Magnetic Resonance Imaging ( MRI ) Contrast Agents for Tumor Diagnosis

This review focuses on MRI contrast agents for tumor diagnosis. Several types of low molecular weight Gd3+-based complexes and dextran-coated superparamagnetic iron oxide (SPIO) nanoparticles have been used for clinical tumor diagnosis as longitudinal relaxation time (T1) and transverse relaxation time (T2) MRI contrast agents, respectively. To further improve the sensitivity of MRI, new types of chelates for T1 MRI contrast agents and combination of low molecular weight T1 MRI contrast agents with different types of carriers have been investigated. Different types of materials for forming secure coating layers of SPIO and novel superparamagnetic particles with higher relaxivity values have been explored. Various types of ligands were applied to improve the capability to target tumor for both T1 and T2 contrast agents. Furthermore, MRI contrast agents for detection of tumor metabolism were also pursued.


INTRODUCTION
In comparison with other diagnosis techniques such as X-ray computed tomography (CT), positron-emission tomography (PET), single photon-emission computed tomography (SPECT) and ultrasound, magnetic resonance imaging (MRI) is noninvasive and can provide tomographic information of whole animals with a high spatial resolution and soft tissue contrast [1,2].There are many types of MRI techniques, including the longitudinal relaxation time (T 1 )-weighted imaging with a hyper-intense signal and the transverse relaxation time (T 2 )-weighted imaging with a hypo-intense signal.The key challenge in MRI technique is its low diagnosis sensitivity.Currently, 40-50% of MRI scans use contrast agents that contain magnetic metal ions to enhance the intensity of signal [1,3].The contrast agents for T 1 -weighted MRI (i.e., T 1 contrast agent) usually contain lanthanide or transitional metal ion (Gd 3+ or Mn 2+ ) that is chelated to reduce serious side effects [4][5][6].The contrast agents for T 2 -weighted MRI (i.e, T 2 contrast agent) normally consists of superparamagnetic nanoparticles with coating layers [6].
Since nuclear magnetic resonance was explored for diagnosis of disease in 1971 [7], MRI has been well developed for diagnosis of various diseases.For tumor diagnosis, MRI contrast agents are useful to obtain good contrast for differentiating tumor from healthy tissues, and indicating tumor malignant status and the treatment efficacy.There are many seminal reviews on MRI contrast agents, most of which are generally about MRI contrast agents for diagnosis of various diseases [4][5][6].In this review, we focus on contrast agents for tumor diagnosis based on T 1 -and T 2 -weighted MRI.The contrast agents used in clinical tumor diagnosis are described first, followed by an update of the progress in developing T 1 MRI contrast agents through exploring new chelates and combining low molecular weight T 1 MRI contrast agents with various types of carriers.The progress in developing T 2 MRI contrast agents through forming secure coating layers for SPIO and preparing new superparamagnetic cores is discussed.The approaches to improving targeting capability of both T 1 and T 2 MRI contrast agents via either passive targeting or active targeting are covered.Also discussed is the research on developing MRI contrast agents for detection of tumor metabolism.

T 1 MRI CONTRAST AGENTS 2.1. T 1 MRI Contrast Agents for Clinical Tumor Diagnosis
As shown in Figure 1, several types of T 1 MRI contrast agents, i.e., Gd-DTPA (Magnevist ® ), Gd-EOB-DTPA (Eovist ® ) and Gd-DTPA-BMA (Omniscan ® ), have been employed for clinical tumor diagnosis.Pettersson et al. showed that Gd-DTPA enhanced only the richly vascularized parts and the surrounding of the soft tissue tumors in 10-15 minutes after injection [8].For the detection of mediastinal lymph nodes, Gd-DTPA-enhanced MRI could provide a diagnosis with a sensitivity of 100%, an accuracy of 97% and a specificity of 91% as compared to 62%, 74% and 100% for non-enhanced MRI, respectively [9].However, Gd-DTPA might not be helpful in screening other types of cancer.Hawnaur et al. demonstrated that it is complicated to identify tumor in bladder using Gd-DTPA-enhanced MRI due to the excretion of Gd-DTPA in urine and changes in bladder volume, which could affect the interpretation of results; it was also not reliable in determining the effectiveness of the radiotherapy due to structural changes in the bladder after radiotherapy [10].
Gd-EOB-DTPA is suitable for liver tumor diagnosis due to its good liver-specificity [11].Vander et al. reported that Gd-EOB-DTPA was taken preferably by an excised and perfused rat liver than Gd-DTPA [12].Shimada et al. showed that Gd-EOB-DTPAenhanced MRI was more accurate and sensitive in detecting small hepatic metastases of a diameter smaller than 2 cm than diffusion-weighted MRI [13].Gd-EOB-DTPA induced a much better tumor enhancement of solid hepatocellular carcinoma lesion of rats than Gd-DTPA and Mn-DPDP.The Gd-EOB-DTPA almost disappeared in 24 hours while a high concentration of Mn-DPDP still remained in the liver [14].
Recently, US FDA approved clinical use of MS-325 in magnetic resonance angiography (MRA).MS-325 can form complex with endogenous serum albumin via hydrophobic interaction without covalent linkages [15][16][17][18], and provide r 1 of a value 10 times higher and a longer vascular residence time than non-protein-binding contrast agents.The reversible bonding between albumin and MS-325 could facilitate the excretion of MS-325 and avoided poor clearance.MS-325 was also used in the assessment of capillary permeability in rat breast tumor [19].
Although several types of T 1 MRI contrast agents have been employed for clinical tumor diagnosis, their sensitivities still need to be improved in terms of higher r 1 value and/or capability to target tumor.

New T 1 MRI Contrast Agents for Tumor Diagnosis Under Investigation
Two approaches are discussed below regarding development of T 1 MRI contrast agents for tumor diagnosis with improved sensitivity, i.e., low molecular weight Gd 3+ complex composed of suitable chelates and targeting ligands, and low molecular weight T 1 MRI contrast agents combined with various carriers.

Low Molecular Weight T1 MRI Contrast Agents
One of the most promising chelates for preparing T 1 MRI contrast agents for tumor diagnosis is porphyrin-based compounds with possible multi-functionality.Porphyrin could function as a ligand and was potentially applicable for cancer photodynamic therapy [20].As the ring of porphyrin is too small to accommodate Gd 3+ ions securely in vivo [21], porphyrin-like synthetic macrocyle, texaphyrin, was explored as a chelate of Gd 3+ instead, which could provide a longer MRI contrast enhancement of the V2 carcinoma than Gd-DTPA [22].Another type of contrast agent obtained from texaphyrin, Motexafin Gd, could provide MRI contrast enhancement of brain tumor and killed the cancer cells via redox cycling simultaneously [23,24].Enhanced targeting of MRI contrast agents to tumor sites can improve the sensitivity significantly.Various types of ligands have been explored to improve the tumor specificity of low molecular weight T 1 MRI contrast agents.Arginine-glycine-aspartic acid (RGD) peptide is well known for its high and specific affinity for α v β 3 -integrins which are over-expressed in endothelial cells during angiogenesis of tumors.Park et al. reported a liver specific contrast agent, cyclic RGD conjugated Gd-DOTA (Gd-DOTA-RGD) [25].Gd-DOTA-RGD could produce a high signal intensity of the tumor, but almost lost this enhancement when the α v β 3 -integrins were blocked [25].Deoxyglucosamine conjugated Gd-DTPA (Gd-DTPA-DG) was developed to target the hypermetabolic cancer cells because deoxyglucosamine was rapidly taken up by tumor due to the over-expressed glucose transporters [26].Gd-DTPA-DG could provide a higher MRI enhancement of A549 tumor than Gd-DTPA and a higher retention rate because the metabolism pathway was blocked by the deoxyglucosamine analog [26].MRI contrast agents were also developed to target the overexpression of estrogen and estrogen related progesterone receptors in breast and ovarian cancers.Sukerkar et al. conjugated progesterone to Gd-DO3A to improve the cellular uptake by around 3 times higher in two breast cancer cell lines and provided a higher contrast enhancement of the xenograft tumors in nude mice [27].Pais et al. developed another type of breast cancer specific MRI contrast agent, EPTA-Gd, by conjugating 17b-estradiol to pyridinetetraacetate-Gd (PTA-Gd) for differentiating estrogen receptors-transfected PR(+) from wild-type PR(-) human breast cancer cells [28].

Low Molecular Weight T 1 MRI Contrast Agents Combined with Carriers
Combination of low molecular weight T 1 MRI contrast agents with carriers including polymers and nanomaterials can produce contrast agents with a high payload of chelated Gd 3+ , normally a higher r 1 value, and enhanced tumor targeting capability.One factor contributing to the enhanced targeting capability is the enhanced permeability and retention (EPR) effect owing to accumulation of complexes of carriers and low molecular weight T 1 contrast agents, which are larger, in tumors with loosely vascular structures [29].However, the possible release of free Gd 3+ was observed from some liposome loaded with low molecular weight T 1 MRI contrast agent, which showed a long retention time [30].Therefore, the safety issues of the complex of carriers and low molecular weight T 1 contrast agent should be taken into account as well.

Water-Soluble Polymer as Carriers
Many types of water soluble polymers, including linear polymers, dendrimers, and proteins, have been explored for carrying low molecular weight T 1 MRI contrast agents.Generally, such conjugation limits the rotation and motion of the chelated Gd 3+ leading to a higher r 1 value [31][32][33][34].
Gd-DTPA conjugated polylysine was able to accumulate in grafted tumor in rat models and therefore provided an enhanced imaging for several days [35].Gd-DTPA conjugated polyaspartamide demonstrated a preferential uptake and therefore an enhanced MRI contrast in hepatoma in mouse models [36].A high molecular weight polyglutamic acid based MRI contrast agent exhibited an improved tumor accumulation [37].Low molecular weight T 1 MRI contrast agents were also conjugated with polysaccharides including dextran, starch, inulin and oligoglucoamines.Conjugates of Gd-DTPA with dextran or oligopolyglucoamines were investigated for delineation of tumor in rabbits [38], while Gd-DO3A conjugated carboxymethyl hydroxyethyl starch showed the ability to image leaky vasculature of tumor [39].Galatose units targeting the lectin asialoglycoprotein receptor (ASGPR) expressed on liver hepatocytes [40] were explored for imaging of hepatocyte carcinoma through combination with either DOTA [41] or DTPA [40,42].
In comparison with linear polymers, dendrimers have well-defined, rigid dendritic structures together with abundant terminal groups.The conjugation to the terminal groups produces dense peripheral layers of low molecular weight T 1 MRI contrast agents which can induce high r 1 values.For example, the r 1 of G6-(C-DOTA-Gd)115 shown in Figure 2, prepared using a preligation technique, could reach 89.1 mM −1 s −1 as compared to 4.2 mM −1 s −1 for DOTA-Gd [31].It was also found that G6 or G7 dendrimers provided the highest r 1 values, while protonation of amines [43,44] and formation of adducts [45,46] could further improve the values by forming more rigid and open structures with a lower internal motion.Therefore, a higher level of contrast enhancement of tumors could be obtained using a lower amount of PAMAM [47][48][49][50] and polylysine dendrimer [51] conjugated with low molecular weight T 1 MRI contrast agents.PEGylated and non-PEGylated Gd labeled dendrimers had a r 1 value higher than 20 mM −1 s −1 together with a longer retention time [52][53][54].Targeting ligands, e.g., OST7 [55], murine monoclonal IgG1, folic acid which targets folate receptor (hFR) [56][57][58][59], and cyclic RGD as an angiogenesis marker [60], were applied to improve active targeting of the conjugates of PAMAM and low molecular weight T 1 MRI contrast agents.Also, dendrimer nanoclusters (DNCs) with folic acid as ligand were developed with a high payload of low molecular weight T 1 MRI contrast agents [61].In order to improve biocompatibility, biodegradable esteramide dendrimer was combined Gd-DOTA conjugated PAMAM [31].Used with permission.
with low molecular weight T 1 MRI contrast agents [32] which showed a low toxicity similar to Gd-DTPA [62,63].
Low molecular weight T 1 MRI contrast agents were also combined with proteins such as albumin [64,65], IgG and fibrinogen [66] and could increase r 1 by 3 folds.Albumin-Gd-DTPA was employed to monitor the histological profile of tumor and abnormal capillary permeability in cancer models [67][68][69][70][71].The changes in capillary permeability could estimate angiogenic activity and the effects of pharmacological stress [72], radiation [73] and toxins [74].The combination with certain types of proteins could improve the tumor targeting capability.Through the interaction between biotin and avidin, Gd 3+ -labeled avidin was used to image the dynamic response of tumors to etoposide treatment in mice [75] and breast cancer [76].Antibody was also explored to deliver MRI contrast agents to tumor specifically.It was shown that antibody labeled Gd-DTPA could visualize melanoma [77,78], human rectal carcinoma [79] and human gastrointestinal cancer [80,81].However, many results have shown that conjugation could destroy the immunereactivity of antibodies; therefore, the targeting capability of these MRI contrast agents was limited [82,83].

Nanomaterials as Carriers
With the advancement in nanotechnology, many types of nanomaterials have been developed, such as polymer micelles and vesicles, liposomes and lipid particles, viral particles, carbon nanotubes and fullerenes, gold nanoparticles, and silica particles; most of them have been explored as carriers of Gd 3+ as MRI contrast agents for tumor diagnosis.
Bui et al. incorporated Gd-DTPA into PEG-coated phospholipid nanoparticles (LNP), which showed a very high r 1 value of 134.8 mM −1 s −1 ; the Gd-DTPA loaded LNP was excreted from the body through the biliary system instead of the renal system due to its lipid nature [93].Low molecular weight T 1 MRI contrast agents were loaded into liposomes in several ways as shown in Figure 3 [6,94].For example, ensomes with reduced r 1 values and memsomes with higher r 1 values were formed when low molecular weight T 1 MRI contrast agents were trapped in the inner parts and the membranes of liposomes, respectively.These systems demonstrated an enhanced passive targeting of tumor such as liver tumor [95].For active targeting, RGD was employed to label PEGylated liposomes encapsulated with Gd-DTPA and provided a higher MRI contrast enhancement of human lung cancer in xenograft mice [96].Transferrin, which is over-expressed in many cancerous cells, was used to label liposomes loaded with Gd-DTPA to image the detailed pathway of the liposomes in the human prostate cancer cells inoculated in nude mice [97].These liposomes entered the peripheral region of the tumor reflected by higher signal intensity observed in 10 minutes after injection of the contrast agent, and then entered the cells via endocytosis where Gd-DTPA was released.Finally liposomes and the released Gd-DTPA were pumped out by the cancer cells and were then accumulated in the necrotic area due to the lack of washout mechanism indicated by the significant increase in signal intensity in 60 minutes after injection [97].Moreover MRI based on chemical exchange saturation transfer (CEST) has a high potential to provide better imaging [6,94,98].MRI contrast agents for this technology can be obtained by loading low molecular weight T 1 MRI contrast agents into non-spherical liposomes to form LipoCEST agents as shown in Figure 3E.Nanosized silica has been explored for loading low molecular weight T 1 MRI contrast agents.Gd-DTPA was conjugated to PEG functionalized mesoporous silica nanospheres (MSN) with anisamide as a targeting ligand via cleavable disulfide linkage, and provided an r 1 value up to 25.7 mM −1 s −1 [99].Such nanospheres could be taken up by AsPC-1 pancreatic cancer cells, and the in vivo results indicated that Gd-DTPA was cut from MSN in 15 minutes after injection, reflected by a strong imaging enhancement of the bladder, due to rapid reduction of the disulfide linkage by plasma thiols [99].The biocompatibility of silica nanoparticles was investigated using Gd 2 O 3 doped mesoporous silica nanocomposite, which indicated that silica particles showed a low toxicity in cell lines and no potential immunotoxicity [100].Silica nanoparticles coated with Gd 2 (CO 3 ) 3 were also prepared and exhibited a low r 1 value of 1.6 mM −1 s −1 [101].
Low molecular weight T 1 MRI contrast agents were also conjugated with other types of nanomateirals.Conjugation with viral capsids could significantly improve r 1 values [102].Anchoring low molecular weight T 1 MRI contrast agent onto Au nanoparticles could improve r 1 value by several times [103,104] and benefit multimodal cell imaging [104].When Gd was loaded into fullerenes, gadofullerene formed with either PEG shells or succinic acid shells provided r 1 50 to 60 times higher than Gd-DTPA, and the gadofullerene was tested for imaging of brain tumor in rat models [105,106].

T 2 MRI CONTRAST AGENTS
The majority of T 2 MRI contrast agents contain superparamagnetic iron oxide (SPIO) nanoparticles which are composed of either maghemite (γ-Fe 2 O 3 ) or magnetite (Fe 3 O 4 ) phases prepared by various methods including co-precipitation and hydrothermal procedures.SPIO can provide a high proton relaxivity with a typical r 2 value of 100 mM −1 s −1 and r 1 value of 30 mM −1 s −1 , together with a prolonged contrast enhancement [107][108][109].Pure SPIO possesses good biocompatibility due to low cytotoxicity and biodegradability with degraded SPIO entering the iron reservoirs such as hemoglobin in red blood cells [110,111].However, a suitable coating layer is necessary to avoid formation of aggregates and provide a long circulation in the blood stream by avoiding uptake by the reticuloendothelial system (RES) and excretion through the renal filtration [112][113][114].

T 2 MRI Contrast Agents for Clinical Tumor Diagnosis
Dextran-coated SPIO, e.g., Ferumoxide ® and Ferrixan/Ferucarbotran ® , have been tested for clinical tumor dignosis [6].Ferumoxide ® nanoparticles with diameters of 80-150 nm are used for MR imaging of liver and spleen, Ferrixan/Ferucarbotran with a diameter of 62 nm are used for liver, and both contain SPIO of a diameter of 4.2 nm.These contrast agents are prepared via copreciptation method in the presence of dextran.The dextran coating layers are formed via multiple cooperative low-energy interactions between dextran and SPIO including van der Waals force, electrostatic and hydrophobic interactions [115].Pharmacokinetic and toxicity studies have revealed that the dextranbased nanomaterials are non-toxic and biodegradable with extended vascular retention times [116].
T 2 MRI contrast agents have been demonstrated to be useful for imaging liver tumors by providing a strong contrast between healthy and cancerous tissues.This is due to a preferential uptake of contrast agents by reticuloendothelial cells such as Kuppfer cells which are absent or in low concentration in tumors [117,118].Clinical studies have shown that Ferumoxide ® could detect hepatic tumors with a high accuracy as the nanoparticles accumulate exclusively in healthy liver tissues [119].Ferumoxide ® tended to exhibit a two-phase blood clearance with a half-life of the first phase and the second phase ranging from 4.4-22.2minutes and 79-309 minutes, respectively, after intravenous injection in patients with liver metastases [120].Reimer et al. have shown that SPIO enhanced T 2 -weighted MRI was more accurate in the detection of focal hepatic lesions than non-enhanced T 1 -and T 2 -weighted MRI and contrast-enhanced spiral computed tomography (CT) [121].Ferumoxide ® has also been used for detecting focal splenic tumors through observing a significant increase in signal intensity of spleen but not the tumor [122].This allow a more accurate identification of lesions than other diagnostic methods such as sonography, contrast-enhanced CT, and unenhanced MRI.The clinical investigation was also extended to lymph nodes, and smaller SPIObased contrast agents were found to accumulate in lymph nodes [123,124].Specifically, smaller SPIO-based contrast agents with a diameter of ca. 30 nm were extravasated from the vasculature to the interstitial space and were then transported to the lymph nodes via the lymphatic vessels.Lymph nodes containing cancerous tissues lack the necessary macrophages to phagocytize SPIO; as a result, the accumulation of SPIO in healthy tissues shortened T 2 signal, and significantly increased the positive predictive values and accuracy of node metastases diagnosis [125].
The drawback of these T 2 MRI contrast agents used in clinical tumor diganosis lies in that the non-crosslinked dextran coating layer can be removed by the surrounding medium.However, there are safety concerns about the cross-linking agents used in forming the cross-linked dextran layer coated on SPIO (CLIO) via reaction with epichlorohydrin [126], although CLIO has been applied in clinical studies of prostate cancers [117,127] and other cancers [128][129][130].

New T 2 MRI Contrast Agents Under Investigation
In order to develop better T 2 MRI contrast agents for tumor diagnosis, superparamagnetic nanoparticles with a higher relaxivity, secure coating layers of SPIO and an improved tumor targeting capability have been pursued.

T 2 MRI Contrast Agents with New Superparamagnetic Nanoparticles
The magnetic properties of superparamagnetic particles are affected by size, shape, and defect concentration.There are many different methods to synthesize superparamagnetic particles [131].The most common fabrication method is the Massart's procedure whereby base is added to an aqueous solution of ferrous (Fe 2+ ) and ferric (Fe 3+ ) ions with a 1:2 stoichiometric ratio under an oxygen free environment [132].However, SPIO produced in this process has a varied size and a low saturation magnetization value of 30-50 emu/g due to impurities and crystal defects [133].In contrast, thermal decomposition of organometallic reagents could yield SPIO with a well-defined size and a high saturation magnetization value > 70 emu/g [134].In order to further improve the magnetic performance, metal-doped SPIO nanoparticles as depicted in Figure 4 were developed, and the MnFe 2 O 4 produced had a low cytotoxicity and exhibited a very high r 2 value of 350 mM −1 s −1 [135].However, most of these particles were prepared in organic solvents, and transformation into aqueous solution was necessary for further coating.In addition, SPIO nanoparticles prepared by precipitation in alkaline solution are more suitable for in vivo purposes, and the SPIO nanoparticles prepared by other procedures are limited to in vitro applications [136].

T 2 MRI Contrast Agents with Ligands
SPIO based contrast agents without targeting capability is only useful for the diagnosis of tumors in RES organs where large quantities of resident macrophages exist.For the detection of cancerous tissues in other parts of the body, it is necessary to integrate ligands for active targeting.Many type of ligands, such as small molecules, proteins, and oligonucleotides, have been conjugated to dextran layers coated on SPIO through conventional chemical methods, click chemistry methods and cycloaddition methods as depicted in Figure 5 [151][152][153].Among 146 different types of small molecules with varied solubility in aqueous solution and chemical diversity, glycine-conjugated CLIO was identified to be capable of targeting active macrophages associated with tumors with proangiogenic and immunosuppressive properties [154], but not resting macrophages [155].On the other hand, CLIO conjugated with 3,3',4,4'benzophenontetracarboxylic dianhydride was able to target resting macrophages [156].
Anti-vascular cell adhesion molecules-1 (VCAM-1) antibodies [157], VHS peptide [158] and VHPKQHR peptide [159,160] were explored to target T 2 MRI contrast agents to cells with VCAM expression which is related to tumor angiogenesis.SPION coated with dextran conjugated with folate showed a rapid and efficient uptake via receptor-mediated endocytosis by both human nasopharyngeal epidermal carcinoma cells (KB cells) overexpressing folate receptors and subcutaneous tumor (xenografts grown from implanted KB cells) in mouse models [161,162].Transferrin-conjugated dextran-coated SPIO was tested to target tumors with a higher level of transferrin receptor expression [163].Furthermore, RGD was also explored to enhance the targeting of SPIO to tumors [164].It was shown that RGD-modified SPIO nanoparticles could significantly enhance the sensitivity of MRI for early stage tumor detection [165].

MRI CONTRAST AGENTS RESPONSIVE TO TUMOR METABOLISM
It is very important to develop diagnosis techniques to reflect tumor metabolism, e.g., apoptosis, glycolysis, pH, redox, and hypoxia, which are related to the malignant status and therapeutic responses of cancers.It is well-known that particular metabolites are produced from tumor with a certain metabolism; therefore, magnetic resonance spectroscopy (MRS), which can identify the particular metabolites, is powerful for monitoring tumor metabolism [1,166].When MRI is applied to detect tumor metabolism, it is a prerequisite to identify the relationships between signal intensity and particular tumor metabolites or biomarkers.However, so far there are very few reports in this area.By exploring interaction with the phosphatidylserine on the surface of apoptotic cells, C2A domain of synaptotagmin I loaded with SPIO [167] and Gd-DTPA [168] were applied for image apoptosis of tumor cells.GdTODA-4AmP5 − , whose proton exchange rate changes with pH, was developed and explored to detect pH of tumor [169].Iwaki et al. also developed a pH-responsive Gd-based contrast agent, 4NO 2 2MeOSAGd.The relaxivity of 4NO 2 2MeOSAGd was increased by 1.8 times after it was reduced to 4NH 2 2MeOSAGd via an enzymatic reaction [170].Conjugation chemistry to attach small molecules to CLIO [152].Used with permission.

CONCLUSION Both
to overcome the hurdles related to the low sensitivity and specificity of current MRI contrast agents.Novel chelating approaches can produce T 1 MRI contrast agents with much high r 1 values, such as forming complexes of Gd 3+ with fullerenes and some proteins, but extensive examinations of their stability, retention behaviors and safety are still needed.The combination of low molecular weight T 1 MRI contrast agents with carriers can yield a higher sensitivity and specificity for MRI.However, suitable carriers, including hyperbranched polymers, dendrimers and nanomaterials, with good biocompatibility and safety are still desired.
T 2 MRI contrast agents for clinical applications use biocompatible SPIO prepared by coprecipitation method, but it is still a challenge to produce biocompatible superparamagnetic nanoparticles with a well-defined size, a high saturation magnetization value, and good batch reproducibility.Meanwhile, it is still crucial to develop secure coating layer for superparamagnetic nanoparticles together with improved targeting capability.
For detection of tumor metabolism using MRI technique, more efforts in developing MRI contrast agents responsive to particular metabolites or biomarkers are needed.

Figure 1 .
Figure 1.Typical low molecular weight T 1 MRI contrast agents used in clinical tumor diagnosis.

Figure 4 .
Figure 4. Metal-doped superparamagnetic iron oxide nanoparticles.(a) TEM images of MnFe 2 O 4 (MnMEIO), Fe 3 O 4 (MEIO), CoFe 2 O 4 (CoMEIO) and NiFe 2 O 4 (NiMEIO).All nanoparticles were of ~12 nm with narrow size distributions (σ < ~8%).Scale bar: 50 nm.(b) Mass magnetization values of MFe 2 O 4 .(c, d) Schematics of spin alignments of magnetic ions in spinel structures under external magnetic field, and magnetic spin moment of MFe 2 O 4 nanoparticles.In face-centered cubic lattices of oxygen, the magnetic spins at O h sites aligned in parallel with the direction of the external magnetic field, whereas those at T d sites aligned antiparallel.MnFe 2 O 4 has the highest mass magnetization value, with a magnetic spin magnitude of 5 µ B .(e, f) T2-weighted spin echo MR images, their color maps and relaxivity (R2) of a series of MEIO nanoparticles at 1.5 T. In (f), the R2 of CLIO is also presented for comparison.Consistent with the mass magnetization results, MnMEIO displays the strongest MR contrast effect (darkest MR image, violet in color map) with the highest R2 coefficient.Mass magnetization value, MR contrast, and R2 coefficient decrease as M 2+ changed from Mn 2+ to Fe 2+ , Co 2+ and Ni 2+ [135].Used with permission.
Figure 5.Conjugation chemistry to attach small molecules to CLIO[152].Used with permission.