*3.2. Overview of the Use of SPIONs as MRI Contrast Agents*

MRI is a powerful imaging modality for soft tissue imaging as it offers high spatial resolution and tissue discrimination without exposing the subject to ionizing radiations. Hydrogen protons are abundant in our bodies. The proton resonance is obtained by the application of short radio frequency (RF) pulses changing their magnetic moment orientation. After the RF is stopped, relaxation occurs, and magnetic moments realign along their original alignments. The reorientations of spins along the B0 axis, i.e., the spin-lattice relaxation, is characterized by the relaxation time T1, the inverse of which is the relaxation rate R1 = 1/T1 (expressed in s−<sup>1</sup> or Hz). The disappearance of the magnetization in the transverse plane, named spin-spin relaxation, is characterized by the relaxation time T2 (or relaxation rate R2 = 1/T2). The relaxivity (usually expressed in s−1mM−<sup>1</sup> or L.mmol−<sup>1</sup> .s−<sup>1</sup> ) expresses the R1 or R2 proton relaxation rate modulation induced by an MRI contrast agent in a biological tissue as a function of its concentration.

SPIONs are commonly employed as the contrast agent to change the tissue relaxation rates of normal or pathological tissues in order to improve the sensitivity and specificity of MRI. SPIONs administered for this purpose should offer high relaxivity and adequate biodistribution without inducing local or systemic toxicity. The effect of SPIONs on T1 and T2 relaxations depends on both the saturation magnetization of the nanoparticles and their interaction with water protons in tissues. The size, shape, and surface coatings of SPIONs strongly modulate their T1 and T2 effects [61]. The physical phenomena resulting in the modification of the spin-lattice and spin-spin relaxation rates in tissues under the influence of SPION nanoparticles are thoroughly detailed elsewhere in the literature [61,75,76]. Briefly, the accumulation of SPIONs in a tissue induces local perturbations of the principal magnetic field of the MRI system, which increase spin dephasing and finally shorten the transverse relaxation time (T2). This arises from the magnetic coupling between protons spin in tissues and the spins of SPIONs. Therefore, the presence of SPIONs causes a negative MRI contrast in tissues.

T2-shortening agents have two major drawbacks: (1) the increased magnetic susceptibility artifacts and (2) the difficult interpretation of low-signal areas which may be confused with bone or vascular structures [77]. This encouraged the development of particles providing contrast in both T1-weighted (T1w) and T2-weighted (T2w) imaging. For instance, gadolinium-labeled magnetite nanoparticles dedicated to positive contrast MR angiography were successfully used in vitro and in vivo by Kellar et al. [78]. More recently, a new class of cubic SPIONs, suitable for use as a dual-mode contrast agent, was presented by Alipour et al. [79]. Although these agents do not yet represent the majority in the literature, their development has been accelerated in recent years, expanding possible diagnostic applications with SPIONs. The typical R2 relaxivity of SPIONs ranges from 100 s−1mM−<sup>1</sup> to a few hundred s−1mM−<sup>1</sup> depending on the characteristics of the particle (composition, coating) and the B0 MRI field [61]. One of the challenges of current studies is to increase the R1 relaxivity) (usually much lower than R2 relaxivity).

The oral administration of SPIONs as a gastrointestinal MRI contrast agent has been considered by several research teams [76]. For instance, Hahn et al. described the improvement of the gastrointestinal tract delineation of MR images provided by a 200 nm SPIO suspended in a low-viscosity food-grade fluid [80]. Their preparation was globally well tolerated by animals and patients. Apart from a few special cases, in the vast majority of studies on the subject since the late 1980s [75], SPIONs are administered intravenously [75]. Unlike low molecular weight water-soluble agents such as gadolinium chelates, SPIONs are usually not transferred to the extracellular-extravascular compartment in healthy subjects and are rapidly eliminated by the RES [67,69,70]. As a consequence, their biodistribution is characterized by a short biological half-life and a significant accumulation in the RES (typically: liver, spleen, bone marrow). As a first approach, SPIONs can thereby be used to enhance malignant lesions within organs of RES [80]. For instance, Weissleder et al. used SPIONs to detect focal splenic tumors with MRI, leading to an important step forward in this domain since the other existing imaging techniques do not provide contrast between such lesions and healthy tissues [67].

On the other hand, the phagocytosis of SPIONs allows the visualization of tissues infiltrated by macrophages during inflammatory processes, which would not be possible with gadolinium-based contrast agents, not internalized by immune cells [81]. Macrophages are the key component of acute inflammation [35]. When an infectious agent is detected, an immune response is set up resulting in vasodilation, higher vascular permeability, and infiltration of free fluid and immune cells (neutrophils and macrophages) in tissues [81,82]. These phenomena are followed by the formation of a fibrotic scar. MRI procedures taking advantage of the phagocytosis of SPIONs for the detection of inflammatory areas and infectious foci, and more generally for the assessment of immune-mediated disorders, were described in the mid-2000s [82,83]. Stoll et al. described the interest in SPIONs in the assessment of central nervous system inflammations [84]. Sillerud et al. detected amyloid-β plaques in a transgenic mouse model of Alzheimer's disease [85]. Ruehm et al. described, in a preclinical assay, the interest in SPIONs as a marker of atherosclerosis (chronic inflammatory response to a vascular wall injury) [86].

Over the last few decades, significant progress has been made in the design of SPIONs, such as the reduction in the average size of these nanoparticles, the improvement of their physico-chemical characteristics, the incorporation of innovative coatings, and especially their surface functionalization. For instance, by decreasing the diameter of their SPIONs to the size range of plasma proteins (i.e., around 10 nm), Weissleder et al. increased their biological half-life and facilitated their transcapillary passage to the interstitium. As a result of these improvements, SPIONs were progressively promoted to the rank of multimodal theranostic nanoprobes with, among others, applications in MHT treatment and immunotherapy. It has been established that MRI examinations performed after the administration of SPIONs offer higher sensitivity and specificity than non-injected MR acquisitions in the diagnosis of lymph node metastasis [87].

SPIONs can also help to distinguish infectious masses from cancerous tumors [81]. Preclinical [88] and clinical studies attested to the benefits of such examinations in axillary node metastases detection in breast cancer patients [87]. Other authors proved the interest in SPIONs for cardiovascular system explorations. Majundar et al. showed the blood-tobackground nuclear magnetic resonance (NMR) signal ratio improvement provided by a 72 nm SPION used in rat brain perfusion imaging [72]. Antonelli et al. reported the use of

SPIONs to image atrioventricular fistulas, chronic venous occlusions, and lower extremity arteries [74]. SPIONs internalized by macrophages were also reported as a possible contrast agent of the vascular phase, providing cardiovascular applications such as perfusion and viability imaging [75,78,89]. Moreover, for assessing the inflammatory microenvironment of primary/metastatic tumors and for monitoring the therapeutic response of cancer patients receiving radiotherapy and immunotherapy, non-invasive imaging of TAMs with SPION may offer considerable potential [90].

Limitations to the SPION-enhanced MRI examinations have been mentioned in the literature [81,91]. These imaging procedures are long compared with other imaging modalities, potentially causing a greater motion sensitivity. The concentration of MR probes must reach 0.01 mM to 10 mM for efficient detection [92]. By comparison, in single photon computed emission computed tomography (SPECT) and positron emission tomography (PET) imaging, the tracer can be detected at the picomolar scale [93]. Increased iron levels in the body can lead to tissue damage through oxidative injury. This must be taken into account for repeated examinations or longitudinal studies [81], or if the iron clearance rate of the subject is altered. Particles remaining after a SPION-enhanced acquisition can also cause major susceptibility artifacts and interfere with other MR acquisitions even several months after the injection for liver MRI [81].
