Recent Advances in Gadolinium Based Contrast Agents for Bioimaging Applications

Gadolinium (Gd) based contrast agents (CAs) (Gd-CAs) represent one of the most advanced developments in the application of Gd for magnetic resonance imaging (MRI). Current challenges with existing CAs generated an urgent requirement to develop multimodal CAs with good biocompatibility, low toxicity, and prolonged circulation time. This review discussed the Gd-CAs used in bioimaging applications, addressing their advantages and limitations. Future research is required to establish the safety, efficacy and theragnostic capabilities of Gd-CAs. Nevertheless, these Gd-CAs offer extraordinary potential as imaging CAs and promise to benefit bioimaging applications significantly.


Introduction
Nanotechnology has paved the way for the development of new therapeutics leading to efficient theragnostics of severe diseases. In the last few decades, the integration of nanoparticles (NPs) has dramatically impacted medical imaging. Significant advances have evolved in synthetic methodologies to develop a variety of nanomaterials with controllable size and shape, physicochemical characteristics, and surface charge of nanomaterials. These nanomaterials could be decorated with other NPs, polymers, and bioactive compounds to enhance their targeting, biosensing, and biocompatibility. These technological advances have exemplified their importance in biological imaging applications [1]. The utilization of NPs in disease diagnosis dates back to 1990, when it was first used commercially as a liver imaging agent and is currently used for bowel imaging under the trade names Gastromarks/Lumirems [2]. Subsequent progress in understanding size-dependent physical and chemical parameters of NPs has led to substantial progress in this research area. Many non-invasive medical imaging methodologies have been developed and are used in clinical diagnosis and drug discovery research. These molecular imaging techniques involve advancement in molecular probes to visualize cellular function and characterize and measure the molecular processes inside the living organisms [3]. The evolution in synthetic technologies of NPs in collaboration with the intense development of imaging modalities has significantly advanced disease detection. Progress in targeted contrast agents (CAs) has advanced the selective imaging of specific biological events and processes considerably with enhanced detection limits and imaging modalities, establishing these

Magnetic Resonance Imaging (MRI)
The MRI technique operates on the basic nuclear magnetic resonance (NMR) principles and generates images using the tissue contrast produced through NMR signals. Gadolinium-based MRI contrast agents operate by shortening the T 1 and T 2 relaxation time of protons present inside the tissues, thus enhance the image contrast. This contrast imaging can be further improved by the use of CAs, which enhances the signal intensity between the tissue of interest (tumor) and the background tissue (normal tissues). There are several parameters such as longitudinal relaxation time (T 1 ), Transverse relaxation time (T 2 ), and Spin density (ρ) which affect the signal intensity and the extent of contrast obtained from a sample. These aspects are crucial in designing MRI contrast agents, which aids in generating contrast images for diagnosis. MRIs are limited by the factors of cost and longer imaging times. Nevertheless, MRI contrast agents can efficiently detect lesions and differentiate them from healthy tissues [9].

Optical Imaging (OI)
Optical imaging utilizes different physical parameters of light interaction with tissues to generate images. Among all the optical imaging techniques, fluorescence microscopy has evolved as a prominent imaging technique that depends on the fluorophore's inherent property (i.e., lanthanide compounds, organic dyes, etc.). Fluorescence imaging (FI), and particularly near-infrared fluorescence (NIRF) imaging, provides the highest spatial resolution on a microscopic level for disease diagnosis. However, this technique is limited by the factors of limited penetration depth and auto-fluorescence in various tissues, which hinders its clinical utility [10]. Several efforts have been made to overcome these limitations by the use of NPs. Surplus loading of fluorescent dyes in NPs can increase signal [11], modified NPs can prevent the NIRF quenching [12], and local lesion concentration of the fluorescent dye can be increased by increasing NPs concentration [13]. There have been several studies where nanoparticle FI was used to detect genes, analyze protein, evaluate enzyme activity, trace elements, and cells and for the early-stage diagnosis of tumors [14][15][16][17][18].

Computed Tomography (CT) Imaging
A CT scan generates tomographic (cross-sectional) images of a tissue using X-ray measurements taken from different angles. Although this technique involves larger radiation exposure, it still has gained importance due to its faster examination speed, improved efficiency, high spatial resolution, and cost-effectiveness. Current CT contrast agents are mainly iodine-based and limited by fast clearance rates and adverse side effects. Nanosized CT contrast agents have emerged as potential substitutes surmounting these limitations. There are two methods to synthesize these contrast agents; the first is iodine-based contrast agents where iodine is loaded with NPs [19], and the second is based on metals NPs having high X-ray attenuation coefficients [20]. These contrast agents based on metal NPs are utilized in multiple areas based on their ability to generate attenuation, cellular uptake and targeting capabilities.

Ultrasound (US) Imaging
Ultrasound (US) imaging is a technique that utilizes pulses of US to generate images of tissue using a probe where the US pulses are echoed off tissues with different reflection properties returning back to the probe, which records and exhibit them as an image. It is one of the most commonly used imaging techniques owing to its high spatial resolution, probability, and cost-effectiveness. Commercial US contrast agents are comprised of microbubbles [21]. These microbubbles are limited by the factors of relatively short circulation lifetime, low stability and provide only blood pool contrast signals [22]. NPs with particle sizes ranging from 100-1000 nm have been investigated to surmount these limitations [23]. These NPs can be attached with several surface ligands to impart specific target binding abilities. These US contrast agents are synthesized in three types based on their composition. Most common agents utilize microbubbles (gas-based) to create acoustic reflections. The second type is smaller than gas-based particles and utilizes a solid-based NP with relatively larger scattering acoustic signals. The third type is based on liquid-based NP, which generates an acoustic signal due to the difference in speeds of sound transmission and water. These NP contrast agents are comparatively much smaller than current US contrast agents used and, due to their smaller size and supplementary surface labelling, provide better lesion targeting.

Positron Emission Tomography (PET), Single Photon Emission Computed Tomography (SPECT) Imaging
PET and SPECT are imaging techniques that provide detailed metabolic information. PET provides real-time quantitative imaging analysis with high tissue penetration and high sensitivity. SPECT possesses similar advantages as PET imaging, but both are disadvantageous in high cost and radioactive exposure. In addition, PET/SPECT NPs imaging tracers require nuclides with a long half-life, where radionuclides used in SPECT generally have longer half-lives. They are most commonly used in tumor imaging, where the images are obtained via specific binding to receptors [24].

Nanoparticles in Molecular Imaging
In the last few decades, nanomaterials have gained considerable attention and have been extensively explored for their potential application in the field of molecular imaging, especially in cancer therapeutics [25]. Various parameters such as particles size, shape, charge, and hydrophilicity of the NPs are crucial for the effectiveness of the designed contrast agents. Medical imaging CAs currently used are mostly the small molecules exhibiting swift metabolism, having a non-specific distribution with undesirable toxicities [26]. NPs owing to their nano size, exhibit greater permeability and retention effects inside the tumor cells [27]. Nanoparticle's size plays a crucial role in biodistribution, cellular uptake, blood circulation half-life and tumor penetration [28]. NPs having a particle size smaller than 10 nm pass through the renal filtration pore having a pore size of 10 nm and thus get rapid clearance through the renal excretion system [29,30], whereas NPs over 100 nm in size can be easily identified by macrophages and accumulate in lung, liver, lymph nodes and spleen [31], while other reports suggested that NPs with size range between 10 and 60 nm have increased cellular uptake [32]. Particle charge is yet another factor; positively charged particles improve endocytosis or phagocytosis for cell labelling [33]. Modification and functionalization of NPs provide a platform for improvement over current contrast media by improving specificity, prolonged circulation half-life, and in vitro and in vivo stability [30,[34][35][36]. The addition of targeting ligands significantly enhances the specificity and increases nanoparticle-tumor interactions for tumorous tissues [37]. NPs functionalized with targeting ligands such as peptides, antibodies, small molecules and proteins for efficient drug delivery and diagnosis [38][39][40][41][42]. Functionalization of NPs have been reported to achieve higher affinity towards certain cell surface receptor proteins overexpressed in various cancer cells [43]. Another approach is modifying NPs using antibodies that impart labelling of cells and tissues but is limited to cost-effectiveness and smaller shelf life [3]. Small organic molecules are conjugated with NPs to impart bioimaging which is an excellent alternative to modifies NPs having limited shelf life [44,45]. Certain factors are crucial in the synthesis of NPs for bioimaging applications. The primary requirement is the synthesis of optical core encapsulating the fluorochrome [46][47][48][49]. To improve the photostability and for the protection of the optical core, a shell is synthesized. For the prevention of coagulation or agglomeration, it is necessary to modify the NPs to maintain a dispersed state which is achieved by using surfactants, polymers, chelating groups, etc. [50,51]. Finally, to achieve specificity and increase the bioconjugation and targeting of NPs, it is necessary to attach them to suitable biomolecules such as enzymes, antibodies, peptides, drugs etc., which also promotes or maintains their dispersion [40,[52][53][54]. All of these factors are required in designing optimum nanoparticulate systems for bio-imaging applications.
Most of the clinically available CAs used in MRI are based on paramagnetic Gd complexes [55]. Magnevist, a Gadolinium (Gd) (III) chelate, is a widely used MRI Cas [56,57]. However, it is associated with potential biotoxicity issues. It is limited by the factors of relatively lower relaxivity and shorter blood circulation time [58][59][60]. Gadolinium (III)-based NPs (Gd-NPs) have found their place in biomedical applications due to their excellent characteristics. They are utilized to formulate enhanced CAs as these NPs are thermodynamically and kinetically stable, possess high relaxivity, have good water solubility and in vivo stability, exhibit low toxicity, and offer better control over the molecular size and functionalization [61][62][63][64][65][66][67]. Free Gadolinium (Gd) ions are toxic in nature and pose significant threats to the human body in their free form. To reduce the toxicity of free Gd ions, they are encapsulated by a ligand or inside a material. Two principal ways are utilized to exploit their properties as nanomaterials: (i) grafting of Gd chelates inside or on the surface of the nanomaterials, (ii) development of Gd crystalline NPs. These methods are advantageous in increasing the number of Gd in the CAs for obtaining a better signal or to increase the therapeutic effects [68]. The trivalent Gd(III) ion possesses the highest electron spin magnetic moment (S = 7/2) of elements in the periodic table as it has the greatest number of unpaired electrons (seven) in its 4f-orbital ( 8 S 7/2 ). Owing to this, Gd-NPs has gained significant attention due to their enhanced r 1 values, owing to a large amount of Gd per nanoparticle, providing stronger contrast [69,70]. The r 1 and transverse water proton spin relaxivity (r 2 ) of Gd-NPs depend on the ligand size coated on its surface. Kim et al. studied this dependence and found that r1 and r2 values decreased with increasing ligand size due to the ligand size effect [71]. In another study, the relaxometric properties of Gd-NPs coated with several ligands such as small diacids with hydrophobic chains, namely, succinic acid, glutaric acid, and terephthalic acid, and large polyethyleneimine (PEI) with hydrophilic chains, namely, PEI-1300 and PEI-10000 were investigated, where ligand-size and ligand-chain hydrophilicity effects were observed. The r 1 and r 2 water proton relaxivities were found to be decreasing with increasing ligand-size. The ligand-size effect was weaker in case of PEI since its hydrophilic chains permitted water molecules to access the NPs (the ligand-chain hydrophilicity effect). This result was explained on the basis of the magnetic dipole interaction between the dipoles of the nanoparticle and water protons [72]. The r 2 /r 1 ratio of T 1 MRI contrast agent is also an important factor while designing contrast agents. An ideal T 1 MRI contrast agent should have a r 2 /r 1 ratio close to 1. Bony et al. synthesized d-glucuronic acid-coated Cu(II)/Gd(III) oxide nanoparticle, which showed r 1 = 13.78 s −1 ·mM −1 and r 2 = 14.48 s −1 ·mM −1 (r 2 /r 1 = 1.05). This is due to the reduction in magnetization of mixed NPs caused by the mixing of NPs [73].
The properties of Gd include the largest spin magnetic moment among all the elements, a high X-ray attenuation coefficient (m) and the highest thermal neutron capture crosssection (s) among all stable radioisotopes. The first property allows Gd agents to have a very high longitudinal water proton relaxivity (r 1 ), useful for positive (T 1 ) MRI. The second property is effective for CT. The third property is beneficial for neutron capture therapy (NCT) of tumors. Given these properties, Gd agents might be used for the theragnosis a malignant tumor (i.e., diagnose the malignant tumor by way of T 1 MRI or CT and treat it by NCT) as outlined in Figure 1 [74]. Table 2 represent the Single/multimodal bioimaging application of Gd-CAs.

Naturally Derived Polymers
Functionalization of Gd compounds with naturally derived polymers such as proteins, liposomes, saccharides etc., is obtained through chemical conjugation, chelation or encapsulation. These polymers enhance relaxivity, provides additional functionalization sites and greatly increases the biocompatibility of the synthesized agents. Albumins are globular proteins, mostly serum albumins. They have been extensively utilized in developing Gd-CAs due to their amphiphilic character and affinity towards a variety of malignant tumors. In a study, Gd-albumin conjugates were synthesized having relaxivity of approximately 9~10.5 s −1 ·mM −1 at 3 tesla (T), which is greater than the reported value for Magnevist ® [110]. In another approach, encapsulation of Gd-NPs inside the albumin-folic acid NPs resulted in reduced toxicity and increased r1 value of 10.8 s −1 mM −1 at 0.47 T [111]. Ahmad et al. synthesized polyethylene glycol diacid (PEGD) coated Gd-NPs (PEGD-Gd-NPs, the average particle diameter (d avg = 2.0 nm)), conjugated with bovine serum albumin (BSA/cleaved-BSA (C-BSA) (i.e., BSA-PEGD-Gd-NPs and C-BSA-PEGD-Gd-NPs) by reacting amine group of albumins with the acidic moiety of PEGD ( Figure 2). Large relaxivities values i.e., r 1 = 6.0 s −1 ·mM −1 and r 2 = 28.0 s −1 mM −1 for BSA-PEGD-Gd-NPs and r 1 = 7.6 s −1 ·mM −1 and r 2 = 22.0 s −1 ·mM −1 for C-BSA-PEGD-Gd-NPs, were observed along with significant negative contrast enhancements [44]. Gd-NPs coated with trans-activator of transcription (TAT) peptide with the cellpenetrating ability (i.e., TAT-Gd-NPs) were synthesized through a one-pot process exhibiting r 1 value greater than those of commercial Gd-chelates. Synthesized TAT-Gd-NPs possessed the d avg of 1.5 nm with a r 1 of 18.2 s −1 ·mM −1 and r 2 /r 1 = 1.6 [112]. There are several cancer-related proteins that are abundantly present in the tumor extracellular matrix. These proteins can be used to functionalize Gd-CAs to impart tumor specificity. Therefore, cyclic RGD-conjugated Gd-NPs have been reported to be employed for tumor-targeting T 1 MRI. Five types of commercial cyclic RGDs (cRGDs) were used as a tumor-targeting ligand to coat Gd-NPs having particle d avg ranging from 1.0-2.5 nm. Synthesized con-jugates exhibited r 1 values of 10.0-18.7 s −1 ·mM −1 , with r 2 /r 1 ratios of 1.4-1.7, which is approximately 3-5 times higher than the values reported for commercial Gd chelates. In addition, approximately 3 times contrast enhancements in the T 1 MR images were observed [25]. In a similar approach, Gd-DOTA-cRGD was used to target tumor correlated α v β 3 -receptor. Synthesized Gd-CAs exhibited relaxivity r 1 of~7.4 s −1 ·mM −1 at 1.5 T (64 MHz) and moderate specificity for the α v β 3 -receptor in hepatocellular carcinoma in in vivo studies [113]. Yang et al. developed a Gd-integrated polypyrrole nano-theragnostic agent (PPy@BSA-Gd) by using BSA as polymerization and biomimetic mineralization stabilizer, possessing high stability and appreciable photothermal property. These agents possess good cytocompatibility and a relaxivity value, r 1 = 10.203 s −1 ·mM −1 , posing them as a potential probe for T 1 MRI and applications in photothermal therapy [114].

Saccharides and Their Derivatives
Saccharides stimulate the cell response and are widely used as tumor targeting agents [115]. Gd-CAs modified with several saccharides or their derivative have gained attention by researchers. For instance, cyclodextrin (CD), glucosamine, chitosan (CS), and dextran conjugation with Gd-CAs have been reported to impart tumor specificity and enhance biocompatibility. Gd-CAs modified with saccharides, and their derivatives have become a common approach, especially CD [116], dextran [117], glucosamine [118], and CS [119] are widely investigated ones. β-CD-based polyester was used to coat Gd-NPs and targeted by folic acid (FA) to develop novel targeted MRI CAs. The developed Gd-NPs@ β-CD-FA MRI CAs revealed no significant cytotoxicity and possessed high biocompatibility up to 500 µg concentration of Gd 3+ /mL. In vitro MRI experiments revealed targeted contrast T 1 and T 2 weighted MR imaging [120]. Bony et al. synthesized D-glucuronic acid coated Ln/Mn (Ln = Gd and Dy) oxide NPs (d avg = 2.0 nm). The D-glucuronic acid coated Gd-NPs displayed strong positive contrast (T 1 MRI) enhancements in 1.5 T [121]. Dextran coated Gd-NPs synthesized through a one-pot synthesis approach were found to be highly water-dispersible and non-toxic as determined in a cellular cytotoxicity test. relaxivities r 1 = 12.2 and r 2 = 29.3 s −1 ·mM −1 with r 2 /r 1 = 2.4 were observed [122]. In another study, dextran-coated Gd-phosphate NPs was employed both as tumor-targeted MRI CAs and as a vehicle for drug delivery to tumors [123]. CS has been widely used to functionalize the Gd-CAs owing to its biocompatibility, bioactivity, biodegradability, and non-toxicity. Recent studies reported hydrophilic and biocompatible chitosan oligosaccharide lactate (COL)-coated ultra-small Gd-NPs synthesized through a one-pot polyol method. The in vitro cellular cytotoxicity assay indicated that the COL-coated Gd-NPs were non-toxic up to 500 µM Gd and their r 1 and r 2 values were estimated to be 13.0 and 27.0 s −1 ·mM −1 , respectively, which are higher than those of commercial MRI CAs [124].

Lipids and Their Derivatives
Lipids and their derivatives, such as liposomes and choline, are used to functionalize Gd-CAs. Phospholipid liposomes impart biocompatibility and increase the functionality of the Gd-CAs [125]. Generally, Gd-chelates are encapsulated inside the liposomes to synthesize tumour-targeting Gd-CAs. Thermosensitive Gd-CAs were synthesized conjugating thermosensitive liposomes with several clinically approved Gd-CAs where the r 2 value was observed to be increased in the temperature range from 38-44 • C [126]. In an effort to develop a high-resolution, 3D tumor evaluation process, a Gd-dendron assembled liposomal NPs CAs was synthesized. A clear image of the tumor micro-vessel structure using the 50-µm isotropic MR angiography was obtained using Gd-liposome. It also facilitated the observation of the differences in the vascular structures of malignant lymphoma grafted models (Colon-26 and SU-DHL6) and their therapeutic alterations using a chemotherapeutic drug (sunitinib) as shown in Figure 3 [127]. Choline is an essential nutrient that can easily break through the blood-brain barrier (BBB) and is used to design targeted imaging agents for diagnosing brain tumors by MRI [128]. Lattuada

Synthetic Polymers
Several synthetic polymers (degradable and non-degradable) have been extensively used to synthesize modified Gd-CAs. Generally, these are synthesized by conjugation, encapsulation and chelation. Degradable synthetic polymers are often preferred owing to their biocompatibility and intrinsic biodegradability, while non-degradable synthetic polymers possessing hydrophilicity are an abundant choice. Poly(amino acid)s (PAAs) are commonly employed to synthesize conjugated Gd-CAs having improved relaxivity, biocompatibility, and biodegradability [131]. Miao et al. reported the synthesis of stable and non-toxic Gd-NPs colloids coated with PAAs as positive T 1 MRI-CAs having optimum particle diameter size for renal excretion. Additionally, they possessed a high r 1 value of 31.0 ± 0.1 s −1 ·mM −1 and r 2 /r 1 ratio of 1.2, where r 1 was approximately eight times higher than that of commercial Gd-chelates. High positive contrast enhancements were observed in the liver, kidneys and bladder of the mouse [113]. Poly(ethylene glycol) (PEG) containing PAAs block copolymers (b-poly) are used to functionalize Gd-CAs. In a study, Gd-DOTA-based CAs were synthesized by conjugating Gd(III) ions to PEG-bpoly(L-lysine)/(PEG-b-P(Lys)) through the ligand DOTA. Synthesized complex PEG-P(Lys-DOTA-Gd displayed r 1 = 5.6-7.3 s −1 ·mM −1 at 9.4 T [132]. A novel theragnostic vesicle exhibiting admirable T 1 -weighted MRI contrast effect was developed by conjugating BSA-Gd with amphiphilic di-block copolymer poly(ethylene glycol)-block-poly(L-lactic-coglycolic acid) (PEG-b-PLGA) [133]. In a similar approach, novel multifunctional polymeric CAs were synthesized by conjugating polylactic acid/PEG (PLA/PEG)-P(Lys)/poly(lactic acid) (PLA) with anti-vascular endothelial growth factor (VEGF) antibody and Gd-DTPA yielding Anti-VEGF-PLA-PEG-PLL-Gd-NPs) for the targeted delivery of Gd-DTPA to the liver cancer. The Anti-VEGF PLA-PEG-PLL-Gd-NPs displayed high T 1 relaxivity and no apparent cytotoxicity in human liver cancer (HepG2) cells under experimental concentrations [134]. Ho et al. developed monodispersed Gd-NPs colloids (d avg = 1.5 nm) coated with hydrophilic polyacrylic acid (PAA) and partly conjugated with rhodamine B (Rho) for an additional functionalization (mole ratio of PAA: Rho = 5:1). The NPs colloids exhibited a very high r 1 of 22.6 s −1 ·mM −1 (r 2 /r 1 = 1.3), which was~6 times higher than those of commercial Gd-chelates. These NPs colloids were applied to Gd-NCT in vitro and exhibited a significant U87MG tumor cell death (28.1% net value) after thermal neutron beam irradiation, which was 1.75 times higher than that obtained using commercial Gadovist. These colloids also exhibited stronger fluorescent intensities in tumor cells than in normal cells owing to conjugated Rho, proving their pH-sensitive fluorescent tumor cell detection ability (Figure 4) [135]. Jang and coworkers synthesized poly(acrylic acid-co-maleic acid) (PAAMA) coated Gd-NPs (d avg = 1.8 nm), which exhibited outstanding colloidal stability, exceptionally low cellular toxicity, and a high r 1 value of 40.6 s −1 ·mM −1 and r 2 /r 1 ratio of 1.56, which is approximately 10 fold higher than those of commercial molecular CAs [136]. In another study, poly(methyl vinyl ether-alt-maleic acid) (PMVEMA) was used as a surface-coating polymer to develop PMVEMA-coated Gd-NPs, which displayed excellent colloidal stability in aqueous solution and appreciable biocompatibility. The r 1 value of 36.2 s −1 ·mM −1 and r 2 /r 1 = 2 under a 3 T MR field was observed (approximately 10 fold higher value than that of commercial molecular CAs) [137].
In a recent study, hybrid polyion complexes (HPICs) were synthesized by mixing the metal ions, zirconium ion (ZrO 2+ ) combined with Gd 3+ ions to a double-hydrophilic poly(ethylene oxide) (PEO)-b-PAA block copolymer solution forming nanostructures with an average radius of 11-16 nm. An increase in the zirconyl mole fraction imparted excellent chemical stability, while the addition of Gd 3+ greatly enhanced magnetic relaxivity. This behavior presents these nanostructures as excellent CAs having good biocompatibility [138]. In order to achieve a multimodal theragnostic agent, Hu et al. developed a water-soluble Gd containing polymer-based theragnostic compound (PFTQ-PEG-Gd-NPs) for the in vivo tri-modal photoacoustic imaging (PA)/MR second near-infrared (NIR-II) imaging-guided tumor photothermal therapy (PTT). PFTQ is a semiconducting conjugated polymer synthesized through a grafting-on method by utilizing thiadiazoloquinoxaline (TQ) and fluorene (F) as the acceptor and donor, respectively, to endow the complex with near-infrared (NIR) absorption and fluorescence emission capabilities. These agents possessed low biotoxicity and outstanding chemical and optical stability. Administration of PQTF-PEG-Gd-NPs in the tumorous sites of the in vivo model of mice displayed en-hancement in PA, NIR-II fluorescence and positive MR signal intensities after 24 h of systemic administration [139]. Grogna and coworkers synthesized Gd 3+ based MRI CAs by grafting 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid, 1,4,7-tris(1,1-dimethyl ethyl) ester (DO3AtBu) onto succinimidyl esters and PEO chains. DO3AtBu-NH 2 was synthesized by dissolving DOTA(tert-butyl ester)-methylic ester in ethylenediamine. This was grafted onto the copolymers bearing activated esters, i.e., succinimidyl esters and PEO chains. Gd 3+ ions were then attached to unprotected carboxyl groups attached DO3AtBu-NH 2 by stirring the GdCl 3 .6H 2 O solution with DO3AtBu-NH 2 to yield the DO3A(Gd 3+ )-NH 2 complex. The synthesized MRI CAs displayed enhanced relaxivity and efficient long blood circulation lifetime [140]. Kang

Organic and Inorganic Molecules
In spite of the employment of several polymers and lipids for the conjugation of Gd-CAs, several organic and inorganic molecules such as chalcones (Chal), fluorescein, carbon, fullerene, GO, silica, and Au have also found their application in imparting enhancement in bioimaging applications. Gd-CAs have been conjugated with several organic and inorganic materials such as GO, silica, Chal, and other NPs to provide additional efficacy.
Carbon is an ideal surface-coating material on NPs for promoting biomedical applications owing to its biocompatibility, nearly chemically inertness, and photoluminescent properties in the visible region. Carbon-coated Gd-NPs were synthesized by Yue and coworkers in an aqueous solution using a simple method. Gd-NPs@C (d avg = 3.1 nm) displayed excellent colloidal stability, very high r 1 value (16.26 s −1 ·mM −1 ; r 2 /r 1 = 1.48) and exhibited photoluminescence in the visible region. In vivo positive (T 1 ) MR images of high contrast indicated that the Gd-NPs@C could prove to be a potential T 1 MRI CAs. Additionally, strong fluorescence in the visible region was observed due to carbon coating on the NPs surfaces, indicating that the synthesized materials are eligible for a dual-modal imaging agent [143]. The GO and fullerenes have been employed to functionalize Gd-CAs for MRI and drug delivery applications. A water dispersible Gd-NPs decorated with GO nanocomposites (NCs) (Gd-NPs/GO-NCs) was developed through a simple solvent evaporation process. The Gd-NPs/GO-NCs displayed a high relaxivity value of 34.48 s −1 ·mM −1 with good biocompatibility [144]. The GQDs, derivatives of graphene exhibit promising applications in the field of biosensors, drug/gene delivery vehicles, and bioimaging agents owing to their easy functionalization, biocompatibility, and intrinsic fluorescence properties. Li et al. developed Mn 2+ or Gd 3+ doped nitrogen-containing GQDs (NGQDs) as biocompatible MRI CAs. Synthesis was performed through a single-step microwaveassisted hydrothermal reaction to get Mn-NGQDs and Gd-NGQDs having dual MRI and fluorescence modalities. The synthesized quasi-spherical 3.9-6.6 nm average-sized nanostructures possessed highly crystalline graphitic lattice structures (0.24 and 0.53 atomic% for Mn 2+ and Gd 3+ doping). These structures possessed high in vitro biocompatibility with values up to 1.3 mg·mL −1 for Mn-NGQDs and 1.5 mg·mL −1 for Gd-NGQDs, having effective internalization in HEK-293 cells traced by intrinsic NGQD fluorescence. Mn-NGQDs exhibited substantial r 2 /r 1 ratios of 11.190. At the same time, Gd-NGQDs possess r 2 /r 1 of 1.148 with a high r 1 value of 9.546 s −1 ·mM −1 These results demonstrate their potential for not only biocompatible alternatives to available T 1 /T 2 and T 1 CAs but also as FI agents [145]. Gd 3+ ions loaded PEG-modified Gd-PEG-GO-QDs were synthesized where the r 1 was effectively enhanced by increasing the proton exchange. A 20-30 folds increase in r 1 values of Gd-PEG-GO-QDs was observed as compared to the commercial CAs. Additionally, Gd-PEG-GO-QDs possessed low biotoxicities while the FA modified Gd-PEG-GO-QDs demonstrated efficiency for MRI-fluorescence bimodal tumor targeting agent in animals having greater than 98.3% specific cellular uptake rate [146].
The chemical inertness, biocompatibility, non-toxicity, stability, and magnetism transparency of silica has made it a favourable choice for the conjugating Gd-CAs. A new strategy to enhance the relaxivity and image contrast was developed by inserting Gd-CAs inside the nanoporous silicon NPs [68]. Theragnostic agents having therapeutic and imaging modalities are prepared by the development of theragnostic nano-vectors having the ability to release therapeutic agents at the pathological sites and provide instantaneous MR images. Carniato et al. developed Gd based MSNPs by selectively functionalizing the external surface of MSNPs loaded with ibuprofen molecules and functionalized with Gd-DOTA-monoamide chelate for the relaxivity and drug release studies of the Gd based MSNPs [147]. In another effort, DOX (an anticancer drug) was incorporated with MSNPs along with Gd 2 O 3 forming Gd-NPs@MSN-DOX. These hybrid NPs were coated with pH-responsive polyelectrolytes, which, upon entering into cells, disassociated from the surfaces of the Gd-NPs @MSN-DOX and thus activated the DOX release [148]. A high relaxivity Au-based bimodal Gd-CAs were synthesized and investigated for the effects of shape on proton relaxation. Gd 3+ were covalently attached to thiolated DNA, forming the nanoconjugates (Gd-DNA), followed by the conjugation onto Au nanostars (DNA-Gd@stars), which displayed efficient Gd(III) delivery and biocompatibility [76]. Liu and others synthesized Gd(III))-dendrimer-AuNPs (AuNPs-DEN) as multimodal CT/MRI agents. Poly(amidoamine) (PAMAM) dendrimers were partly conjugated with carboxybetanie acrylamide (CBAA), 2-methacryloyloxyethyl phosphorylcholine (MPC), and 1,3propane sultone (1,3-PS), respectively, and then the Au NPs were entrapped within while the remaining amine terminal was covered by acetylation. Zwitterionic Gd(III)-loaded AuNPs-DEN modified with arginine-glycine-aspartic acid peptide were then synthesized for targeted dual-mode CT/MR imaging. It was observed that AuNP-DEN (AuNPs core size of 2.7 nm and a surface potential of 7.6 ± 0.9 mV) displayed good X-ray attenuation properties, relatively higher r 1 values (13.17 s −1 ·mM −1 ), satisfactory cytocompatibility, and targeting specificity to α v β 3 integrin-expressing cancer cells [149]. In another study, mixed Gd-dysprosium oxide nanoparticles (Gd-DONPs) were synthesized as a dual-mode T 1 and T 2 MRI contrast agent. The D-glucuronic acid coated Gd-DONPs (d avg = 1.0 nm) exhibited large r 1 and r 2 values (r 2 /r 1 ≈ 6.6), displaying obvious dose-dependent contrast enhancements in R 1 and R 2 map images. In vivo T 1 and T 2 MR images revealed the dual-mode imaging capability of the NPs [150]. Choi et al. developed surface-doped with manganese oxide (MnO) Gd-NPs abbreviated as Gd-NPs@MnO ranging from 1 to 2 nm in diameter, coated with hydrophilic biocompatible compound, lactobionic acid (LA). In-vitro studies revealed dose-dependent contrast enhancements in both T 1 and T 2 map images, establishing their potential as both T 1 and T 2 MRI CAs [151]. Mixed Zn(II)/Gd(III) oxide NPs with approximately 8 mole% of Zn with an average diameter of 2.1 nm were synthesized by Tegafaw et al. in their studies. The D-glucuronic acid coated Zn(II)/Gd(III) oxide NPs showed a r 1 value of 12.3 s −1 ·mM −1 with r 2 /r 1 ratio of 1.1, corresponding to an ideal condition for T 1 MRI CAs. This could be attributed to the reduced magnetization of the mixed NPs because of non-magnetic Zn in the NPs [152]. A LA coated Gd-europium oxide NPs (d avg 1.75 nm) with good water solubility and biocompatibility were synthesized and evaluated for T 1 , T 2 MRI-FI in vitro and in vivo. The r 1 value of 11.9 and r 2 values of 38.7 s −1 ·mM −1 were observed, displaying clear dose-dependent contrast images. In addition, they showed both positive and negative contrast enhancements in 3 T and fluorescent confocal images in both DU145 cells and C. elegans (a small nematode). This study demonstrates the T 1 , T 2 MRI-FI multi-functionality of LA coated mixed Gd-europium oxide nanoparticles [153].
Kim et al. reported a new concept of neuroprognostic agents, which combines molecular diagnostic imaging and targeted neuroprotection to treat reperfusion injury after stroke. These neuroprognostic agents are inflammation-targeted Gd compounds conjugated with nonsteroidal anti-inflammatory drugs (NSAIDs). It was found that Gd-based MRI CAs conjugated with NSAIDs suppressed the increase in cyclooxygenase-2 (COX-2) levels, ameliorated glial activation, and neuron damage that is phenotypic for stroke by mitigating neuroinflammation, which prevented reperfusion injury. In addition, it was also observed that the neuroprognostic agents were promising T 1 molecular MRI CAs for detecting precise reperfusion injury locations at the molecular level [154]. Multifunctional imaging of the deposition of amyloid-beta (Aβ) aggregates in the brain is of great importance in diagnosing Alzheimer's disease. A novel multifunctional Aβ-targeting Gd-CAs comprised of Gd-chelate conjugated with Chal (Gd-DO3A-Chal) was synthesized by Choi and coworkers. Studies revealed that Gd-DO3A-Chal showed 8 times higher binding affinity to Aβ aggregates than a previously reported Gd-chelate conjugated with Pittsburgh compound B. Gd-DO3A-Chal showed multimodal imaging capability [155]. Park et al. utilized organic molecules for the surface coating Gd-NPs where all the samples exhibited large r 1 and r 2 water proton relaxivities with r 2 /r 1 ratios that were close to one, corresponding to ideal conditions for T 1 MRI CAs. Finally, in-vivo T 1 MR images were acquired to prove the effectiveness of the surface-coated ultrasmall Gd-NPs as T 1 MRI CAs [156]. Baek et al. synthesized a macrocyclic Gd chelate based on DO3A coordination cage having an ethoxybenzyl (EOB) moiety and studied it as a T 1 hepatobiliary MRI con-trast agent. Synthesized agents displayed high chelation stability and high r 1 relaxivity as compared with the linear-type Gd chelates (currently clinically approved liver agents). In addition, they also displayed high tumor detection sensitivity [157]. Fluorescein is an organic compound and dye and is most frequently used in optical contrast media for the synthesis of organic dyes-based MRI/OI dual-modal Gd-CAs. Miao et al. synthesized a dye-coated Gd-NPs [dye = fluorescein and fluorescein isothiocyanate (FITC)] in a one-pot synthesis approach and investigated their dual imaging properties. The dye-coated Gd-NPs exhibited excellent relaxometric properties suitable for T 1 MRI: r 1 = 9.8 s −1 ·mM −1 (r 2 /r 1 = 2.6) for fluorescein-coated NPs (d avg = 1.6 ± 0.1 nm) and r 1 = 12.3 s −1 ·mM −1 (r 2 /r 1 = 2.3) for FITC-coated NPs (d avg = 1.4 ± 0.1 nm), and strong photoluminescence (PL) in the green region (around 514 nm) suitable for FI. The dye-coated NPs exhibited strong fluorescence in cellular confocal images and high contrast in T 1 MR images in mice, suggesting that they are potential dual T 1 MRI-FI agents [158,159]. Macrocyclic diethylenetriamine penta-acetic acid (DTPA) conjugates of 2,2 -diaminobiphenyl and their Gd complexes were synthesized and studied for their potential use as new MRI blood-pool CAs (MRI-BPCAs). The r 1 relaxivity values were found to be 10.9 s −1 ·mM −1 , which is approximately 3 times as high as that of structurally related Gd-DOTA (r 1 = 3.7 s −1 ·mM −1 ). The r 1 relaxivity in human serum albumin (HSA) goes up to 37.2 s −1 ·mM −1 , almost twice as high as that of MS-325, a leading BPCAs, demonstrating a strong blood pool effect [160]. In a similar approach, Gd-complexes consisting of DOTA conjugates of tranexamic acid and tranexamic esters were prepared as a new class of MRI-BPCAs. The r 1 -relaxivity was significantly higher than those of any of the clinically used MRI CAs [161]. Table 3 briefly summarizes r 1 , particle size, biotoxicity, specific properties and modality of Gd-CAs. Table 3. Summary of magnetic relaxivity (r 1 ), particle size (d avg ), specific properties and modalities of reported CAs.

Conclusions and Future Perspectives
Gd-CAs have been widely studied for their magnetic properties and have been utilized as positive MRI CAs. In this review, we have outlined the basic imaging techniques and their limitations. Functionalization of Gd-CAs from single-modality to multi-modality imaging has emerged as a major aspect in overcoming the limitations of current commercially available CAs. Currently, major publications are based on the application of the NPs on the enhancement of relaxivity values and biocompatibility of the designed Gd-CAs.
Although several studies have reported, agents having relaxivities higher than that of commercially available CAs, the prospect of the potential toxicity of nanomaterials and probable risk of release of Gd limits their clinical applications. Factors such as the size and shape of NPs, and their surface charge and surface labelling impact the biodistribution of NPs. Encapsulation and surface labelling can reduce the toxic effects of synthesized NPs, but still, more studies are required to understand these aspects. Therefore, the development of Gd-CAs conjugated with ultra-small NPs having rapid elimination can somewhat overcome their toxicity issue. Imparting multi-modality to Gd-CAs has become a fascinating ideology, and it has been widely accepted as the next generation CAs. Due to the outstanding MRI enhancement characteristic of Gd, Gd-CAs can be coupled with other modalities to enhance or complement their imaging abilities. CT imaging, most widely used to afford detailed information of skeletons, lacks in yielding information on soft tissues, whereas MRI can offer high spatial anatomic information of tissues. Therefore Gd-CAs having the combination of MRI and CT could be of great significance in clinical applications. Gd-CAs augmented with optical imaging CAs complements their applications. PET can be incorporated with MR imaging of Gd-CAs to yield high-resolution images with anatomical details. The Gd agents for theragnostics have a potential role that incorporates diagnostics with therapeutics in the same platform. This strategy involves targeted delivery, controlled release and simultaneous diagnosis and treatment at the molecular level. Smart carriers or delivery vehicles NPs encapsulate various therapeutic agents and deliver them to target locations at the time of bioimaging.
In spite of these developments, most of the CAs failed to attain clinical applications, and only a handful of CAs have reached clinical trials. Current studies should be more focused on target specificity and reduced toxicity, exploring the Gd NPs to their full potential. Gd-CAs can be conjugated with highly specific tumor-targeting ligands, such as antibodies, aptamers, and peptides. Second, to increase the biocompatibility, renal excretion and non-toxicity, Gd-CAs should be conjugated to hydrophilic and biocompatible ligands, small enough to be excreted through the renal system. In addition, understanding the pharmacokinetics of these agents in humans should be emphasized. Effective development of Gd-CAs for bioimaging applications will require multidisciplinary research efforts. Despite these challenges, developing such Gd-CAs can prove to be a breakthrough in bioimaging CAs in the future.