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Inflammation is signaled by the overexpression of epitopes on the vascular endothelium that primarily aim at recruiting immune cells into the inflamed area. The intravascular localization of these biomarkers makes them suitable targets for the MRI visualization of inflammation. Phospholipid-based nanosystems appear excellent candidates in virtue of their good biocompatibility, ability to deliver a high number of imaging units at the target site, and for the easy functionalization with targeting vectors. In this work, phospholipid-based micelles (hydrodynamic diameter of 20 nm) loaded with the amphiphilic Gd(III)-complex Gd-DOTAMA(C18)2 were vectorized with a small peptide able to specifically bind VCAM-1 receptors. The micelles displayed a high longitudinal relaxivity (36.4 s(-1)mmolGd(-1) at 25 °C and 0.7 T). A (1)H- and (17)O-water relaxometry study indicated that the paramagnetic complex embedded in the nanoparticles adopted two isomeric conformations, likely reflecting the well-known square antiprismatic (SAP) and twisted square antiprismatic (TSAP) configurations typically observed in DOTA-like lanthanide complexes. Interestingly, the TSAP structure, showing a much faster exchange rate for the water molecule coordinated to the metal ion, was the most abundant, thus explaining the high relaxivity of the micellar agent. The systemic administration of the micelles into a lipopolysaccharide-induced murine model of acute inflammation successfully demonstrated the ability of the targeting agents to detect the diseased area by T1 contrast enhanced MRI.

From the early days of CEST agents' disclosure, it was evident that their potential for in vivo applications was strongly hampered by the intrinsic low sensitivity. Therefore, much work has been devoted to seek out suitable routes to achieve strong CEST contrast enhancement. The use of nanosized systems turned out to be a strategic choice, because a very large amount of CEST agents can be delivered at the site of interest. However, the breakthrough innovation in term of increase of sensitivity was found by designing the lipoCEST agents. The naturally inspired, liposomes vesicles, when loaded with paramagnetic lanthanide-based shift reagents, can be transformed into CEST probes. The large number of water molecules entrapped inside the inner cavity of the nanovesicles represents an enormous pool of exchanging protons for the generation of CEST contrast, whereas the presence of the shift reagent increases the separation in chemical shift of their nuclear magnetic resonance signal from that of the bulk water, thus allowing for a proper exchange regime for the activation of CEST contrast. From lipoCEST, it has been rather straightforward to evolve to cellCEST in order to exploit the cytoplasmatic water molecules as source of the CEST effect, once cells have been loaded with the proper shift reagent. The red blood cells were found to be particularly suitable for the development of the cellCEST concept. Finally, an understanding of the main determinants of the CEST effects in nanosized and cellular-sized agents has allowed the design of innovative lipoCEST/RBC aggregates for potential theranostic applications. WIREs Nanomed Nanobiotechnol 2016, 8:602-618. doi: 10.1002/wnan.1385 For further resources related to this article, please visit the WIREs website., (© 2016 Wiley Periodicals, Inc.)

This work addresses the possibility of using Magnetization Transfer Contrast (MTC) for an improved MRI detection of T1 relaxation agents. The need to improve the detection threshold of MRI agents is particularly stringent when the contrast agents failed to accumulate to the proper extent in targeting procedures. The herein reported approach is based on the T1 dependence of MT contrast. It has been assessed that MT contrast can allow the detection of a Gd-containing agent at a lower detection threshold than the one accessible by acquiring T1W images. Measurements have been carried out either in TS/A cells or in vivo in a syngeneic murine breast cancer model. The reported data showed that in cellular experiments the MTC method displays a better sensitivity with respect to the common T1W experiments. In particular, the reached detection threshold allowed the visualization of samples containing only 2% of Gd-labeled cells diluted in unlabeled cells. In vivo experiments displayed a more diversified scheme. In particular, the tumor region showed two distinct behaviors accordingly with the localization of the imaging probe. The probe located in the tumor core could be detected to the same extent either by T1w or MTC contrast. Conversely, the agent located in the tumor rim was detected with a larger sensitivity by the MTC method herein described., (Copyright © 2015 John Wiley & Sons, Ltd.)

Purpose: To describe and quantify the different relaxation mechanisms operating in suspensions of liposomes that encapsulate paramagnetic lanthanide(III) complexes., Theory and Methods: The transverse relaxation rate of lanthanide-loaded liposomes receives contribution from the exchange between intraliposomal and bulk water protons, and from magnetic susceptibility effects. Phospholipids vesicles encapsulating different Ln(III)-HPDO3A complexes (Ln = Eu, Gd, or Dy) were prepared using the conventional thin film rehydration method. Relaxation times (T1 , T2 , and T2*) were measured at 14 Tesla (T) and 25 °C. The effect of compartmentalization of the paramagnetic agent inside the liposomal cavity was evaluated by means of an IRON-modified MRI sequence., Results: NMR measurements demonstrated that Curie spin relaxation is the dominant contribution (> 90%) to the observed transverse relaxation rate of paramagnetic liposomes. This was further confirmed by MRI that showed the ability of the liposome entrapped lanthanide complexes to generate IRON-MRI positive contrast in a size dependent manner., Conclusion: The Curie spin relaxation mechanism is by far the principal mechanism involved in the T2 shortening of the water protons in suspension of paramagnetic liposomes at 14T. The access to IRON contrast extends the potential of such nanosystems as MRI contrast agents., (© 2014 Wiley Periodicals, Inc.)

Purpose: The correlation between glutamine metabolism and oncogene expression in cancers has led to a renewed interest in the role of glutamine in cancer cell survival. Hyperpolarized [5-(13) C]glutamine is evaluated as a potential biomarker for noninvasive metabolic measurements of drug response in prostate cancer cells., Methods: Hyperpolarized [5-(13) C]glutamine is used to measure glutamine metabolism in two prostate cancer cell lines (PC3 and DU145) before and after treatment with the two natural anticancer drugs resveratrol and sulforaphane. An invasive biochemical assay simulating the hyperpolarized experiment is used to independently quantify glutamine metabolism., Results: Glutamine metabolism is found to be 4 times higher in the more glutaminolytic DU145 cells compared with PC3 cells under proliferating growth conditions by using hyperpolarized [5-(13) C]glutamine as a noninvasive probe. A significant decrease in glutamine metabolism occurs upon apoptotic response to treatment with resveratrol and sulforaphane., Conclusion: Hyperpolarized NMR using [5-(13) C]glutamine as a probe permits the noninvasive observation of glutaminolysis in different cell lines and under different treatment conditions. Hyperpolarized [5-(13) C]glutamine metabolism thus is a promising biomarker for the noninvasive detection of tumor response to treatment, as it directly monitors one of the hallmarks in cancer metabolism - glutaminolysis - in living cells., (© 2014 Wiley Periodicals, Inc.)

The work aimed at developing a MRI-guided protocol for the visualization of the release of material entrapped in liposomes stimulated by the local application of pulsed low-intensity non-focused ultrasound (pLINFU). The task was achieved by formulating liposomes filled up with the clinically approved paramagnetic agent gadoteridol, because the release of the agent from the nanovesicles is accompanied by a significant MRI signal enhancement. The protocol was validated in vivo on mice-bearing subcutaneous syngeneic B16 melanoma and i.v. injected with the paramagnetic liposomes. Upon exposing tumor to pLINFU (3MHz, insonation time 2min, duty cycle 50%) few minutes after liposomes injection, a signal enhancement of ca. 35% was detected. The effective release of the agent was confirmed by the strong enhancement measured in kidneys calyx and bladder due to the rapid renal excretion of the agent released in the tumor., From the Clinical Editor: In this paper, a pulsed low-intensity non-focused ultrasound-based technique was used to release a paramagnetic MRI contrast agent from liposomes, demonstrating the feasibility of this triggered release system in a mouse melanoma model for future research applications., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Fluorine MRI ((19) F MRI) is receiving an increasing attention as a viable alternative to proton-based MRI ((1) H MRI) for dedicated application in molecular imaging. The (19) F nucleus has a high gyromagnetic ratio, a 100% natural abundance and is furthermore hardly present in human tissues allowing for hot spot MR imaging. The applicability of (19) F MRI as a molecular and cellular imaging technique has been exploited, ranging from cell tracking to detection and imaging of tumors in preclinical studies. In addition to applications, developing new contrast materials with improved relaxation properties has also been a core research topic in the field, since the inherently low longitudinal relaxation rates of perfluorocarbon compounds result in relatively low imaging efficiency. Borrowed from (1) H MRI, the incorporation of lanthanides, specifically Gd(III) complexes, as signal modulating ingredients in the nanoparticle formulation has emerged as a promising approach to improvement of the fluorine signal. Three different perfluorocarbon emulsions were investigated at five different magnetic field strengths. Perfluoro-15-crown-5-ether was used as the core material and Gd(III)DOTA-DSPE, Gd(III)DOTA-C6-DSPE and Gd(III)DTPA-BSA as the relaxation altering components. While Gd(III)DOTA-DSPE and Gd(III)DOTA-C6-DSPE were favorable constructs for (1) H NMR, Gd(III)DTPA-BSA showed the strongest increase in (19F) R(1). These results show the potential of the use of paramagnetic lipids to increase (19F) R(1) at clinical field strengths (1.5-3 T). At higher field strengths (6.3-14 T), gadolinium does not lead to an increase in (19F) R(1) compared with emulsions without gadolinium, but leads to an significant increase in (19F) R(2). Our data therefore suggest that the most favorable situation for fluorine measurements is at high magnetic fields without the inclusion of gadolinium constructs., (Copyright © 2014 John Wiley & Sons, Ltd.)

Purpose: A novel method based on the use of Yb-HPDO3A as MRI Para-CEST agent for in vivo pH mapping of the tumor region in a melanoma murine model is reported. This method does not require the knowledge of the concentration of the imaging agent., Methods: C57BL/6-mice were inoculated with B16-F10 cells. CEST-MR images of tumor and bladder were acquired upon the i.v. administration of Yb-HPDO3A (1.2 mmol/Kg). pH was assessed by the use of a ratiometric method., Results: Yb-HPDO3A distributes well in the extracellular space of the tumor allowing the detection of good levels of saturation transfer (ST). It is excreted throughout kidneys and accumulated in the bladder thus yielding a strong CEST signal from urine. By comparing the ST% obtained upon selective irradiation of the two OH resonances belonging to the two isomeric forms of Yb-HPDO3A, it has been possible to measure the extracellular pH for each voxel (0.22 mm(3) ). The obtained pH-maps of tumors show a great heterogeneity. Marked differences are associated to tumor staging., Conclusion: The application of Yb-HPDO3A to measure extracellular tumor pH provides a good spatio-temporal resolution and it does not require the prior knowledge of the contrast agent concentration. The herein reported data support the potential clinical translation of Yb-HPDO3A., (Copyright © 2013 Wiley Periodicals, Inc.)

A Gd(III)-DOTA-like complex bearing two aliphatic chains on adjacent acetic arms was designed, synthesized, and compared with its analogous monofunctionalized complex. A 1/T(1) NMR relaxometric study of the two amphiphilic complexes incorporated into micelles and liposomes showed an unprecedented relaxivity enhancement for the complex with two lipophilic side arms. This remarkable result, which is attributed to a favorable water exchange rate and increased rigidity of the system resulting from limiting of the local motion of the gadolinium center, represents an important advance in the development of highly efficient nanosystems for MRI applications.

Contrast in magnetic resonance imaging (MRI) arises from changes in the intensity of the proton signal of water between voxels (essentially, the 3D counterpart of pixels). Differences in intervoxel intensity can be significantly enhanced with chemicals that alter the nuclear magnetic resonance (NMR) intensity of the imaged spins; this alteration can occur by various mechanisms. Paramagnetic lanthanide(III) complexes are used in two major classes of MRI contrast agent: the well-established class of Gd-based agents and the emerging class of chemical exchange saturation transfer (CEST) agents. A Gd-based complex increases water signal by enhancing the longitudinal relaxation rate of water protons, whereas CEST agents decrease water signal as a consequence of the transfer of saturated magnetization from the exchangeable protons of the agent. In this Account, we survey recent progress in both areas, focusing on how MRI is becoming a more competitive choice among the various molecular imaging methods. Compared with other imaging modalities, MRI is set apart by its superb anatomical resolution; however, its success in molecular imaging suffers because of its intrinsic insensitivity. A relatively high concentration of molecular agents (0.01-0.1 mM) is necessary to produce a local alteration in the water signal intensity. Unfortunately, the most desirable molecules for visualization in molecular imaging are present at much lower concentrations, in the nano- or picomolar range. Therefore, augmenting the sensitivity of MRI agents is key to the development of MR-based molecular imaging applications. In principle, this task can be tackled either by increasing the sensitivity of the reporting units, through the optimization of their structural and dynamic properties, or by setting up proper amplification strategies that allow the accumulation of a huge number of imaging reporters at the site of interest. For Gd-based agents, high sensitivities can be attained by exploiting a range of nanosized carriers (micelles, liposomes, microemulsions, and the like, as well as biological structures such as apoferritin and lipoproteins) properly loaded with Gd-based chelates. Furthermore, the sensitivity of Gd-based agents can be markedly affected either by their interactions with biological structures or by their cellular localization. For CEST agents, a huge sensitivity enhancement has been obtained by using the water molecules contained in the inner cavity of liposomes as the exchangeable source of protons for magnetization transfer. Several "tricks" (for example, the use of multimeric lanthanide(III) shift reagents, changes in the shape of the liposome container, and so forth) have been devised to improve the chemical shift separation between the intraliposomal water and the "bulk" water resonances. Overall, excellent sensitivity enhancements have been obtained for both classes of agents, enabling their use in MR molecular imaging applications.

Magnetically oriented liposomes can be prepared by exposing unilamellar spherical systems loaded with paramagnetic lanthanide(III) complexes to hyperosmotic stress. The resulting nonspherical, lens-shaped, nanoparticles can orient within a static magnetic field, depending on the magnetic properties of their membrane bilayer. The orientation of the vesicles can be easily determined by measuring the paramagnetic contribution to the (1)H chemical shift of the intraliposomal water proton resonance. As the latter shift is dominated by the bulk magnetic susceptibility contribution, its sign (negative or positive) reports about the preferred orientation adopted by the nanovesicles. The alignment within the field depends upon the magnetic susceptibility anisotropy of the liposome membrane, Delta(chi)(LIPO). When Delta(chi)(LIPO) has a negative value (e.g., for nonspherical liposomes made of conventional phospholipids), the nanoparticles align with their long axis parallel to the field, whereas when Delta(chi)(LIPO)>0 the vesicles flip by 90 degrees . The sign of the chemical shift of the intraliposomal water resonance is positive in the former case and negative in the latter, respectively. The liposome orientation can be switched by incorporating in the liposome bilayer suitable amphiphilic paramagnetic lanthanide(III) complexes. The sign of Delta(chi)(LIPO), and consequently the magnetic alignment, will correspond to the sign of the magnetic susceptibility anisotropy of the metal complex. The magnetic susceptibility anisotropy is dependent on both the electronic configuration of the lanthanide ion and the structural characteristics of the amphiphilic complex incorporated in the liposome membrane. The magnetic orientation of such vesicles is maintained in vivo, thus opening promising perspectives for the application of nonspherical liposomes in medical imaging.

The peculiar properties of osmotically shrunken liposomes acting as magnetic resonance imaging-chemical exchange saturation transfer (MRI-CEST) contrast agents have been investigated. Attention has been primarily devoted to assessing the contribution arising from encapsulated and incorporated paramagnetic lanthanide(III)-based shift reagents in determining the chemical shift of the intraliposomal water protons, which is a relevant factor for generating the CEST contrast. It is demonstrated that a highly shifted resonance for the encapsulated water can be attained by increasing the percentage of the amphiphilic shift reagent incorporated in the liposome bilayer. It is also demonstrated that the shift contribution arising from the bulk magnetic susceptibility can be optimized through the modulation of the osmotic shrinkage. In terms of sensitivity, it is shown that the saturation transfer efficiency can be significantly improved by increasing the size of the vesicle, thus allowing a high number of exchangeable protons to be saturated. In addition, the role played by the intensity of the saturating radiofrequency field has also been highlighted.

One of the major advantages of the CEST methodology deals with the possibility of visualizing more probes in the same MR image voxels. This is a unique property within the contrast media that act on the (1)H-NMR signal of water protons, and it might considerably improve the potential of the technique. In addition to displaying sufficiently different resonance frequencies of their mobile protons, it is also important that the CEST agents designed for this application are highly sensitive. LIPOCEST agents represent the most sensitive class of CEST systems developed so far. On this basis, two LIPOCEST samples, a spherical one and an osmotically shrunken nonspherical one, endowed with markedly different resonance frequencies of their intraliposomal water protons, 3 ppm and 15 ppm, respectively, were prepared and tested both in vitro and in ex-vivo on a bovine muscle used as tissue-surrogate. The response of the two agents did not interfere each other, thus allowing the multiple visualization of the two agents present at nanomolar concentrations in the same image voxels., ((c) 2008 John Wiley & Sons, Ltd.)

Dramatic relaxation enhancements of L-lactate resonances have been observed upon formation of ternary adducts with Gd(III) complexes of heptadentate DO3A and DO3A-like ligands (DO3A = 1,4,7,10-tetraazaciclododecane-1,4,7-triacetic acid). Detailed 1H and 17O NMR relaxometry investigations allow us to obtain structural, dynamic and thermodynamic information on the ternary complexes in which L-lactate acts as a bidentate ligand replacing two water molecules in the inner coordination sphere of the Gd(III) ion. It has been found that the exchange rate of the coordinated L-lactate is modulated by the structural and electronic properties of the parent Gd-heptacoordinated macrocyclic chelate. In addition to the characterisation of the relaxation behaviour of the 1H methyl resonance of L-lactate, this study has been extended to its 13C isomer (fully enriched at the three positions) and to the trifluoro-L-lactate. The obtained results may be relevant to the development of relaxation agents able to promote the relaxation enhancement of specific substrates detectable by in vivo magnetic resonance spectroscopy.

The (1)H and (17)O NMR relaxometric properties of two cationic complexes formed by Gd(III) with a macrocyclic heptadentate triamide ligand, L(1), and its Nmethylated analogue, L(2), have been investigated in aqueous media as a function of pH, temperature and magnetic field strength. The complexes possess two water molecules in their inner coordination sphere for which the rate of exchange has been found to be sensibly faster for the Nmethylated derivative and explained in terms of electronic effects (decrease of the charge density at the metal center) and perturbation of the network of hydrogen-bonded water molecules in the outer hydration sphere. The proton relaxivity shows a marked dependence from pH and decreases of about six units in the pH range 6.5 to 9.0. This has been accounted for by the displacement of the two water molecules by dissolved carbonate which acts as a chelating anion. The formation of ternary complexes with lactate, malonate, citrate, acetate, fluoride and hydrogenphosphate has been monitored by (1)H NMR relaxometric titrations at 20 MHz and pH 6.3 and the value of the affinity constant, K, and of the relaxivity of the adducts could be obtained. Lactate, malonate and citrate interact strongly with the complexes (log K > or =3.7) and coordinate in a bidendate mode by displacing both water molecules. Larger affinity constants have been measured for GdL(2). Acetate, fluoride and hydrogenphosphate form monoaqua ternary complexes which were investigated in detail with regard to their relaxometric properties. The NMR dispersion (NMRD) profiles indicate a large contribution to the relaxivity of the adducts from water molecules belonging to the second hydration shell of the complexes and hydrogen-bonded to the anion. A VT (17)O NMR study has shown a marked increase of the rate of water exchange upon binding which is explained by coordination of the anion in an equatorial site, thus leaving the water molecule in an apical position, more accessible for interactions with the solvent molecules of the second hydration shell which facilitate the exchange process.