accordingly, surface-coating ligands should be properly selected. Gd 2 O 3 nanoparticles act as positive (T 1 ) MRI contrast agents due to the high spin magnetic moment of Gd 3+ (s = 7/2); they have high r 1 values and r 2 /r 1 ratios close to one [39][40][41] (T 1 MRI contrast agents exhibit low r 2 /r 1 ratios, whereas T 2 MRI contrast agents have high r 2 /r 1 ratios [23]). The r 1 and r 2 values of PAAMA-coated Gd 2 O 3 nanoparticles at 3.0 T MR field were determined to be 40.6 and 63.4 s −1 mM −1 (r 2 /r 1 = 1.56), respectively, using the 1/T 1 and 1/T 2 plots versus Gdconcentration depicted in Figure 9 [40]. This r 1 value is approximately 10 times higher than those [21][22][23] of commercial molecular T 1 MRI contrast agents, such as Dotarem, ProHance, Gadovist, Magnevist, and Omniscan [21,22]. The r 1 value also depends on the particle diameter of Gd 2 O 3 nanoparticles, for which the optimal value was suggested to be 1.0 to 2.5 nm [24]. The observed particle diameter of the PAAMA-coated Gd 2 O 3 nanoparticles is within this size range. As a reference, the r 1 and r 2 values of Gadovist as a commercial molecular agent are provided at the bottom of Figure 9, which shows that this nanoparticle sample is more powerful than Gadovist as a T 1 MRI contrast agent. On the other hand, other Ln 2 O 3 nanoparticles (Ln = Tb, Dy, and Ho) show appreciable to high r 2 values and negligible r 1 values with very large r 2 /r 1 ratios [66]. Therefore, these nanoparticles are suitable as T 2 MRI contrast agents. The r 2 values of these nanoparticles increased with increasing the MR field [66] because r 2 is proportional M 2 [59,60]. Therefore, these nanoparticles are expected to provide appreciable T 2 MR contrast images at high MR fields. For example, PAA-coated Ln 2 O 3 nanoparticles (Ln = Tb and Ho) exhibit appreciable T 2 MR contrast images at 9.4 T MR Field [43]. For comparison, this T 2 contrast is lower than that of superparamagnetic iron oxide nanoparticles possessing high saturation magnetizations (50-80 emu/g) and high r 2 values [67,68].

MR Imaging Properties: r1 and r2 Values
The r1 and r2 values are affected by the number of water molecules around the nanoparticles and the distance between them and, therefore, depend on the water-attracting ability of the ligands around the nanoparticles [59,60,65]. Accordingly, surface-coating ligands should be properly selected. Gd2O3 nanoparticles act as positive (T1) MRI contrast agents due to the high spin magnetic moment of Gd 3+ (s = 7/2); they have high r1 values and r2/r1 ratios close to one [39][40][41] (T1 MRI contrast agents exhibit low r2/r1 ratios, whereas T2 MRI contrast agents have high r2/r1 ratios [23]). The r1 and r2 values of PAAMA-coated Gd2O3 nanoparticles at 3.0 T MR field were determined to be 40.6 and 63.4 s −1 mM −1 (r2/r1 = 1.56), respectively, using the 1/T1 and 1/T2 plots versus Gd-concentration depicted in Figure 9 [40]. This r1 value is approximately 10 times higher than those [21−23] of commercial molecular T1 MRI contrast agents, such as Dotarem, ProHance, Gadovist, Magnevist, and Omniscan [21,22]. The r1 value also depends on the particle diameter of Gd2O3 nanoparticles, for which the optimal value was suggested to be 1.0 to 2.5 nm [24]. The observed particle diameter of the PAAMA-coated Gd2O3 nanoparticles is within this size range. As a reference, the r1 and r2 values of Gadovist as a commercial molecular agent are provided at the bottom of Figure 9, which shows that this nanoparticle sample is more powerful than Gadovist as a T1 MRI contrast agent. On the other hand, other Ln2O3 nanoparticles (Ln = Tb, Dy, and Ho) show appreciable to high r2 values and negligible r1 values with very large r2/r1 ratios [66]. Therefore, these nanoparticles are suitable as T2 MRI contrast agents. The r2 values of these nanoparticles increased with increasing the MR field [66] because r2 is proportional M 2 [59,60]. Therefore, these nanoparticles are expected to provide appreciable T2 MR contrast images at high MR fields. For example, PAA-coated Ln2O3 nanoparticles (Ln = Tb and Ho) exhibit appreciable T2 MR contrast images at 9.4 T MR Field [43]. For comparison, this T2 contrast is lower than that of superparamagnetic iron oxide nanoparticles possessing high saturation magnetizations (50-80 emu/g) and high r2 values [67,68].  Figure 9. r1 and r2 values of PAAMA-coated Gd2O3 nanoparticles [40]. Adapted from [40]. Copyright 2021 MDPI.

Tumor-Targeting T1 MRI Contrast Agents
Gd2O3 nanoparticles are the most widely applied Ln2O3 nanoparticles as in vivo tumor-targeting T1 MRI contrast agents because of the high spin magnetic moment (s = 7/2) of Gd 3+ ( 8 S7/2), which is the largest value among the elements in the periodic table and thus provides very high T1 MR contrast. Therefore, various studies on the synthesis and application of tumor-targeting Gd2O3 nanoparticles have been reported, which are discussed below according to the type of tumor-targeting ligands used.

Tumor-Targeting T 1 MRI Contrast Agents
Gd 2 O 3 nanoparticles are the most widely applied Ln 2 O 3 nanoparticles as in vivo tumor-targeting T 1 MRI contrast agents because of the high spin magnetic moment (s = 7/2) of Gd 3+ ( 8 S 7/2 ), which is the largest value among the elements in the periodic table and thus provides very high T 1 MR contrast. Therefore, various studies on the synthesis and application of tumor-targeting Gd 2 O 3 nanoparticles have been reported, which are discussed below according to the type of tumor-targeting ligands used.

Chlorotoxin (CTX)
Gu et al. developed a mouse brain tumor-targeting contrast agent using CTX as a tumor-targeting ligand [45]. CTX is a peptide consisting of a 36-amino acid sequence found in the venom of the deathstalker scorpion [69]. CTX preferentially binds to glioma cells, enabling the development of therapeutic and diagnostic protocols for several types of tumors [70]. In this study, CTX-PEG-TETT-coated Gd 2 O 3 nanoparticles with a core diameter of 3.46 nm and r 1 value of 8.41 s −1 mM −1 at 7.0 T were applied to tumortargeting imaging in vivo. Figure 10 shows a series of in vivo T 1 MR images of mice with brain glioma tumor after mice tail intravenous injection of PEG-TETT-Gd 2 O 3 and CTX-PEG-TETT-Gd 2 O 3 nanoparticle samples at the same Gd dose (6 mg Gd/kg). The PEG-TETT-Gd 2 O 3 nanoparticles slightly improved the contrast of glioma sites (labeled with arrows) compared with the preinjection image. However, the CTX-PEG-TETT-Gd 2 O 3 nanoparticles exhibited highly enhanced T 1 MR images in brain tumors (labeled with arrows) after injection, clearly supporting that the CTX-PEG-TETT-Gd 2 O 3 nanoparticles act as a tumor-targeting T 1 MRI contrast agent. Gu et al. developed a mouse brain tumor-targeting contrast agent using CTX as a tumor-targeting ligand [45]. CTX is a peptide consisting of a 36-amino acid sequence found in the venom of the deathstalker scorpion [69]. CTX preferentially binds to glioma cells, enabling the development of therapeutic and diagnostic protocols for several types of tumors [70]. In this study, CTX-PEG-TETT-coated Gd2O3 nanoparticles with a core diameter of 3.46 nm and r1 value of 8.41 s -1 mM -1 at 7.0 T were applied to tumor-targeting imaging in vivo. Figure 10 shows a series of in vivo T1 MR images of mice with brain glioma tumor after mice tail intravenous injection of PEG-TETT-Gd2O3 and CTX-PEG-TETT-Gd2O3 nanoparticle samples at the same Gd dose (6 mg Gd/kg). The PEG-TETT-Gd2O3 nanoparticles slightly improved the contrast of glioma sites (labeled with arrows) compared with the preinjection image. However, the CTX-PEG-TETT-Gd2O3 nanoparticles exhibited highly enhanced T1 MR images in brain tumors (labeled with arrows) after injection, clearly supporting that the CTX-PEG-TETT-Gd2O3 nanoparticles act as a tumortargeting T1 MRI contrast agent.

Cyclic RGD (cRGD)
Ahmad et al. investigated Gd2O3 nanoparticles that were directly conjugated with cRGDs [47]. Prior to measuring in vivo T1 MR images to confirm tumor targeting, an in vitro cellular incubation experiment was conducted with cRGD-Gd2O3 nanoparticles to confirm the internalization of the nanoparticles in the tumor cells, which is particularly important in the case of tumor theragnosis. To investigate this, human brain glioma (U87MG) tumor cells were incubated with cRGD-Gd2O3 nanoparticles (0.01 mM Gd). Untreated cells as a control group are shown in Figure 11a. The treated cells exhibited different surface morphologies from that of the control cells due to nanoparticle attachment on the tumor cell surfaces (Figure 11b). The internalization of cRGD-Gd2O3 nanoparticles in the tumor cells was confirmed by analyzing the TEM images of the cells, where no cRGD-Gd2O3 nanoparticles were found in the TEM image of the control cells (Figure 11c), whereas they were found in the treated tumor cells (Figure 11d). In addition, an energydispersive X-ray (EDX) spectroscopy analysis of the circled region in Figure 11d confirmed the presence of Gd (Figure 11e), supporting the internalization of cRGD-Gd2O3 nanoparticles in the U87MG tumor cells.

Cyclic RGD (cRGD)
Ahmad et al. investigated Gd 2 O 3 nanoparticles that were directly conjugated with cRGDs [47]. Prior to measuring in vivo T 1 MR images to confirm tumor targeting, an in vitro cellular incubation experiment was conducted with cRGD-Gd 2 O 3 nanoparticles to confirm the internalization of the nanoparticles in the tumor cells, which is particularly important in the case of tumor theragnosis. To investigate this, human brain glioma (U87MG) tumor cells were incubated with cRGD-Gd 2 O 3 nanoparticles (0.01 mM Gd). Untreated cells as a control group are shown in Figure 11a. The treated cells exhibited different surface morphologies from that of the control cells due to nanoparticle attachment on the tumor cell surfaces (Figure 11b). The internalization of cRGD-Gd 2 O 3 nanoparticles in the tumor cells was confirmed by analyzing the TEM images of the cells, where no cRGD-Gd 2 O 3 nanoparticles were found in the TEM image of the control cells (Figure 11c), whereas they were found in the treated tumor cells (Figure 11d). In addition, an energy-dispersive Xray (EDX) spectroscopy analysis of the circled region in Figure 11d confirmed the presence of Gd (Figure 11e To demonstrate the occurrence of in vivo tumor targeting via T1 MRI, a sample solution containing cRGD-Gd2O3 nanoparticles was injected into the tail vein of a U87MG tumor-bearing mouse inoculated into the liver (injection dose = ~0.1 mmol Gd/kg) [47]. T1 MR images were obtained before and after injection ( Figure 12a). As shown in the figure, tumor targeting was confirmed via positive contrast enhancement in the liver tumor 5 min after injection, indicating the accumulation of the nanoparticles in the tumor. Figure 12b shows a color map of the middle T1 MR image depicted in Figure 12a, in which the contrast enhancement in the liver tumor (i.e., the widely spread brighter part) can be clearly observed. The signal-to-noise ratios (SNRs) of the three regions of interest (ROIs) in the liver, i.e., the normal region (filled square), the tumor region (filled circle), and the necrotic region (filled triangle), as labeled in the inserted T1 MR image, are plotted versus time in Figure 12c. The T1 contrast enhancement in the liver tumor was approximately three times higher than in the normal region of the liver, as shown in the percentage SNR plots in Figure 12d. These results suggest that the cRGD-Gd2O3 nanoparticles act as a tumor-targeting T1 MRI contrast agent. To demonstrate the occurrence of in vivo tumor targeting via T 1 MRI, a sample solution containing cRGD-Gd 2 O 3 nanoparticles was injected into the tail vein of a U87MG tumor-bearing mouse inoculated into the liver (injection dose =~0.1 mmol Gd/kg) [47]. T 1 MR images were obtained before and after injection ( Figure 12a). As shown in the figure, tumor targeting was confirmed via positive contrast enhancement in the liver tumor 5 min after injection, indicating the accumulation of the nanoparticles in the tumor. Figure 12b shows a color map of the middle T 1 MR image depicted in Figure 12a, in which the contrast enhancement in the liver tumor (i.e., the widely spread brighter part) can be clearly observed. The signal-to-noise ratios (SNRs) of the three regions of interest (ROIs) in the liver, i.e., the normal region (filled square), the tumor region (filled circle), and the necrotic region (filled triangle), as labeled in the inserted T 1 MR image, are plotted versus time in Figure 12c. The T 1 contrast enhancement in the liver tumor was approximately three times higher than in the normal region of the liver, as shown in the percentage SNR plots in Figure 12d. These results suggest that the cRGD-Gd 2 O 3 nanoparticles act as a tumor-targeting T 1 MRI contrast agent.

TAT Peptide
Ahmad et al. explored the tumor-imaging application of transactivator of transcription (TAT) peptide-conjugated Gd 2 O 3 nanoparticles [48]. The TAT peptide, which contains 48−57 fragments of the basic domain of the human immunodeficiency virus type 1 TAT protein, exhibits cell-penetrating properties [71]. To date, various nanomaterials grafted with TAT peptides have been applied in gene delivery, drug delivery, and tumor-cell imaging [72][73][74]. Ahmad et al. observed a greater accumulation of TAT peptide-grafted Gd 2 O 3 nanoparticles in tumor cells than in normal cells in a mouse via T 1 MRI in vivo [48]. The TAT peptide allowed cell penetration of the nanoparticles during circulation through angiogenesis via the EPR effect [26,75].

TAT Peptide
Ahmad et al. explored the tumor-imaging application of transactivator of transcription (TAT) peptide-conjugated Gd2O3 nanoparticles [48]. The TAT peptide, which contains 48−57 fragments of the basic domain of the human immunodeficiency virus type 1 TAT protein, exhibits cell-penetrating properties [71]. To date, various nanomaterials grafted with TAT peptides have been applied in gene delivery, drug delivery, and tumor-cell imaging [72][73][74]. Ahmad et al. observed a greater accumulation of TAT peptide-grafted Gd2O3 nanoparticles in tumor cells than in normal cells in a mouse via T1 MRI in vivo [48]. The TAT peptide allowed cell penetration of the nanoparticles during circulation through angiogenesis via the EPR effect [26,75].

Linear RGD
Recently, Ho et al. synthesized highly stable Gd2O3 nanoparticles coated with PAA and then conjugated with liner RGD (Figure 2) to target tumors by binding to αvβ3 and αvβ5 integrins, which are overexpressed in tumor angiogenic sites and tumor cells [44]. The RGD-PAA-Gd2O3 nanoparticles accumulated at the tumor sites, showing enhanced T1 contrast at the tumor sites after injection ( Figure 13). This result was probably due to the combination of various outstanding properties of the RGD-PAA-Gd2O3 nanoparticles, such as the delivery of hundreds of Gd atoms per nanoparticle to the tumor site, tumortargeting ability, and ultrasmall size, which provides them with good transport properties during circulation through blood vessels and tumor-cell penetration. This study indicated that the RGD-PAA-Gd2O3 nanoparticles are applicable to in vivo tumor diagnosis.  (Figure 2) to target tumors by binding to α v β 3 and α v β 5 integrins, which are overexpressed in tumor angiogenic sites and tumor cells [44]. The RGD-PAA-Gd 2 O 3 nanoparticles accumulated at the tumor sites, showing enhanced T 1 contrast at the tumor sites after injection ( Figure 13). This result was probably due to the combination of various outstanding properties of the RGD-PAA-Gd 2 O 3 nanoparticles, such as the delivery of hundreds of Gd atoms per nanoparticle to the tumor site, tumor-targeting ability, and ultrasmall size, which provides them with good transport properties during circulation through blood vessels and tumor-cell penetration. This study indicated that the RGD-PAA-Gd 2 O 3 nanoparticles are applicable to in vivo tumor diagnosis.

MRI-Guided Therapy
One of the main advantages of Gd 2 O 3 nanoparticles as T 1 MRI contrast agents is that they can be used for MRI-guided therapy. For example, Le Duc et al. developed gadolinium-based nanoparticles encapsulated in a polysiloxane shell and then grafted with diethylenetriaminepentaacetic dianhydride for nanoparticle colloidal stabilization [76]. They used the nanoparticles for tumor therapy using X-ray microbeam radiation [77,78], which improved the survival of mice against an aggressive brain tumor. The efficacy of this treatment depended on how accurately MRI contrast agents locate the tumor position and shape. In the T 1 MR images recorded a few minutes after intravenous injection, the delineation of the tumor was clearly distinguished from normal tissues in a rat brain as shown in Figure 14, and X-ray microbeam radiation was then conducted to kill the brain tumor cells.
Pharmaceutics 2021, 13, x FOR PEER REVIEW 13 of 22 Figure 13. T1 MR images before and 20 min after (labeled "post 20 min") intravenous injection into the mouse tail: the bright contrast at the tumor (labeled with arrows) was due to the accumulation of nanoparticles at the tumor site. Adapted from [44]. Copyright 2020 The Royal Society of Chemistry.

MRI-Guided Therapy
One of the main advantages of Gd2O3 nanoparticles as T1 MRI contrast agents is that they can be used for MRI-guided therapy. For example, Le Duc et al. developed gadolinium-based nanoparticles encapsulated in a polysiloxane shell and then grafted with diethylenetriaminepentaacetic dianhydride for nanoparticle colloidal stabilization [76]. They used the nanoparticles for tumor therapy using X-ray microbeam radiation [77,78], which improved the survival of mice against an aggressive brain tumor. The efficacy of this treatment depended on how accurately MRI contrast agents locate the tumor position and shape. In the T1 MR images recorded a few minutes after intravenous injection, the delineation of the tumor was clearly distinguished from normal tissues in a rat brain as shown in Figure 14, and X-ray microbeam radiation was then conducted to kill the brain tumor cells. Figure 14. T1-weighted images of the brain of a rat having intracerebral 9L gliosarcoma (9LGS) before and after (5, 20, and 45 min) intravenous injection of gadolinium-based nanoparticles. Adapted from [76]. Copyright 2011 American Chemical Society.

T2-Weighted MRI
Recently, Ln2O3 (Ln = Tb, Dy, and Ho) nanoparticles with appreciable r2 and negligible r1 values, suitable for T2 MRI were developed [42,43,50,66,79,80].  [43], and negligible r1 values at all MR fields. With such r1 and r2 values, only the exclusive induction of T2 relaxations by the nanoparticles occurs at all MR fields. The appreciable T2 MR contrast enhancements at 9.4 T MR field confirmed the effectiveness of the nanoparticles as T2 MRI contrast agents at high MR fields in vivo [43]. Dy2O3 nanoparticles constitute another example with application in T2 MRI. Thus, D-glucuronic acid-coated Dy2O3 nanoparticles exhibited T2 MR contrast enhancements in a mouse liver at 3.0 T MR field in vivo [79] Figure 13. T 1 MR images before and 20 min after (labeled "post 20 min") intravenous injection into the mouse tail: the bright contrast at the tumor (labeled with arrows) was due to the accumulation of nanoparticles at the tumor site. Adapted from [44]. Copyright 2020 The Royal Society of Chemistry. Figure 13. T1 MR images before and 20 min after (labeled "post 20 min") intravenous injection into the mouse tail: the bright contrast at the tumor (labeled with arrows) was due to the accumulation of nanoparticles at the tumor site. Adapted from [44]. Copyright 2020 The Royal Society of Chemistry.

MRI-Guided Therapy
One of the main advantages of Gd2O3 nanoparticles as T1 MRI contrast agents is that they can be used for MRI-guided therapy. For example, Le Duc et al. developed gadolinium-based nanoparticles encapsulated in a polysiloxane shell and then grafted with diethylenetriaminepentaacetic dianhydride for nanoparticle colloidal stabilization [76]. They used the nanoparticles for tumor therapy using X-ray microbeam radiation [77,78], which improved the survival of mice against an aggressive brain tumor. The efficacy of this treatment depended on how accurately MRI contrast agents locate the tumor position and shape. In the T1 MR images recorded a few minutes after intravenous injection, the delineation of the tumor was clearly distinguished from normal tissues in a rat brain as shown in Figure 14, and X-ray microbeam radiation was then conducted to kill the brain tumor cells. Figure 14. T1-weighted images of the brain of a rat having intracerebral 9L gliosarcoma (9LGS) before and after (5, 20, and 45 min) intravenous injection of gadolinium-based nanoparticles. Adapted from [76]. Copyright 2011 American Chemical Society.

T2-Weighted MRI
Recently, Ln2O3 (Ln = Tb, Dy, and Ho) nanoparticles with appreciable r2 and negligible r1 values, suitable for T2 MRI were developed [42,43,50,66,79,80]. For example, PAAcoated Ln2O3 (Ln = Tb and Ho) nanoparticles exhibited appreciable r2 values at 3.0 T MR field (3.19 s −1 mM −1 for Ln = Tb and 1.44 s −1 mM −1 for Ln = Ho), enhanced r2 values at 9.4 T MR field (16.40 s −1 mM −1 for Ln = Tb and 9.20 s −1 mM −1 for Ln = Ho) [43], and negligible r1 values at all MR fields. With such r1 and r2 values, only the exclusive induction of T2 relaxations by the nanoparticles occurs at all MR fields. The appreciable T2 MR contrast enhancements at 9.4 T MR field confirmed the effectiveness of the nanoparticles as T2 MRI contrast agents at high MR fields in vivo [43]. Dy2O3 nanoparticles constitute another example with application in T2 MRI. Thus, D-glucuronic acid-coated Dy2O3 nanoparticles exhibited T2 MR contrast enhancements in a mouse liver at 3.0 T MR field in vivo [79] Figure 14. T 1 -weighted images of the brain of a rat having intracerebral 9L gliosarcoma (9LGS) before and after (5, 20, and 45 min) intravenous injection of gadolinium-based nanoparticles. Adapted from [76]. Copyright 2011 American Chemical Society.

T 2 -Weighted MRI
Recently, Ln 2 O 3 (Ln = Tb, Dy, and Ho) nanoparticles with appreciable r 2 and negligible r 1 values, suitable for T 2 MRI were developed [42,43,50,66,79,80]. For example, PAA-coated Ln 2 O 3 (Ln = Tb and Ho) nanoparticles exhibited appreciable r 2 values at 3.0 T MR field (3.19 s −1 mM −1 for Ln = Tb and 1.44 s −1 mM −1 for Ln = Ho), enhanced r 2 values at 9.4 T MR field (16.40 s −1 mM −1 for Ln = Tb and 9.20 s −1 mM −1 for Ln = Ho) [43], and negligible r 1 values at all MR fields. With such r 1 and r 2 values, only the exclusive induction of T 2 relaxations by the nanoparticles occurs at all MR fields. The appreciable T 2 MR contrast enhancements at 9.4 T MR field confirmed the effectiveness of the nanoparticles as T 2 MRI contrast agents at high MR fields in vivo [43]. Dy 2 O 3 nanoparticles constitute another example with application in T 2 MRI. Thus, D-glucuronic acid-coated Dy 2 O 3 nanoparticles exhibited T 2 MR contrast enhancements in a mouse liver at 3.0 T MR field in vivo [79] (Figure 15a), proving the potential of Dy 2 O 3 nanoparticles as T 2 MRI contrast agents. Recently, Yue et al. investigated carbon-coated Dy 2 O 3 nanoparticles as a new class of T 2 MRI contrast agent at 3.0 T MR field [80]. The nanoparticles were nearly non-toxic via an in vitro cellular cytotoxicity assay. The presence of numerous hydroxyl groups on the carbon-coating layer conferred the colloidal nanoparticles with stability in aqueous media. The nanoparticles exhibited T 2 contrast enhancement in the mice kidneys after intravenous administration, acting as a T 2 MRI contrast agent (Figure 15b). (Figure 15a), proving the potential of Dy2O3 nanoparticles as T2 MRI contrast agents. Recently, Yue et al. investigated carbon-coated Dy2O3 nanoparticles as a new class of T2 MRI contrast agent at 3.0 T MR field [80]. The nanoparticles were nearly non-toxic via an in vitro cellular cytotoxicity assay. The presence of numerous hydroxyl groups on the carbon-coating layer conferred the colloidal nanoparticles with stability in aqueous media. The nanoparticles exhibited T2 contrast enhancement in the mice kidneys after intravenous administration, acting as a T2 MRI contrast agent (Figure 15b). Figure 15. (a) A series of 3.0 T in vivo T2 MR images: coronal views of kidneys and liver (labeled as "K" and "L", respectively) before and after intravenous injection of an aqueous solution of D-glucuronic acid-coated Dy2O3 nanoparticles into the mouse tail [79]. (b) In vivo coronal 3.0 T T2 MR images of the mice kidneys (indicated with arrows) as a function of time before and after intravenous injection of an aqueous solution of carbon-coated Dy2O3 nanoparticles into the mouse tail [80]. Adapted from [79,80]. Copyrights 2012 Elsevier and 2020 MDPI.

CT Imaging
Lanthanide elements exhibit higher X-ray attenuation coefficients [16] than iodine, which is currently used as a CT contrast agent in its organic molecular forms. Consequently, Ln2O3 nanoparticles can be applied as CT contrast agents [17,18,81,82]. For example, an aqueous solution of Gd2O3 nanoparticles coated with iodine compounds was used to investigate CT imaging in vivo [81]. Brighter contrast enhancements were observed in mouse bladder (labeled B in Figure 16a) after intravenous tail injection. The X-ray absorption in the ROI of the bladder (indicated by a small, dotted circle in Figure 16a) indicated that the contrast reached a maximum at ~30 min after injection and then decreased with time (Figure 16b), suggesting that the sample solution was excreted through the bladder as urine. This result showed that an aqueous solution of Gd2O3 nanoparticles might serve as a CT contrast agent.   [80]. Adapted from [79,80]. Copyrights 2012 Elsevier and 2020 MDPI.

CT Imaging
Lanthanide elements exhibit higher X-ray attenuation coefficients [16] than iodine, which is currently used as a CT contrast agent in its organic molecular forms. Consequently, Ln 2 O 3 nanoparticles can be applied as CT contrast agents [17,18,81,82]. For example, an aqueous solution of Gd 2 O 3 nanoparticles coated with iodine compounds was used to investigate CT imaging in vivo [81]. Brighter contrast enhancements were observed in mouse bladder (labeled B in Figure 16a) after intravenous tail injection. The X-ray absorption in the ROI of the bladder (indicated by a small, dotted circle in Figure 16a) indicated that the contrast reached a maximum at~30 min after injection and then decreased with time (Figure 16b), suggesting that the sample solution was excreted through the bladder as urine. This result showed that an aqueous solution of Gd 2 O 3 nanoparticles might serve as a CT contrast agent.
cently, Yue et al. investigated carbon-coated Dy2O3 nanoparticles as a new class of T2 MRI contrast agent at 3.0 T MR field [80]. The nanoparticles were nearly non-toxic via an in vitro cellular cytotoxicity assay. The presence of numerous hydroxyl groups on the carbon-coating layer conferred the colloidal nanoparticles with stability in aqueous media. The nanoparticles exhibited T2 contrast enhancement in the mice kidneys after intravenous administration, acting as a T2 MRI contrast agent (Figure 15b). Figure 15. (a) A series of 3.0 T in vivo T2 MR images: coronal views of kidneys and liver (labeled as "K" and "L", respectively) before and after intravenous injection of an aqueous solution of D-glucuronic acid-coated Dy2O3 nanoparticles into the mouse tail [79]. (b) In vivo coronal 3.0 T T2 MR images of the mice kidneys (indicated with arrows) as a function of time before and after intravenous injection of an aqueous solution of carbon-coated Dy2O3 nanoparticles into the mouse tail [80]. Adapted from [79,80]. Copyrights 2012 Elsevier and 2020 MDPI.

CT Imaging
Lanthanide elements exhibit higher X-ray attenuation coefficients [16] than iodine, which is currently used as a CT contrast agent in its organic molecular forms. Consequently, Ln2O3 nanoparticles can be applied as CT contrast agents [17,18,81,82]. For example, an aqueous solution of Gd2O3 nanoparticles coated with iodine compounds was used to investigate CT imaging in vivo [81]. Brighter contrast enhancements were observed in mouse bladder (labeled B in Figure 16a) after intravenous tail injection. The X-ray absorption in the ROI of the bladder (indicated by a small, dotted circle in Figure 16a) indicated that the contrast reached a maximum at ~30 min after injection and then decreased with time (Figure 16b), suggesting that the sample solution was excreted through the bladder as urine. This result showed that an aqueous solution of Gd2O3 nanoparticles might serve as a CT contrast agent.  [81]. Adapted from [81]. Copyright 2015 Springer Nature. Figure 16. (a) In vivo CT images of a mouse bladder (labeled B) and (b) plot of the X-ray absorption of the ROI in the bladder (indicated by the small, dotted circle in (a)) before and after intravenous injection of an aqueous sample into the mouse tail [81]. Adapted from [81]. Copyright 2015 Springer Nature.

Ln2O3 Nanoparticle Toxicity
As shown in Figure 17a, bare Gd2O3 nanoparticles exhibit toxicities in both NCTC1469 normal and U87MG tumor-cell lines, whereas PAA-coated Gd2O3 nanoparticles are nearly non-toxic up to 500 µM Gd with cell viabilities of ∼93% in DU145, ∼99% in NCTC1469, and ∼80% in U87MG cell lines (Figure 17b) [83]. Other PAA-coated Ln2O3 nanoparticles (Ln = Dy, Tb, and Ho) also exhibit very low cytotoxicities in both DU145 and NCTC1469 cell lines (Figure 17c-e) [42,43], showing good biocompatibilities. These examples demonstrate that Ln2O3 nanoparticles must be well protected with watersoluble and biocompatible ligands for biomedical applications. Lanthanides are relatively non-toxic elements [37,38]. For example, for lanthanide chlorides, the lethal dose causing the death of 50% of a group of 10 animals (LD50) is Lanthanides are relatively non-toxic elements [37,38]. For example, for lanthanide chlorides, the lethal dose causing the death of 50% of a group of 10 animals (LD50) is higher than 10 and 450 mg per kg bodyweight for intravenous and intraperitoneal injections, respectively [84]. In the case of Ln 2 O 3 nanoparticles, other properties, either alone or in concert, must be considered to evaluate the possible toxic effects of the nanoparticles. These properties include chemical composition, doping, hydrodynamic size, shape, redox properties, tendency for aggregation, composition of the shell or coating material, surface modifications, colloidal stability, solubility, biodegradability, concentration, and duration of exposure [85]. Although several studies have investigated the toxicity of lanthanide-based nanoparticles having different properties, such as chemical composition, size, surface ligands, and lanthanide concentration [86][87][88], the lack of literature data has prevented drawing firm conclusions for the assessment of the potential toxicity of Ln 2 O 3 nanoparticles. Furthermore, several aspects of the biological interaction of Ln 2 O 3 nanoparticles in living systems still need to be unveiled. For example, despite in vitro cytotoxicity studies being conducted using various cell cultures, these studies did not provide information about long-term safety and cytotoxicity [89,90], for which in vivo cytotoxicity studies would be needed.
Nevertheless, several important conclusions can be extracted from the currently available literature data. In the case of biomedical applications, Ln 2 O 3 nanoparticles are usually introduced into the body by intravenous injections and circulated by the bloodstream primarily to organs, such as the liver and spleen, kidneys, heart, lungs, and brain [91]. The possible retention or uptake in blood and organs strongly depends on the surface properties of the nanoparticles. Ligand coating can promote the interaction of the Ln 2 O 3 nanoparticles with the cell membranes, favoring the internalization of the nanoparticles by various types of cells. However, biologically inert coating ligands, such as PEG, may prolong the circulation in the bloodstream of nanoparticles [11]. In addition, nanoparticle size is an important factor for the excretion route [92,93]. Thus, nanoparticles smaller than 3 nm can be excreted by renal filtration [19,20], whereas those larger than 3 nm are enclosed by a phagocyte system. As a part of the immune system, the phagocyte system is composed of several types of phagocytic cells in the reticular tissue within the body, whose main function is to remove undesired species, such as bacteria, viruses, and foreign materials, including nanoparticles.
The toxicity of Ln 2 O 3 nanoparticles differs depending on the lanthanide ions. For example, Er 2 O 3 shows higher toxicity than Gd 2 O 3 and La 2 O 3 and is highly toxic to zebrafish embryos at a concentration of 50 ppm Er, causing significant mortality and morphological malformations [93]. Meanwhile, the toxicity of Gd 2 O 3 nanoparticles is primarily attributed to the release of Gd 3+ ions [94][95][96]. The toxicity of Gd 3+ ions has been addressed not only for Gd 2 O 3 nanoparticles but also for a variety of molecular Gd 3+ -chelates. In the case of Gd 2 O 3 nanomaterials, Eu 3+ -doped Gd 2 O 3 (Gd 2 O 3 :Eu 3+ ) nanotubes can adversely affect bone marrow stromal cells (BMSCs) [97]. Yang et al. reported the application of 153 Sm-doped Gd(OH) 3 nanorods as a potential MRI contrast agent [98]. In vitro cell toxicity tests revealed that Gd(OH) 3 nanorods have no toxic effect on cellular proliferation and viability. Furthermore, an in vivo toxicity test using Kunming mice showed that injection up to 100 mg/kg of Gd(OH) 3 nanorods had no toxic effect up to 150 days after exposure. However, this in vivo test investigated the short-term toxic effect of Gd(OH) 3 nanorods. In contrast, the long-term toxicity of clinically used Gd 3+ -chelates was reported. When used in patients with severely compromised kidney function, Gd 3+ -chelates promoted the development of nephrogenic systemic fibrosis (NSF), which is a rare disease affecting different parts of the body that can lead to thickening or hardening of the skin and deposits [99] (Figure 18). Moreover, NSF is a progressive condition that can be fatal because it may cause multiple organ failure [99][100][101][102]. Gd 3+ retention in the body, which is greater with linear structured Gd 3+ -chelates than with macrocyclic structured Gd 3+ -chelates, was demonstrated to be associated with NSF.
Compared with clinically used Gd 3+ -chelates, the toxicity of Ln 2 O 3 nanoparticles, including Gd 2 O 3 nanoparticles, has been less explored. For instance, the extent, mechanism, chemical form, and clinical implications of chronic lanthanide retention for Ln 2 O 3 nanoparticles remain unknown. Therefore, more comprehensive investigations are required to improve our understanding of Ln 2 O 3 nanoparticle toxicity and its clinical importance. In this context, the development of new experimental techniques may play a significant role in unveiling lanthanide nanotoxicity. For example, nanotoxicogenomics [103], which uses DNA microarray technologies to investigate the impact of nanoparticles on global gene expression profiles of cells and tissues, has emerged as a new field of toxicology to provide new and important insights into the toxicity of Ln 2 O 3 nanoparticles.
Ln2O3 nanoparticles remain unknown. Therefore, more comprehensive investigations are required to improve our understanding of Ln2O3 nanoparticle toxicity and its clinical importance. In this context, the development of new experimental techniques may play a significant role in unveiling lanthanide nanotoxicity. For example, nanotoxicogenomics [103], which uses DNA microarray technologies to investigate the impact of nanoparticles on global gene expression profiles of cells and tissues, has emerged as a new field of toxicology to provide new and important insights into the toxicity of Ln2O3 nanoparticles.

Conclusions and Perspectives
Recent studies on the synthesis, surface modification, tumor-targeting ligand conjugation, toxicity, and novel biomedical applications to tumor-targeting T1 MRI and image-guided tumor therapy of Ln2O3 nanoparticles were reviewed here. In addition, T2 MRI and CT imaging applications were also discussed. High-quality Ln2O3 nanoparticles can be synthesized via the polyol method and organic-phase thermal decomposition method and then surface-coated with hydrophilic and biocompatible ligands for colloidal stability and biocompatibility. Polymers are more efficient than small molecules as surface-coating ligands because of their many -COOH, -NH2, -OH groups, which can be attached to nanoparticles and conjugated with tumor-targeting ligands and drugs.
The interest in Ln2O3 nanoparticles in the field of medical imaging lies principally in the excellent imaging properties of lanthanides, which arises from their appreciable paramagnetic moments at room temperature; Gd 3+ has the highest 4f-electron spin magnetic moment (s = 7/2) among the elements in the periodic table, which is extremely useful for T1 MRI, and other Ln 3+ ions (Ln = Tb, Dy, and Ho) have a very high 4f-electron spin-orbital magnetic moment, which is suitable for T2 MRI. In addition, lanthanide elements possess higher X-ray attenuation coefficients than iodine, which is currently used as a CT contrast agent in its organic compound forms. The potential of Ln2O3 nanoparticles as CT contrast agents has been confirmed by recording in vivo CT images. In the case of tumor targeting, Gd2O3 nanoparticles are the most widely investigated Ln2O3 nanoparticles due to their very high T1 MRI sensitivity. Various tumor-targeting ligands have been conjugated to Gd2O3 nanoparticles for tumor imaging and T1 MRI-guided tumor therapy. Especially, the enhanced accumulation of tumor-targeting ligand-coated Gd2O3 nanoparticles at tumor cells compared with that at normal cells allowed the development of precise image-guided tumor therapies via clear distinction and delineation of tumors from normal tissues.
As reviewed here, further intensive research is essential to achieve the ultimate goal of using Ln2O3 nanoparticles for tumor-targeting diagnosis and therapy and nontumortargeting medical imaging. In addition, sophisticated toxicological and pharmacological improvements are required to demonstrate the safety of nanoparticle formulations prior to clinical trials. We hope that this review will guide the future development of biomedical applications of Ln2O3 nanoparticles.

Conclusions and Perspectives
Recent studies on the synthesis, surface modification, tumor-targeting ligand conjugation, toxicity, and novel biomedical applications to tumor-targeting T 1 MRI and imageguided tumor therapy of Ln 2 O 3 nanoparticles were reviewed here. In addition, T 2 MRI and CT imaging applications were also discussed. High-quality Ln 2 O 3 nanoparticles can be synthesized via the polyol method and organic-phase thermal decomposition method and then surface-coated with hydrophilic and biocompatible ligands for colloidal stability and biocompatibility. Polymers are more efficient than small molecules as surface-coating ligands because of their many -COOH, -NH 2 , -OH groups, which can be attached to nanoparticles and conjugated with tumor-targeting ligands and drugs.
The interest in Ln 2 O 3 nanoparticles in the field of medical imaging lies principally in the excellent imaging properties of lanthanides, which arises from their appreciable paramagnetic moments at room temperature; Gd 3+ has the highest 4f-electron spin magnetic moment (s = 7/2) among the elements in the periodic table, which is extremely useful for T 1 MRI, and other Ln 3+ ions (Ln = Tb, Dy, and Ho) have a very high 4f-electron spin-orbital magnetic moment, which is suitable for T 2 MRI. In addition, lanthanide elements possess higher X-ray attenuation coefficients than iodine, which is currently used as a CT contrast agent in its organic compound forms. The potential of Ln 2 O 3 nanoparticles as CT contrast agents has been confirmed by recording in vivo CT images. In the case of tumor targeting, Gd 2 O 3 nanoparticles are the most widely investigated Ln 2 O 3 nanoparticles due to their very high T 1 MRI sensitivity. Various tumor-targeting ligands have been conjugated to Gd 2 O 3 nanoparticles for tumor imaging and T 1 MRI-guided tumor therapy. Especially, the enhanced accumulation of tumor-targeting ligand-coated Gd 2 O 3 nanoparticles at tumor cells compared with that at normal cells allowed the development of precise image-guided tumor therapies via clear distinction and delineation of tumors from normal tissues.
As reviewed here, further intensive research is essential to achieve the ultimate goal of using Ln 2 O 3 nanoparticles for tumor-targeting diagnosis and therapy and nontumortargeting medical imaging. In addition, sophisticated toxicological and pharmacological improvements are required to demonstrate the safety of nanoparticle formulations prior to clinical trials. We hope that this review will guide the future development of biomedical applications of Ln 2 O 3 nanoparticles.