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][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][4,5,6,7]. Moreover, nanoparticles can be easily surface-functionalized for advanced imaging and tumor targeting ^[8][9][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][10,11].

Lanthanide oxide (Ln₂O₃) 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]}[12,13,14,15], and high X-ray attenuation power, which is useful for X-ray computed tomography (CT) ^[16][17][18][16,17,18]. In addition, surface-modified Ln₂O₃ nanoparticles exhibit improved properties, such as high-water proton spin relaxivities, high colloidal stabilities, and low toxicities ^[12][13][14][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 intravenous injection ^[19][20][19,20]. Ln₂O₃ nanoparticles meet such requirements, displaying excellent MRI and CT imaging properties at ultrasmall particle diameters ^{[12][13][14][15]}[12,13,14,15].

The development of tumor-targeting Ln₂O₃ nanoparticles is challenging. Especially when compared with commercial molecular Gd-chelates ^[21][22][23][21,22,23], Gd₂O₃ nanoparticles are more efficient longitudinal relaxation promoters ^{[12][13][14][15]}[12,13,14,15] because their longitudinal relaxivity (r₁) values are higher than those (i.e., 3–5 s^‒1mM^‒1) ^[21][22][23][21,22,23] of commercial molecular MRI contrast agents. Therefore, Gd₂O₃ nanoparticles can provide very high contrast T₁ MR images and thus, are ideal candidates for tumor-targeting T₁ MRI contrast agents. In particular, their r₁ value is optimal at ultrasmall nanoparticle size (1.0–2.5 nm) ^[24][25][24,25]. Meanwhile, other Ln₂O₃ nanoparticles (Ln = Dy, Tb, and Ho) are eligible as T₂ MRI contrast agents ^[12][13][15][12,13,15].

Nanoparticles alone accumulate nonspecifically in tumors via passive targeting, i.e., the enhanced permeability and retention (EPR) effect [26]. The accumulation amount and specificity to tumors can be enhanced by active targeting, which is commonly achieved by modifying contrast agents with tumor-targeting ligands that can selectively bind to receptors overexpressed on tumor-cell membranes. Such tumor-targeting ligands include small molecules, such as arginylglycylaspartic acids (Arg-Gly-Asp or RGDs) ^[27][28][29][27,28,29] and folic acid ^[30][31][30,31], peptides, such as chlorotoxin (CTX) ^[32][33][32,33], and biological molecules, such as antibodies [34]. Nanoparticles can provide a flexible platform to attach tumor-targeting ligands, thereby improving their specificity and effectiveness for tumor treatment. In addition, anticancer drugs can also be attached to nanoparticle surfaces for chemotherapy. Generally, in this type of treatment, most anticancer drugs cannot differentiate between tumor and normal cells, causing toxic side effects ^[35][36][35,36]. However, such side effects can be minimized or eliminated by delivering drugs via tumor-targeting nanoparticles. However, the cytotoxicity and biocompatibility of lanthanides during and after endocytosis by cells are still largely unknown, although numerous reports describe lanthanides as relatively non-toxic elements ^[37][38][37,38].

Among the various methods currently available for the synthesis of ultrasmall Ln₂O₃ nanoparticles, the synthesis in a polyol solvent is preferred for biomedical applications because ultrasmall nanoparticles are obtained (average particle diameter = 2.0 nm) and subsequent surface coating of the nanoparticles with hydrophilic and biocompatible ligands can be performed in one pot ^[17][31][17,31]. A general reaction scheme for the polyol synthesis is provided in Figure 1.

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]}[39,40,41,42,43]. In addition, hydrophilic polymers can provide higher r₁ values than small molecular ligands ^[39][40][41][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][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 Gd₂O₃ nanoparticles were reported by Ho et al. [44] (Figure 2). 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, CTX-poly(ethylene glycol)-N-(trimethoxysilylpropyl) ethylenediamine triacetic acid silane-coated 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]. Figure 3. Schematic illustration for the synthesis of CTX-PEG-TETT-Gd₂O₃ nanoparticles. TETT = N-(trimethoxysilylpropyl) ethylenediamine triacetic acid trisodium salt and OA = oleic acid. Reproduced from [45], The Royal Society of Chemistry, 2014.

3. Ln₂O₃ Nanoparticle Toxicity

As shown in Figure 417a, 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 417b) ^[46][83]. Other PAA-coated Ln₂O₃ nanoparticles (Ln = Dy, Tb, and Ho) also exhibit very low cytotoxicities in both DU145 and NCTC1469 cell lines (Figure 417c–e) ^[42][43][42,43], showing good biocompatibilities. These examples demonstrate that Ln₂O₃ nanoparticles must be well protected with water-soluble and biocompatible ligands for biomedical applications. Lanthanides are relatively non-toxic elements ^[37][38][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 ^[47][84]. In the case of Ln₂O₃ 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 ^[48][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 ^[49][50][51][86,87,88], the lack of literature data has prevented drawing firm conclusions for the assessment of the potential toxicity of Ln₂O₃ nanoparticles. Furthermore, several aspects of the biological interaction of Ln₂O₃ 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 ^[52][53][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, Ln₂O₃ 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 ^[54][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 Ln₂O₃ 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 ^[55][56][92,93]. Thus, nanoparticles smaller than 3 nm can be excreted by renal filtration ^[19][20][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 Ln₂O₃ nanoparticles differs depending on the lanthanide ions. For example, Er₂O₃ shows higher toxicity than Gd₂O₃ and La₂O₃ and is highly toxic to zebrafish embryos at a concentration of 50 ppm Er, causing significant mortality and morphological malformations ^[56][93]. Meanwhile, the toxicity of Gd₂O₃ nanoparticles is primarily attributed to the release of Gd³⁺ ions ^[57][58][59][94,95,96]. The toxicity of Gd³⁺ ions has been addressed not only for Gd₂O₃ nanoparticles but also for a variety of molecular Gd³⁺-chelates. In the case of Gd₂O₃ nanomaterials, Eu³⁺-doped Gd₂O₃ (Gd₂O₃:Eu³⁺) nanotubes can adversely affect bone marrow stromal cells (BMSCs) ^[60][97]. Yang et al. reported the application of ¹⁵³Sm-doped Gd(OH)₃ nanorods as a potential MRI contrast agent ^[61][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 Gd³⁺-chelates was reported. When used in patients with severely compromised kidney function, Gd³⁺-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 ^[62][99] (Figure 518). Moreover, NSF is a progressive condition that can be fatal because it may cause multiple organ failure ^{[62][63][64][65]}[99,100,101,102]. Gd³⁺ retention in the body, which is greater with linear structured Gd³⁺-chelates than with macrocyclic structured Gd³⁺-chelates, was demonstrated to be associated with NSF.

Figure 518. Clinical pictures of the legs of two patients with NSF for (a) about three years and (b) four weeks. Adapted from ^[62][99]. Copyright 2007 International Society of Nephrology.

Compared with clinically used Gd³⁺-chelates, the toxicity of Ln₂O₃ nanoparticles, including Gd₂O₃ nanoparticles, has been less explored. For instance, the extent, mechanism, chemical form, and clinical implications of chronic lanthanide retention for 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 ^[66][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.

4. 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 introduced 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; Gd³⁺ 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 Ln³⁺ 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.

Further intensive research is essential to achieve the ultimate goal of using Ln₂O₃ nanoparticles for tumor-targeting diagnosis and therapy and nontumor-targeting 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.