Immuno Magnetic Thermosensitive Liposomes For Cancer Therapy

The present work describes the encapsulation of the drug doxorubicin (DOX) in immuno paramagnetic thermosensitive liposomes. DOX is the most common chemotherapeutic agent for the treatment of a variety of carcinomas. However, the pure drug has high cytotoxicity and therefore requires a targeted and biocompatible delivery system. The introduction includes concepts, modalities, and functionalities of the project. First, a detailed description of the cell type (triple-negative breast cancer) is given. Furthermore, the importance of liposomal doxorubicin is explained and the current state of research is shown. The importance of modification to achieve thermosensitive properties and the procedure for co-encapsulation with Gd chelate to achieve paramagnetic properties is also discussed. In addition, the first part describes the surface modification with ADAM8 antibodies, which leads to improved targeting. The second part of the thesis covers the different materials and methods used in this paper. The production of the liposomes LipTS, LipTS-GD, LipTS-GD-CY, LipTS-GD-CY-MAB and the loading of DOX using an ammonium sulfate gradient method were described in detail. The results part deals with the physicochemical characterization using dynamic light scattering and laser Doppler velocimetry, which confirmed a uniform monodisperse distribution of the liposomes. These properties facilitate the approach of liposomes to target cancer cells. The influence of lipid composition of liposomes, co-encapsulation with Gd chelate and surface modification of liposomes was evaluated and described accordingly. The size and structure of the individual liposomal formulations were determined by atomic force microscopy and transmission electron microscopy. Morphological examination of the liposomes confirmed agreement with the sizes obtained by dynamic light scattering. Temperature-dependent AFM images showed an intact liposome structure at 37 °C, whereas heating by UHF-MRI led to a lipid film indicating the destruction of the lipid bilayer. Furthermore, TEM images showed the morphological properties of the liposomes and gave a more precise indication of how Gd-chelate accumulates within the liposomes. Liposomes with Gd-chelate showed well-separated vesicles, suggesting that Gd- chelate is deposited in the lipid bilayer of the liposomes. Gd was encapsulated in the hydrophilic core whereas chelate was extended into the lipid bilayer. By differential scanning calorimetry and drug release, the heat-sensitive functionality of the liposomes could be determined. Liposomes showed a beginning of phase transition temperature at about 38 °C, which can be achieved by UHF-MRI exposure. The maximum phase transition temperature in the case of LipTS-GD and LipTS-GD-CY-MAB was 42 °C and 40 °C, respectively. A proof of concept study for the thermosensitive properties of liposomes and a time-dependent DOX release profile in hyperthermia was performed. Gd-chelate is encapsulated in both LipTS-GD and LipTS-GD-CY-MAB and led to paramagnetic properties of the liposomes. This facilitates imaging mediated DOX delivery and diagnosis of the solid tumor and metastatic cells. The change in relaxation rate R1 of liposomes was quantified before and after heating above Tm (T> Tm). The relaxivity of the liposomes was obtained from the adapted slope of the relaxation rate against the Gd concentration. Remarkably, the relaxation rate and relaxivity increased after heating the liposomes above Tm (T> Tm), suggesting that the liposomes opened, released Gd chelate, and the exchange of water molecules became faster and more practicable. Toxicity studies describe the different mechanisms for induced DOX toxicity. The increased cytotoxic effect at elevated temperatures showed that the induced toxicity is thermally dependent, i.e. DOX was released from the liposomes. The high viability of the cells at 37 °C indicates that the liposomes were intact at normal physiological temperatures. Under UHF-MRI treatment, cell toxicity due to elevated temperature was observed. The cellular uptake of liposomes under UHF-MRI was followed by a confocal laser scanning microscope. An increase in fluorescence intensity was observed after UHF-MRI exposure. The study of the uptake pathway showed that the majority of liposomes were mainly uptake by clathrin-mediated endocytosis. In addition, the liposomes were modified with anti-ADAM8 antibodies (MAB 1031) to allow targeted delivery. The cellular binding capabilities of surface-modified and non-modified liposomes were tested on cells that had ADAM8 overexpression and on ADAM8 knockdown cells. Surface-modified liposomes showed a significant increase in binding ability, indicating significant targeting against cells that overexpress ADAM8 on their surface. In addition, cells with knockdown ADAM8 could not bind a significant amount of modified liposomes. The biocompatibility of liposomes was assessed using a hemolysis test, which showed neglected hemolytic potential and an activated thromboplastin time (aPTT), where liposomes showed minimal interference with blood clotting. Hemocompatibility studies may help to understand the correlation between in vitro and in vivo. The chorioallantois model was used in ovo to evaluate systematic biocompatibility in an alternative animal model. In the toxicity test, liposomes were injected intravenously into the chicken embryo. The liposomes showed a neglectable harmful effect on embryo survival. While free DOX has a detrimental effect on the survival of chicken embryos, this confirms the safety profile of liposomes compared to free DOX. LipTS-GD-CY-MAB were injected into the vascular system of the chicken embryo on egg development day 11 and scanned under UHF-MRI to evaluate the magnetic properties of the liposomes in a biological system with T2-weighted images (3D). The liposomal formulation had distinct magnetic properties under UHF MRI and the chick survived the scan. In summary, immunomagnetic heat-sensitive liposomes are a novel drug for the treatment of TNBC. It is used both for the diagnosis and therapy of solid and metastasizing tumors without side effects on the neighboring tissue. Furthermore, a tumor in the CAM model will be established. Thereafter, the selective targeting of the liposomes will be visualized and quantitated using fluorescence and UHF-MRI. Liposomes are yet to be tested on mice as a xenograft triple-negative breast cancer model, in which further investigation on the effect of DOX-LipTS-GD-CY-MAB is evaluated. On one hand, the liposomes will be evaluated regarding their targetability and their selective binding. On the other hand, the triggered release of DOX from the liposomes after UHF-MRI exposure will be quantitated, as well as evaluate the DOX-Liposomes therapeutic effect on the tumor

Osmotic- and Stroke-Induced Blood-Brain Barrier Disruption Detected by Manganese-Enhanced Magnetic Resonance Imaging

Manganese (Mn2+) has recently gained acceptance as a magnetic resonance imaging (MRI) contrast agent useful for generating contrast in the functioning brain. The paramagnetic properties of Mn2+, combined with the cell\u27s affinity for Mn2+ via voltage-gated calcium channels, makes Mn2+ sensitive to cellular activity in the brain. Compared with indirect measures of brain function, such as blood oxygenation level dependent (BOLD) functional MRI, manganese-enhanced MRI (MEMRI) can provide a direct means to visualize brain activity. MEMRI of the brain typically involves osmotic opening of the blood-brain barrier (BBB) to deliver Mn2+ into the interstitial space prior to initiation of a specific neuronal stimulus. This method assumes that the BBB-disruption process itself does not induce any apparent stimuli or cause tissue damage that might obscure any subsequent experimental observations. However, this assumption is often incorrect and can lead to misleading results for particular types of MRI applications. One aspect of these studies focused on characterizing the confounding effects of the BBB-opening process on MRI measurements typically employed to characterize functional activity or disease in the brain (Chapters 4 and 5). The apparent diffusion coefficient (ADC) of tissue water was found to decrease (relative to the undisrupted contralateral hemisphere) following BBB opening, obscuring similar ADC changes associated with ischemic brain tissue following stroke. Brain regions exhibiting reduced ADC values following osmotic BBB disruption also experienced permanent tissue damage, as validated by histological measures in the same vicinity of the brain. Non-specific MEMRI-signal enhancement was also observed under similar conditions and was found to be correlated to regions with BBB opening as verified by Evans Blue histological staining. In this case, MEMRI may prove to be a useful alternative for monitoring BBB-permeability changes in vivo. MEMRI was also investigated as a method for visualizing regions of BBB damage following ischemic brain injury (Chapter 6). BBB disruption following stroke has been investigated using gadolinium-based MRI contrast agents (e.g., Gd-DTPA). However, as an extracellular MRI contrast agent, Gd-DTPA is not expected to provide information regarding cell viability or function as part of MR image contrast enhancement. By comparison, brain regions with ischemia-induced BBB damage, and blood-flow levels sufficient to deliver Mn2+, show MEMRI-signal enhancement that correlates to regions with tissue damage as verified by histological staining. This approach should allow us to better understand the factors responsible for ischemia-induced BBB damage. Furthermore, MEMRI should be a useful tool for monitoring therapeutic interventions that might mitigate the damage associated with BBB disruption following stroke

Diamonds On The Inside: Imaging Nanodiamonds With Hyperpolarized MRI

Nontoxic nanodiamonds (NDs) have proven useful as a vector for therapeutic drug delivery to cancers and as optical bioprobes of subcellular processes. Despite their potential clinical relevance, an effective means of noninvasively imaging NDs in vivo is still lacking. Recent developments in hyperpolarized MRI leverage an over 10 000 times increase in the nuclear polarization of biomolecules, enabling new molecular imaging applications. This work explores hyperpolarization via intrinsic paramagnetic defects in nanodiamond. We present the results of MRI experiments that enable direct imaging of nanodiamond via hyperpolarized 13C MRI and indirect imaging of nanodiamonds via Overhauser-enhanced MRI. The construction of custom hardware for these experiments is detailed and the path to future in vivo experiments outlined. As nanodiamond has been established as a biocompatible platform for drug delivery, our results will motivate further research into hyperpolarized MRI for tracking nanoparticles in vivo

Lipid-based nanoparticles for magnetic resonance molecular imaging : design, characterization, and application

In this thesis research is described which was aimed to develop lipidic nanoparticles for the investigation and visualization of atherosclerosis and angiogenesis with both magnetic resonance molecular imaging and optical techniques. The underlying rationale for this is that conventional MR imaging techniques are only capable of visualizing physiological and morphological changes, while magnetic resonance molecular imaging aims to depict cellular and molecular processes that are associated with or lie at the basis of pathological processes. This may lead to earlier detection, and improved diagnosis and prognosis of disease processes. Furthermore this technique may be very useful for the evaluation of a given therapy. The introduction of MRI as a molecular imaging modality is hampered by its low sensitivity compared to nuclear methods like PET and SPECT. With recent developments in chemistry and the synthesis of powerful, innovative, specific, and multimodal contrast agents, e.g. by introducing fluorescent properties as well, MRI is becoming increasingly important for molecular imaging. Therefore, the first aim of the research described in this thesis was to develop biocompatible nanoparticles that can be made target specific and can be detected by both MRI and optical techniques to allow the investigation of disease processes with two highly complementary imaging methods. Chapter 1 gives a general introduction in magnetic resonance molecular imaging and its potential use for the investigation of several pathological processes. Furthermore, contrast enhanced MRI based on differences in T1 and T2 relaxation times is explained. Lastly, different classes of contrast agents and their contrast generating properties are described. Amphphilic molecules are widely applied to serve as building blocks for nanoparticles in biomedical applications. In the field of drug targeting for example, liposomes comprised of amphiphilic molecules hold great promise and have been used extensively the last several decades. Furthermore, micelles, microemulsions, and other amphiphilic aggregates are also under investigation to serve as drug carriers. A relatively new application of lipidic nanoparticles is their use as contrast generating materials for MRI. In Chapter 2 the properties of amphiphilic molecules and their assembly in a wide range of aggregated structures are described. This is followed by an overview of different strategies that are employed to conjugate targeting ligand to such lipid based nanoparticles. The emphasis of this chapter is a literature overview of what has been realized in this research field thus far. Chapter 3 describes the physical characterization of novel liposomal contrast agents. The morphology of different formulations was investigated with electron microscopy, which revealed the necessity of incorporating cholesterol in the liposomal bilayer. Furthermore the relaxation properties of these contrast agent were measured as a function of temperature and magnetic field strength. In Chapter 4 a liposomal contrast agent with both fluorescent and magnetic properties is described. The liposomes were made target specific by conjugating multiple E-selectin specific antibodies to the surface of the nanoparticle. Its feasibility to serve as molecular imaging contrast agent for the detection of the inflammation marker E-selectin is demonstrated in vitro. The specific uptake of the liposomes by human endothelial cells stimulated to express E-selectin was visualized by MRI and fluorescence microscopy. Chapter 5 describes a superparamagnetic nanoparticle encapsulated in a micellular shell. Fluorescent properties were introduced to this contrast agent by the incorporation of fluorescent lipids in the lipid layer. The contrast agent has a very high r2/r1 ratio and therefore is especially suitable to be used for T2 (*) enhanced MRI. The nanoparticle can be made target specific by covalently linking targeting ligands distally to the PEG chains of lipids incorporated in the micellular shell via maleimide-sulfhydryl coupling. Specificity for apoptotic cells was realized by conjugating multiple Annexin A5 proteins. The feasibility to use this contrast agent for molecular imaging purposes was demonstrated in vitro on apoptotic Jurkat cells. In Chapter 6 the synthesis and characterization of a novel bimodal nanoparticle based on semiconductor nanocrystals encapsulated within the corona of paramagnetic micelles is described. The CdSe nanoparticle, also referred to as quantum dot, serves as the contrast generating material for fluorescence imaging, while the paramagnetic micellular coating is employed for contrast enhanced MRI. The in vitro association of this nanoparticle with isolated cells by either conjugating multiple avß3-integrin specific RGDpeptides or multiple phosphatidyl serine specific Annexin A5 proteins was demonstrated with both fluorescence microscopy and MRI. The second aim of the research described in this thesis was to apply the novel nanoparticles for the investigation of atherosclerosis and tumor angiogenesis in mouse models with magnetic resonance molecular imaging. Chapter 7 describes the application of non targeted paramagnetic liposomes for the improved and sustained visualization of neointimal lesions induced after placing a constrictive collar around the right carotid artery of apoE-KO mice. Commercially available Gd-DTPA (Magnevist) showed little potential for the detection of such lesion. In Chapter 8 pegylated micelles conjugated with macrophage scavenger receptor (MSR) specific antibodies were employed for improved atherosclerotic plaque detection and characterization. Existing nanoparticulate agents that are used to detect macrophages, such as USPIO or lipophilic micelles, show little specificity. The micelles used for this study have a hydrophilic PEG coating, and therefore show minimal non-specific interaction with plaque, which results in negligible background signal. In case of the MSR micelles a pronounced enhancement of atherosclerotic plaque was observed. Furthermore, the micelles exhibit fluorescent properties by the incorporation of either quantum dots or fluorescent lipids. This allowed the detection of macrophages with optical techniques as well. Chapter 9 and Chapter 10 describe the application of avß3 targeted bimodal liposomes for the visualization of angiogenically activated tumor blood vessels with both MRI in vivo and fluorescence microscopy ex vivo. The specificity of the contrast agent was demonstrated with an MRI competition experiment, while the exclusive association with endothelial cells was demonstrated with fluorescence microscopy. The follow-up study demonstrates the usefulness of contrast enhanced MRI after applying this contrast agent for the evaluation of angiostatic therapies, i.e. using endostatin and anginex, at two time points after onset of therapy. Most importantly, the in vivo MRI data show very good correlation with ex vivo microvessel density determinations. In the last experimental Chapter 11 of this thesis a sophisticated method for the parallel visualization of angiogenic tumor blood vessels with both intravital microscopy (IVM) and MRI is described. The nanoparticulate contrast agent conjugated with avß3-specific RGDpeptides described in Chapter 6 was administrated to tumor bearing mice. IVM allowed the investigation of the disease process at the cellular level, while MRI was used to investigate angiogenesis at the anatomical level. The contrast agent possesses excellent contrast generating properties for these complementary imaging techniques. Widespread angiogenic activity within the rim of the tumor, and up to 1 cm from the tumor boundary could be observed by using both techniques

In vivo magnetic resonance imaging for assessing the integrity of the blood-tumour barrier in a mouse model of melanoma brain metastasis

Melanoma is the deadliest form of skin cancer. Metastasis to the brain is a life-threatening complication of melanoma in which the clinical incidence is 6-43%. Few animal models exist for melanoma brain metastases, and many are not clinically relevant. MRI was implemented to examine the development of tumors in a clinically relevant model of melanoma brain metastases. Balanced steady-state free precession (b-SSFP) sequence was used to assess total metastasis burden, T1wSE MRI using Gd-DTPA was used to assess blood-tumour barrier (BTB) integrity in vivo and dextran perfusion was used to assess BTB leakiness in situ. This model produced low tumour burden ranging from 5 to 19 metastases at endpoint, many nonenhancing metastases were detected at early time points and there was considerable heterogeneity in permeability of the BTB for melanoma brain metastases. This clinically relevant model can be applied in future studies involving testing efficacy of chemotherapeutic agents

Impact of biopolymer matrices on relaxometric properties of MRI contrast agents and their application to Nanotechnology

Magnetic Resonance Imaging (MRI) represents the first-line diagnostic imaging modality for numerous indications. It is a clinically well-established, non-invasive technique providing three dimensional whole body anatomical and functional imaging. It takes advantage of the magnetic properties of water protons present in the body and their tissue-dependent behaviour. High magnetic fields (1.5 T and above) are clinically favoured because of their higher signal-to-noise ratio, capability for MR spectroscopy, and other forms of functional MRI, high speed imaging, and high resolution imaging. Signal intensity in MRI is related to the relaxation rate of in vivo water protons and can be enhanced by the administration of a contrast agent (CA) prior to scanning. These CAs utilize paramagnetic metal ions and enhance the contrast in an MR image by positively influencing the relaxation rates of water protons in the immediate surroundings of the tissue in which they localize. Among different CAs, Gadolinium contrast medium is used in up to 30% of MRI scans and the most clinically-used MRI. However, Gadolinium (Gd), like most of the clinically-used CAs, is characterized by a relaxivity well below its theoretical limit, lacks in tissue specificity and, in addition, it causes heavy allergic effects and serious nephrotoxicity. In this framework, Port et al. have reported that the rigidification of MRI CAs, obtained through covalent or non-covalent binding to macromolecules, could be favourable to an increase in relaxivity of the metal-chelate. Later, Decuzzi et al. have proved that it is possible to modify through the geometrical confinement the magnetic properties of MRI CAs by controlling their characteristic correlation times without the chemical modification of the chelate structure. Furthermore, Courant et al. have highlighted the capability of combined hydrogels to boost the relaxivity of Gd-based CAs. Despite several experimental studies addressed in this field, a comprehensive knowledge of the mechanisms involved in the relaxation enhancement due to the entrapment of CAs in polymer-based architectures is still missing. In particular, the role played by the water at the interface between polymer chains and MRI CAs has not been fully investigated and could lead to the availability of tailored models that accurately describe these novel complex systems. In this work, we aim to demonstrate that a more in-depth knowledge about the interference between macromolecules and MRI CAs and an understanding of their physicochemical properties can significantly to impact in the design strategies of the nanostructures and, consequently, to overcome the limitations of clinically used MRI CAs, particularly linked to the low relaxivity. In this perspective, it is of primary importance to study the main phenomena involved in the formation of polymer matrices and how their properties can influence the relaxivity of MRI CAs. For this reason, we proposed a general strategy based on formation of nanostructures for boosting the efficacy of commercial Gd-based CAs by using FDA approved biopolymers, providing also tissue specificity and reduced nephrotoxicity. Indeed, we want to take advantage not only by the use of nanotechnologies for enhanced MRI but only by their capability to reach a specific target and to accumulate only in the site of interest. The implemented strategy has consisted in the control of the relaxometric properties by tuning the water dynamics, the physicochemical interactions and, therefore, the polymer conformation. Effectively, we primary investigated, in bulk, the impact of hydrogel solutions on the relaxometric properties of commercial CAs, highlighting the key role of hydrogel structural parameters (mesh size and crosslink density) in the relaxation enhancement. In this part, chemical and thermodynamic interactions involved in the complexation between biopolymers and CAs have been investigated through Isothermal Titration Calorimetry. Furthermore, characterizations of water dynamics and mobility and measurement of the relaxometric properties in hydrogel solutions containing CAs have been carried out by NMR and Time-Domain relaxometer. The main outputs were summarized in a concept called Hydrodenticity and defined as the equilibrium between the water osmotic pressure, the elastodynamic forces of the polymer chains and the hydration degree of the CA which is able to increase the relaxivity of the CA itself. Indeed, hydrogel nanostructures made of hydrophilic polymer chains held together by chemical or physical crosslinking, have the ability to swell in water, forming elastic gels that retain a large quantity of fluid in their mesh-like structures. The presence of hydrophilic polymer interfaces and the control of water behaviour in hydrogels play a fundamental role in the relaxation enhancement of the Gadolinium-based CAs by influencing the characteristic correlation times defined by the theory of Solomon and Bloembergen. Then, starting from the acquired knowledge, we moved to observe the role of Hydrodenticity in the design of biopolymer nanostructures for enhanced MRI. For the nanostructures’ synthesis, we used two different processing techniques: (1) High Pressure Homogenization; (2) Microfluidic Flow Focusing. These techniques were selected because of their ability to control process parameters enabling the tuning of the interaction between the biopolymers and the CA. Indeed, by easily adjusting concentrations, pressure of the Homogenizer and/or flow rates of the Microfluidic platform, we can modulate the crosslinking degree of the nanostructures and tune their hydrophilicity, size, shape and surface charge, impacting on the relaxometric properties. These approaches allow us to load MRI CAs into functional nanostructures and obtain nanocarries with tunable relaxometric properties. The powerful aspect and the novelty of our approach lies in the definition of Hydrodenticity and in its application to several architectures, biopolymers, lipids and mixture of them., preserving the main properties of nanoparticles for drug delivery. As future perspective, the nanostructures can also be engineered to carry more than one agent, accumulate in specific tissues or to act as probes for simultaneous diagnosis and therapy (theranostic or multimodal imaging agents), thereby facilitating targeted treatments and precision medicine

Magnetic Resonance Imaging of Susceptibility Effects in Carotid Atherosclerosis

This thesis explores the use of susceptibility-weighted imaging (SWI) and quantitative susceptibility mapping (QSM), to characterize carotid artery plaques with and without the use of ultrasmall superparamagnetic iron oxide (USPIO) nanoparticle contrast agents. The overall hypothesis is that QSM can serve to differentiate carotid artery plaque features of different susceptibility and provide a positive contrast mechanism for imaging the uptake of USPIOs. Chapter 1 describes the pathophysiology of carotid atherosclerosis. Vulnerable plaques, i.e. those at risk of rupture, can be characterized by the presence of a lipid rich necrotic core (LRNC), intraplaque haemorrhage (IPH), and inflammation. In addition, plaques may develop calcifications that may be protective of rupture. The chapter describes the established multi-contrast imaging protocols used for characterizing plaques. Furthermore, the use of USPIO-contrast agents to image inflammation is described. Chapter 2 describes the physical principles of MR image generation including the sensitivity to magnetic susceptibility. The principles of T2*w imaging, and susceptibility weighted imaging (SWI) are explained. Chapter 3 reviews the principles and post-processing steps involved in commonly used algorithms for QSM in terms of the underlying physical and mathematical principles which are then demonstrated in the form of numerical simulations. Chapter 4 presents the application of SWI to a group of patients who underwent USPIO enhanced MRI on a 1.5T MRI system. Images were acquired prior to infusion and 48 hours post infusion. SWI and gradient echo phase images were used to depict the field inhomogeneities generated by diamagnetic and paramagnetic materials within the plaques, calcification and USPIO-uptake. These results were then compared to a conventional carotid multi-contrast protocol, which includes R2*-mapping and T2*w imaging, and, where available, CT and histology. In chapter 5 QSM is performed in the carotid artery wall of a cohort of normal volunteers on a 1.5T MRI system. Unlike the brain, the neck contains fat which can cause severe errors in the field estimate, which propagate into the susceptibility map. Therefore, QSM was combined with water-fat separation for application in the neck to correct for these artifacts. This correctly estimated a high fat-fraction in fatty tissue in the neck and allowed for a detailed depiction of the anatomy of healthy volunteers. The susceptibility value measured in fatty tissue agreed with literature values. Chapter 6 applies QSM with water-fat separation to a subset of the patient group on a 1.5T MRI system. On pre-contrast scans QSM successfully identified calcification as diamagnetic tissue and the water-fat separation identified a lipid core. On the post-contrast susceptibility maps, USPIO-uptake was identified as hyperintense signal. This allows QSM to provide quantitative contrast in carotid imaging that can identify multiple features simultaneously and to simplify the imaging of USPIO-contrast. The results were confirmed using the multi-contrast carotid MRI protocol and, where available, histology and CT. Chapter 7 discusses the limitations of the current studies and the potential future improvements of the current methodology in terms of MR acquisition, post-processing algorithms and MR protocols. Future studies could serve to further evaluate the potential of QSM in carotid imaging and use it as a novel tool to quantify USPIO uptake in atherosclerotic carotid arteries

Manganese-Enhanced Magnetic Resonance Imaging: Overview and Central Nervous System Applications With a Focus on Neurodegeneration

Manganese-enhanced magnetic resonance imaging (MEMRI) rose to prominence in the 1990s as a sensitive approach to high contrast imaging. Following the discovery of manganese conductance through calcium-permeable channels, MEMRI applications expanded to include functional imaging in the central nervous system (CNS) and other body systems. MEMRI has since been employed in the investigation of physiology in many animal models and in humans. Here, we review historical perspectives that follow the evolution of applied MRI research into MEMRI with particular focus on its potential toxicity. Furthermore, we discuss the more current in vivo investigative uses of MEMRI in CNS investigations and the brief but decorated clinical usage of chelated manganese compound mangafodipir in humans