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Breast cancer is the most common cancer among women worldwide. For high-risk women, contrast enhanced (CE)-magnetic resonance imaging (MRI) is recommended as supplemental screening together with mammography. The development of new MRI contrast agents is an active field of research, which requires efficacy tests on appropriate preclinical pathological models. In this work, a refined method to orthotopically induce breast cancer in BALB/c mice was developed using ultrasound (US) as a guide for the precise localisation of the tumour induction site and to improve animal welfare. The method was coupled with CE-MRI to characterise the evolution of the tumoural lesion.

Cardiovascular diseases (CVD) account for nearly half of all deaths in Europe and almost 30% of global deaths. Despite the improved clinical management, cardiovascular mortality is predicted to rise in the next decades due to the increasing impact of aging, obesity, and diabetes. The goal of emerging cardiovascular nanomedicine is to reduce the burden of CVD using nanoscale medical products and devices. However, the development of novel multicom-ponent nano-sized products poses multiple technical, ethical, and regulatory challenges, which often obstruct their road to successful approval and use in clinical practice. This review discusses the rational design of nanoparticles, including safety considerations and regulatory issues, and highlights the steps needed to achieve efficient clinical translation of promising nanomedicinal products for cardiovascular applications.

Atherosclerosis is a major cardiovascular disease worldwide, that could benefit from innovative nanomedicine imaging tools and treatments. In this perspective, we here studied, by fluorescence imaging in ApoE-/- mice, the biodistribution of non-functionalized and RXP470.1-targeted nanostructured lipid carriers (NLC) loaded with DiD dye. RXP470.1 specifically binds to MMP12, a metalloprotease that is over-expressed by macrophages residing in atherosclerotic plaques. Physico-chemical characterizations showed that RXP-NLC (about 105 RXP470.1 moieties/particle) displayed similar features as non-functionalized NLC in terms of particle diameter (about 60-65 nm), surface charge (about &minus, 5 &mdash, &minus, 10 mV), and colloidal stability. In vitro inhibition assays demonstrated that RXP-NLC conserved a selectivity and affinity profile, which favored MMP-12. In vivo data indicated that NLC and RXP-NLC presented prolonged blood circulation and accumulation in atherosclerotic lesions in a few hours. Twenty-four hours after injection, particle uptake in atherosclerotic plaques of the brachiocephalic artery was similar for both nanoparticles, as assessed by ex vivo imaging. This suggests that the RXP470.1 coating did not significantly induce an active targeting of the nanoparticles within the plaques. Overall, NLCs appeared to be very promising nanovectors to efficiently and specifically deliver imaging agents or drugs in atherosclerotic lesions, opening avenues for new nanomedicine strategies for cardiovascular diseases.

Purpose To dissect the contributions to the longitudinal relaxivity (r1) of two commercial contrast agents (CAs), Gd-DOTA and Gd-HP-DO3A, and to synthesize/characterize a novel macrocyclic agent (Gd-Phen-DO3A) having superior r1. Methods Longitudinal relaxation rates R1 of the CAs in saline with/without human serum albumin (HSA), ionized simulated body fluid (i-SBF), viscous simulated body fluid (v-SBF), and human plasma were measured. Results have been interpreted to evince the main determinants to the observed r1 values. Results In v-SBF or in the presence of HSA, r1 is enhanced for all complexes, reflecting the viscosity increase and a weak interaction with proteins. The CAs further differentiate in plasma, with a relaxivity increase (versus saline) of approximately 1, 1.5, and 2.5 mM−1s−1 for Gd-DOTA, Gd-HPDO3A, and Gd-Phen-DO3A, respectively. R1 versus pH curves in i-SBF indicates that prototropic exchange sizably contributes to the relaxivity of Gd-HP-DO3A and Gd-Phen-DO3A. Conclusion The major contributions to r1 in the physiological environment have been highlighted, namely, increased viscosity, complex-protein interaction, and prototropic exchange. The control of these terms allows the design of novel macrocyclic structures with enhanced r1 as a result of an improved interaction with plasma's macromolecules and the shift of the prototropic exchange to physiological pH. Magn Reson Med 78:1523–1532, 2017. © 2016 International Society for Magnetic Resonance in Medicine.

Meningioma in vivo research is hampered by the difficulty of establishing an easy and reproducible orthotopic model able to mimic the characteristics of a human meningioma. Moreover, leptomeningeal dissemination and high mortality are often associated with such orthotopical models, making them useless for clinical translation studies. An optimized method for inducing meningiomas in nude mice at two different sites is described in this paper and the high reproducibility and low mortality of the models are demonstrated. Skull base meningiomas were induced in the auditory meatus and convexity meningiomas were induced on the brain surface of 23 and 24 nude mice, respectively. Both models led to the development of a mass easily observable by imaging methods. Dynamic contrast enhanced MRI was used as a tool to monitor and characterize the pathology onset and progression. At the end of the study, histology was performed to confirm the neoplastic origin of the diseased mass.

International audience; Nanostructured lipid carriers (NLC) might represent an interesting approach for the identification and targeting of rupture-prone atherosclerotic plaques. In this study, we evaluated the biodistribution, targeting ability and safety of 64Cu-fonctionalized NLC in atherosclerotic mice. 64Cu-chelating-NLC (51.8±3.1 nm diameter) with low dispersity index (0.066±0.016) were produced by high pressure homogenization at tens-of-grams scale. 24 h after injection of 64Cu-chelated particles in ApoE-/- mice, focal regions of the aorta showed accumulation of particles on autoradiography that colocalized with Oil Red O lipid mapping. Signal intensity was significantly greater in aortas isolated from ApoE-/- mice compared to wild type (WT) control (8.95 [7.58, 10.16]×108 vs 4.59 [3.11, 5.03]×108 QL/mm2, P < 0.05). Moreover, NLC seemed safe in relevant biocompatibility studies. NLC could constitute an interesting platform with high clinical translation potential for targeted delivery and imaging purposes in atherosclerosis.

An increased prevalence of liver diseases such as hepatitis C and nonalcoholic fatty liver results in an augmented incidence of the most common form of liver cancer, hepatocellular carcinoma (HCC). HCC is most often found in the cirrhotic liver and it can therefore be challenging to rely on anatomical information alone when diagnosing HCC. Valuable information on specific cellular metabolism can be obtained with high sensitivity thanks to an emerging magnetic resonance (MR) technique that uses 13C labeled hyperpolarized molecules. Our interest was to explore potential new high contrast metabolic markers of HCC using hyperpolarized 13C-MR. This work led to the identification of a class of substrates, low molecular weight ethyl-esters, which showed high specificity for carboxyl esterases and proved in many cases to possess good properties for signal enhancement. In particular, hyperpolarized [1,3-13C2]ethyl acetoacetate (EAA) was shown to provide a metabolic fingerprint of HCC. Using this substrate a liver cancer implanted in rats was diagnosed as a consequence of an ∼4 times higher metabolic substrate-to-product ratio than in the surrounding healthy tissue, (p = 0.009). Unregulated cellular uptake as well as cosubstrate independent enzymatic conversion of EAA, made this substrate highly useful as a hyperpolarized 13C-MR marker. This could be appreciated by the signal-to-noise (SNR) obtained from EAA, which was comparable to the SNR reported in a literature liver cancer study with state-of-the-art hyperpolarized substrate, [1-13C]pyruvate. Also, the contrast-to-noise (CNR) in the EAA based metabolic ratio images was significantly improved compared with the CNR in equivalent images reported using [1-13C]pyruvate.

Glutamine metabolism is, with its many links to oncogene expression, considered a crucial step in cancer metabolism and it is thereby a key target for alteration in cancer development. In particular, strong correlations have been reported between oncogene expression and expression and activity of the enzyme glutaminase. This mitochondrial enzyme, which is responsible for the deamidation of glutamine to form glutamate, is overexpressed in many tumour tissues. In animal models, glutaminase expression is correlated with tumour growth rate and it is readily possible to limit tumour growth by suppression of glutaminase activity. In principle, hyperpolarized 13C MR spectroscopy can provide insight to glutamine metabolism and should hence be a valuable tool to study changes in glutaminase activity as tumours progress. However, no such successful in vivo studies have been reported, even though several good biological models have been tested. This may, at least partly, be due to problems in preparing glutamine for hyperpolarization. This paper reports a new and improved preparation of hyperpolarized [5-13C]glutamine, which provides a highly sensitive 13C MR marker. With this preparation of hyperpolarized [5-13C]glutamine, glutaminase activity in vivo in a rat liver tumour was investigated. Moreover, this marker was also used to measure response to drug treatment in vitro in cancer cells. These examples of [5-13C]glutamine used in tumour models warrant the new preparation to allow metabolic studies with this conditionally essential amino acid.

To dissect the contributions to the longitudinal relaxivity (rLongitudinal relaxation rates RIn v-SBF or in the presence of HSA, rThe major contributions to r

The current study was performed to evaluate the in vivo efficiency of a new nano-sized contrast agent called paramagnetic Solid Lipid Nanoparticles, pSLNs, having promising relaxivity properties for Magnetic Resonance Imaging application. Good stability and stealth properties toward macrophage uptake have been demonstrated. An in vivo MRI study resulted in an improved signal enhancement in the tumor tissue particularly when folate as targeting ligand was used to decorate the nanoparticles surface. Afterward, the biodistribution of pSLNs in several organs was investigated. The accumulation of pSLNs in kidneys, femoral bones, spleen and brain was quite low while high tropism of pSLNs was found for the liver. In this regard, approaches to improve the rate of the hepatic clearance have been proposed.

Dysprosium (Dy)-loaded liposomes act as excellent T(2)-susceptibility agents at high magnetic field strength. The R(2)-enhancement increases with the size of the liposomes and the concentration of entrapped paramagnetic metal complexes. Neuro-2a tumor cells are readily labeled when Dy-loaded liposomes, suitably functionalized with glutamine residues (Gln), are added to the culture medium as glutamine receptors are highly expressed in such proliferating tumor cells. By using fluorescent liposomes doped with fluorescent dyes (either incorporated in the membrane or included in the inner cavity), confocal microscopy experiments showed that targeted liposomes are taken up much more avidly than non-targeted vesicles. In vivo studies showed that glutamine-functionalized and non-functionalized liposomes accumulate in the tumor region to a similar extent. Confocal images of the excised tumor showed extensive co-localization of liposomes and macrophages in both cases. It is suggested that the loss of tumor specificity, shown by Gln-functionalized liposomes in vivo, has to be associated with the efficient removal of liposomes operated by the RES (reticulo endoplasmatic system) or tumor associated macrophages.

The present paper describes the detection of a magnetic resonance imaging (MRI) contrast agent by matrix-assisted laser desorption ionization imaging mass spectrometry (MALDI-IMS). The contrast agent was analyzed in both frozen and paraformaldehyde-fixed mouse livers explanted after its in vivo administration, and its identity was confirmed by fragmentation experiments. Moreover, a semiquantitative analysis was performed, evaluating its content in livers from mice sacrificed at different postadministration times. To the best of our knowledge, this is the first description of a MALDI-IMS analysis of MRI contrast agents and the first time that results obtained by MALDI-IMS are validated by both an in vivo (MRI) and an ex vivo (inductively coupled plasma atomic emission spectroscopy, ICP-AES) technique. Results shown in the present paper demonstrate the possibility of using MALDI-IMS for drug biodistribution analysis. Obviously, this application is particularly interesting in the case of unlabeled compounds, which cannot be detected by any of the other imaging techniques.

This article illustrates some innovative applications of liposomes loaded with paramagnetic lanthanide-based complexes in MR molecular imaging field. When a relatively high amount of a Gd(III) chelate is encapsulated in the vesicle, the nanosystem can simultaneously affect both the longitudinal (R(1)) and the transverse (R(2)) relaxation rate of the bulk H2O H-atoms, and this finding can be exploited to design improved thermosensitive liposomes whose MRI response is not longer dependent on the concentration of the probe. The observation that the liposome compartmentalization of a paramagnetic Ln(III) complex induce a significant R(2) enhancement, primarily caused by magnetic susceptibility effects, prompted us to test the potential of such agents in cell-targeting MR experiments. The results obtained indicated that these nanoprobes may have a great potential for the MR visualization of cellular targets (like the glutamine membrane transporters) overexpressing in tumor cells. Liposomes loaded with paramagnetic complexes acting as NMR shift reagents have been recently proposed as highly sensitive CEST MRI agents. The main peculiarity of CEST probes is to allow the MR visualization of different agents present in the same region of interest, and this article provides an illustrative example of the in vivo potential of liposome-based CEST agents.

An early diagnosis of cancer is crucial in the battle against this disease and the in vivo visualization of tumors at cellular level is still the most challenging goal. In order to target tumor cells, we took into account their increased metabolism and amino acid nutrients or pseudo-nutrients, which are actively transported through the cell membrane, have been chosen as vectors for new MRI contrast agents. For this reason new gadolinium complexes conjugated to agmatine, arginine, and glutamine have been synthesized and studied.

Gd-HPDO3A has been internalized into rat hepatocarcinoma cells in the cytoplasm (by electroporation) or in intracellular vesicles (by pinocytosis), respectively. In the former case, the observed relaxation rates are likely dependent upon the amount of internalized paramagnetic complex, whereas in the latter case the relaxation enhancement is "quenched" to a plateau value (about 3 s(-1)) when the entrapped amount of Gd-chelate is higher than 1 x 10(10) Gd/cell. The observed behavior has been accounted in terms of a theoretical treatment based on equations formally derived by Labadie et al. (J Magn Reson B 1994;105:99-102). On this basis, entrapment into intracellular vesicles has been treated as a three-site water exchange (extracellular/cytoplasm/vesicle compartments), whereas the cell pellets containing the paramagnetic agent spread out in the cytoplasm can be analyzed by a two-site exchange system.

MR-labeling of cells may be carried out by adding a Gd-based contrast agent to the incubation media. The amount of gadolinium internalized in HTC and C6 cells upon incubation with Gd–DTPA–BMA is circa one order of magnitude higher than those found with Gd–DTPA, Gd–DOTA and Gd–HPDO3A, respectively. The comparison of relaxometric and mass spectrometry determinations allows us to establish that only a minor fraction of intact Gd–DTPA–BMA is internalized into the cells. Moreover the binding/uptake behavior shown by Gd–DTPA–BMA resembles that found when GdCl3 is added to the incubation medium. We suggest that the lower stability of Gd–DTPA–BMA is responsible for a shift in the dissociation equilibrium that results in the net transfer of Gd3+ ions on the cell membrane followed by a slower internalization process. The transmetallation process is mediated by components of the incubation media, among which a dominant role is represented by phosphate anions. The uptake of Gd3+ ions is clearly reflected in the drastic decrease of cell viability observed for cells labeled with Gd–DTPA–BMA. Copyright © 2006 John Wiley & Sons, Ltd.

Under HR-MAS conditions, cells are subjected to high centrifugal forces that may cause irreversible cell damage. First, conditions have been defined to monitor and keep to a minimum unwanted effects in HR-MAS spectra arising from the loss of cell integrity. Then, the HR-MAS spectra of reasonably intact cells have been analyzed. Cell suspensions subjected to MAS rates as low as 1 kHz split into a two-compartment system that is composed of a cell-rich phase (H2Oi) and a cell-free phase (H2Oo). Each of these phases is characterized by its own water 1H-NMR signal. Transport of water molecules between the cell-rich and cell-free compartments is limited by the very low contact area between the two compartments, and water exchange dynamics consequently fall into the slow exchange limit on the NMR timescale. Since the exchange between the two water populations is “frozen,” the separation between the H2Oo and H2Oi water signals (Δνwater) detected in an HR-MAS experiment is not affected by chemical exchange but reflects only chemical differences in the two environments. Different cell lines show a different Δνwater, leading to the concept of “cellular water shift.” This shift roughly correlates with the cellular protein content, supporting the view that the most important determinant of the cellular water shift is the interaction between water and proteins in the intracellular compartment. Magn Reson Med, 2005. © 2005 Wiley-Liss, Inc.

The low sensitivity is the major disadvantage of MRI as compared to PET. Therefore, amplification strategies are necessary for specific pathway labeling. This survey is aimed at exploring different routes to the entrapment of Gd(III) chelates in various type of cells at amounts sufficiently large to allow MRI visualization. Namely, the obtained results have been summarized in terms of internalization via i) pinocytosis; ii) phagocytosis; iii) receptors; iv) receptor mediated endocytosis; v) transporters; vi) transmembrane carrier peptides. MRI visualization of cells appears possible when the number of internalized Gd(III) chelates is of the order of 10 7- 10 8/cell. Pinocytosis shows to be particularly useful for labeling cells that can be incubated for several hours in the presence of high concentrations of Gd-agent. This approach appears very effective for labeling stem cells. Nanoparticles filled with Gd-chelates can be used for an efficient loading of cells endowed with a good phagocytic activity. Entrapment via receptors most often results in receptor mediated endocytosis. Suitably functionalized monomeric and multimeric Gdchelates can be considered for being internalized by this route as well as supramolecular systems such as those formed between Avidin and biotinylated Gd-complexes. Exploitation of up-regulated transporters of nutrients in tumor cells appears to be a promising route for their differentiation from healthy cells. Finally, properly designed systems entering the cells by means of penetrin-like peptides deserve great attention.

Agmatine has been proposed as the physiological ligand for the imidazoline receptors. It is not known whether it is also involved in the homoeostasis of intracellular polyamine content. To show whether this is the case, we have studied the effect of agmatine on rat liver cells, under both periportal and perivenous conditions. It is shown that agmatine modulates intracellular polyamine content through its effect on the synthesis of the limiting enzyme of the interconversion pathway, spermidine/spermine acetyltransferase (SSAT). Increased SSAT activity is accompanied by depletion of spermidine and spermine, and accumulation of putrescine and N1-acetylspermidine. Immunoblotting with a specific polyclonal antiserum confirms the induction. At the same time S-adenosylmethionine decarboxylase activity is significantly increased, while ornithine decarboxylase (ODC) activity and the rate of spermidine uptake are reduced. This is not due to an effect on ODC antizyme, which is not significantly changed. All these modifications are observed in HTC cells also, where they are accompanied by a decrease in proliferation rate. SSAT is also induced by low oxygen tension which mimics perivenous conditions. The effect is synergic with that promoted by agmatine.

Rat hepatocytes in culture take up [14C]-agmatine by both a high-affinity transport system [KM = 0.03 mM; Vmax = 30 pmol x min x (mg protein)-1] and a low-affinity system. The high-affinity system also transports putrescine, but not cationic amino acids such as arginine, and the polyamines spermidine and spermine. The rate of agmatine uptake is increased in cells deprived of polyamines with difluoromethylornithine. Of the agmatine taken up, 10% is transformed into polyamines and 50% is transformed into 4-guanidinobutyrate, as demonstrated by HPLC and MS. Inhibition by aminoguanidine and pargyline shows that this is due to diamine oxidase and an aldehyde dehydrogenase. 14C-4-aminobutyrate is also accumulated in the presence of an inhibitor of 4-aminobutyrate transaminase.

A series of diamines with the general structure NH2(CH2) x NH2, x=2–12, was tested for their potential effects on cell proliferation of cultured rat C6 glioma cells in comparison to natural polyamines. Long chain diamines reduced cell number after 48 h in culture with a sequence of 1,12-diaminododecane (1,12-DD) >1,10-diaminodecane >1,9-diaminononane. Polyamines (putrescine, spermidine and spermine) as well as diamines up to a CH2-chain length of x=8 were found to be ineffective. The spermine analogue 1,12-DD was the most effective molecule in reducing cell number in an irreversible, dose-dependent manner (EC50=3 µM under serum-free conditions). In further experiments we investigated the mechanisms of action of 1,12-DD. The compound had only a minor effect on cell cycle and did not affect free internal calcium concentration. Under physiological conditions 1,12-DD interacts with triplex DNA but not with duplex DNA. Ornithine decarboxylase activity as well as the concentration of internal polyamines were found to be reduced by 1,12-DD. Polyamine application, however, was not able to reverse the effect of 1,12-DD, indicating a polyamine-independent or non-competitive mechanism of action. 1,12-DD reduced cell number by induction of apoptosis as well as necrosis. In molecular modeling studies it was found that a minimal hydrophobic intersegment of at least 4 A was required to make a diamine an effective drug in respect to cellular growth. A hydrophobic gap of this size fits the minimum requirement expected from molecular modeling to provide space for hydrophobic interactions with parts of proteins like a CH3-group. Our results show that 1,12-DD acts as a potent drug, reducing the number of C6 glioma cells, and suggest that its spatial and hydrophobic properties are responsible for its mechanism of action.

An increased prevalence of liver diseases such as hepatitis C and nonalcoholic fatty liver results in an augmented incidence of the most common form of liver cancer, hepatocellular carcinoma (HCC). HCC is most often found in the cirrhotic liver and it can therefore be challenging to rely on anatomical information alone when diagnosing HCC. Valuable information on specific cellular metabolism can be obtained with high sensitivity thanks to an emerging magnetic resonance (MR) technique that uses 13C labeled hyperpolarized molecules. Our interest was to explore potential new high contrast metabolic markers of HCC using hyperpolarized 13C-MR. This work led to the identification of a class of substrates, low molecular weight ethyl-esters, which showed high specificity for carboxyl esterases and proved in many cases to possess good properties for signal enhancement. In particular, hyperpolarized [1,3-13C2 ]ethyl acetoacetate (EAA) was shown to provide a metabolic fingerprint of HCC. Using this substrate a liver cancer implanted in rats was diagnosed as a consequence of an ∼4 times higher metabolic substrate-to-product ratio than in the surrounding healthy tissue, (p=0.009). Unregulated cellular uptake as well as cosubstrate independent enzymatic conversion of EAA, made this substrate highly useful as a hyperpolarized 13C-MR marker. This could be appreciated by the signal-to-noise (SNR) obtained from EAA, which was comparable to the SNR reported in a literature liver cancer study with state-of-the-art hyperpolarized substrate, [1-13C]pyruvate. Also, the contrast-to-noise (CNR) in the EAA based metabolic ratio images was significantly improved compared with the CNR in equivalent images reported using [1-13C]pyruvate.

The aim of this study is to describe the synthesis of, relaxometric characterization of, pharmacokinetic properties of, and animal imaging experiments with a new, low molecular weight gadolinium complex with high binding affinity toward serum albumin. The gadolinium(III) chelate (B25716/1) is based on the structure of the heptadentate ligand 1,4-bis(hydroxycarbonylmethyl)-6-[bis(hydroxycarbonylmethyl)]amino-6 methylperhydro-1,4-diazepine (AAZTA) covalently conjugated to an analogue of deoxycholic acid. The study was conducted as a comparison with that of an analogous complex based on the octadentate diethylenetriaminepentaacetic acid ligand B22956/1 (whose albumin binding properties were previously assessed). The structural modification with respect to B22956/1 leads to a system that can host two coordinated water molecules in fast exchange with bulk water with potential higher efficiency as an MRI contrast agent. On interaction with human serum albumin the expected-field-independent-relaxation enhancement is not observed, possibly as a consequence of the displacement of one of the two inner-sphere water molecules of the gadolinium complex. At clinically relevant magnetic fields, however, the plasma relaxivity of B25716/1 is markedly higher than that shown by B22956/1, owing to concomitant synergistic contributions from the electronic correlation time and water molecules in the second coordination sphere. The capability of B25716/1 to enhance tumor regions in magnetic resonance images was assessed in vivo at 3 T on a xenograft tumor mouse model prepared with PC-3 cells. B25716/1 displays signal enhancements approximately double those observed for B22956/1, in agreement with the findings of the in vitro relaxivity investigations.

Highly efficient magnetic resonance imaging (MRI) probes have been prepared by loading Gd(III) complexes on the surface of solid lipid nanoparticles (pSLNs). Applicability as molecular imaging probes is demonstrated by an in vitro model study with targeted pSLNs.

We report the synthesis of novel chelates of Gd and (68)Ga with DPTA, DOTA, HP-DOA3, as well as with AAZTA, a novel chelating agent developed by our research group. These chelating agents were appropriately conjugated, prior to metal complexation, with DB58, an RGD peptidomimetic, conformationally constrained on an azabicycloalkane scaffold and endowed with high affinity for integrin α(ν)β(3) . Because α(ν)β(3) is involved in neo-angiogenesis in solid tumors and is also directly expressed in cancer cells (e.g. glioblastomas, melanomas) and ovarian, breast, and prostate cancers, these constructs could prove useful as molecular imaging probes in cancer diagnosis by MRI or PET techniques. Molecular modeling, integrin binding assays, and relaxivity assessments allowed the selection of compounds suitable for multiple expression on dendrimeric or nanoparticulate structures. These results also led us to an exploratory investigation of (68)Ga complexation for the promising (68)Ga-PET technique; the AAZTA complex 15((68)Ga) exhibited uptake in a xenograft model of glioblastoma, suggesting potentially useful developments with new probes with improved affinity.

Three new magnetic resonance imaging probes that target glutamine transporters have been synthesized. They consist of a Gd-DOTA-monoamide moiety (DOTA=1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) linked through a six carbon atom chain to a vector represented by a glutamine residue bound through alpha-carboxylic, gamma-carboxamidic, or alpha-amino functionalities. Their uptake by HTC (rat hepatocarcinoma) and healthy rat hepatocytes has shown that the system containing the glutamine vector bound through the alpha-carboxylic group displays a markedly higher affinity for tumor cells. The observed behavior is rationalized in terms of the exploitation of an additional glutamine transporter active in hepatic tumor cells.

Dysprosium (Dy)-loaded liposomes act as excellent T(2)-susceptibility agents at high magnetic field strength. The R(2)-enhancement increases with the size of the liposomes and the concentration of entrapped paramagnetic metal complexes. Neuro-2a tumor cells are readily labeled when Dy-loaded liposomes, suitably functionalized with glutamine residues (Gln), are added to the culture medium as glutamine receptors are highly expressed in such proliferating tumor cells. By using fluorescent liposomes doped with fluorescent dyes (either incorporated in the membrane or included in the inner cavity), confocal microscopy experiments showed that targeted liposomes are taken up much more avidly than non-targeted vesicles. In vivo studies showed that glutamine-functionalized and non-functionalized liposomes accumulate in the tumor region to a similar extent. Confocal images of the excised tumor showed extensive co-localization of liposomes and macrophages in both cases. It is suggested that the loss of tumor specificity, shown by Gln-functionalized liposomes in vivo, has to be associated with the efficient removal of liposomes operated by the RES (reticulo endoplasmatic system) or tumor associated macrophages.

Chemical exchange saturation transfer (CEST) imaging is an emerging MRI technique relying on the use of endogenous or exogenous molecules containing exchangeable proton pools. The heterogeneity of the water resonance frequency offset plays a key role in the occurrence of artifacts in CEST-MR images. To limit this drawback, a new smoothing-splines-based method for fitting and correcting Z-spectra in order to compensate for low signal-to-noise ratio (SNR) without any a priori model was developed. Global and local voxel-by-voxel Z-spectra were interpolated by smoothing splines with smoothing terms aimed at suppressing noise. Thus, a map of the water frequency offset (‘zero’ map) was used to correctly calculate the saturation transfer (ST) for each voxel. Simulations were performed to compare the method to polynomials and zero-only-corrected splines on the basis of SNR improvement. In vitro acquisitions of capillaries containing solutions of LIPOCEST agents at different concentrations were performed to experimentally validate the results from simulations. Additionally, ex vivo investigations of bovine muscle mass injected with LIPOCEST agents were performed as a function of increasing pulse power. The results from simulations and experiments highlighted the importance of a proper ‘zero’ correction (15% decrease of fictitious CEST signal in phantoms and ex vivo preparations) and proved the method to be more accurate compared with the previously published ones, often providing a SNR higher than 5 in different simulated and experimentally noisy conditions. In conclusion, the proposed method offers an accurate tool in CEST investigation. Copyright © 2008 John Wiley & Sons, Ltd.

Purpose To investigate the potential value of MRI for noninvasive assessment of angiogenesis in a murine model exploiting the properties of two contrast agents, gadoteridol (ProHance) and gadocoletic acid trisodium salt (B22956/1). Materials and Methods Biocompatible sponges were implanted in both mice flanks. Stimulated sponges contained human recombinant basic fibroblast growth factor (bFGF) as the angiogenic agent; control sponges contained vehicle. Angiogenesis was evaluated by MRI after injection of extravascular (ProHance) or blood-pool (B22956/1) contrast agents at different times after sponge implantation. Sponges signal intensity enhancement was calculated both as the relative enhancement and the rate of relative enhancement. Results from MRI were validated by classic biochemical (hemoglobin level and protein content) and morphological (histology) assays. Results The intrinsic different properties of ProHance and B22956/1 in wash-in and wash-out kinetics were useful to detect progressive vascularization and the establishment of a functional vascular network in the implants. Moreover, MRI allowed the appreciation of differences in neovessel colonization between bFGF-treated sponges and controls. Hemoglobin level, protein content, and histology confirmed the sponge vascularization and MRI results. Conclusion Contrast-enhanced MRI is a reliable tool to study vascular characteristics in animal models of angiogenesis. The different kinetic properties of contrast agents can provide evidence of different functional neovascularization aspects and levels. J. Magn. Reson. Imaging 2008;27:872–880. © 2008 Wiley-Liss, Inc.

The binding affinities of a selected series of Gd(III) chelates bearing bile acid residues, potential hepatospecific MRI contrast agents, to a liver cytosolic bile acid transporter, have been determined through relaxivity measurements. The Ln(III) complexes of compound 1 were selected for further NMR structural analysis aimed at assessing the molecular determinants of binding. A number of NMR experiments have been carried out on the bile acid-like adduct, using both diamagnetic Y(III) and paramagnetic Gd(III) complexes, bound to a liver bile acid binding protein. The identified protein "hot spots" defined a single binding site located at the protein portal region. The presented findings will serve in a medicinal chemistry approach for the design of hepatocytes-selective gadolinium chelates for liver malignancies detection. © 2007 American Chemical Society.

The glutamine transporting system is up-regulated in tumor cells because cell proliferation requires the uptake of large quantities of glutamine. It has been found that the paramagnetic magnetic resonance imaging (MRI) reporter Gd-DOTAMA-C6-Gln, where the glutamine residue is covalently bound to the Gd chelate through a C6 spacer, accumulates in tumor cells both "in vitro" and "in vivo" experiments. The observation that the relaxivity of cellular pellets does not increase with the increase in the amounts of entrapped Gd chelate is taken as an indication that the internalization has occurred through receptor mediated endocytosis. The iv administration of Gd-DOTAMA-C6-Gln allowed the MRI visualization of tumor masses in A/J mice grafted with the murine neuroblastoma cell line Neuro-2a and in Her-2/neu transgenic mice developing multiple mammary carcinoma, respectively.

The high binding affinity between avidin and biotin has been exploited to develop a procedure for magnetic resonance imaging (MRI) visualization of target cells. SHIN3 and PANC1 tumor cell lines have been used as target cells because they possess on their membranes galactosyl receptors able to bind avidin molecules. Avidin-Gd chelate adducts have been built by using two Gd complexes containing one (Gd-I) and two (Gd-II) biotin residues, respectively. The relaxivities of such supramolecular adducts are significantly higher than those shown by free Gd-I and Gd-II. There is evidence of the occurrence of multilayered adducts in which the bis-biotinylated Gd(3+) complex acts as a bridge between adjacent avidin molecules. MRI differentiation of labeled versus unlabeled cells has been attained when approximately 6 x 10(8) Gd units were internalized in each cell. Furthermore, there is a marked decrease in the measured intracellular T(1) relaxivity as the number of internalized Gd complexes increases, probably owing to too short relaxation times of endosomic water protons with respect to their diffusion lifetime.

Gadolinium (III) complexes are under intense scrutiny as contrast agents for magnetic resonance imaging. Although currently used mainly as extracellular agents, there is a growing interest to exploit their contrast enhancing ability in the intracellular environment. To ascertain the preservation of their chemical integrity upon the intracellular entrapment, it is necessary to have a method for their dosage in the cell lysates. Herein, a mass spectrometric method for detection and quantification of gadolinium complexes in cell lysates is reported. The detection by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was carried out by using a non-acidic matrix (2,4,6-trihydroxyacetophenone), which does not allow any leakage of gadolinium from the complex. Quantification has been possible by using as an internal standard an ytterbium complex with the same ligand of the analyte. Ytterbium was chosen because, among the lanthanides, it is the one with the isotopic distribution pattern the most similar to that of gadolinium. Sensitivity was enough to detect low micromolar quantities of a cationic complex and high micromolar quantities of a neutral complex in cell lysates of rat hepatoma cells. In the case of anionic complexes, sensitivity was too low for quantitative analysis. To the best of our knowledge, this is the first report concerning the quantification of metal complexes by MALDI-TOF-MS.

Can gadolinium III [Gd(III)] complexes be considered good candidates for magnetic resonance (MR)-molecular imaging studies? In this review article, we examine the principal issues that are the basis of successful use of Gd-based protocols in molecular imaging applications. High relaxivity is the primary requisite. Therefore, the design of such paramagnetic probes has to be pursued keeping in mind the relationships between structure, dynamics, and the relevant parameters involved in paramagnetic relaxation processes. Moreover, the limited number of target molecules on cellular membranes makes it necessary to define strategies aimed at delivering many Gd-containing moieties to the sites of interest. Examples are reported for the attainment of very high relaxivities for the design of new routes for pursuing the accumulation of small sized Gd(III) complexes at the targeting sites. An efficient cellular uptake of Gd-containing probes is the key step for attaining the visualization of targeted cells by MR imaging, and selected examples are reported. In this context, the problem of the assessment of the minimum amount of Gd(III) complexes necessary for the MR imaging-visualization of cells has been addressed by reporting the authors' observations on the cell-internalization of Gd(III) complexes. A particularly efficient delivery system is represented by Gd-loaded apoferritin, which allows the MR visualization of hepatocytes when the number of Gd-complexes per cell is 4 +/- 1 x 10(7). Finally, the potential of responsive systems is considered by outlining the exploitation of the amplification effect brought about by the action of a specific enzymatic activity on the relaxivity of a suitably functionalized Gd(III) complex.

Background/Aims : Agmatine, the compound formed by decarboxylation of arginine, is believed to be an endogenous neurotransmitter through interaction with the imidazoline receptors. However, it also appears to regulate rat hepatocyte polyamines by modifying both their synthesis and their catabolism. As the decrease in polyamine content has been correlated with apoptosis, we examined the possibility that agmatine has an effect on this phenomenon. Methods : Apoptotic cells were detected by visualizing nuclear shrinkage/fragmentation in hepatocytes cultured at 21 and 5% oxygen tension. Caspase-3 activity, cleavage of PARP, release of cytochrome c and mitochondrial swelling were therefore measured in the two conditions and in the presence or not of agmatine. Results : In rat hepatocytes agmatine promoted apoptosis, procaspase 3 processing and increase of caspase-3 like activity. This occurred through mitochondria swelling and release of cytochrome c . Cyclosporin A and catalase blocked the swelling. Conclusions : Our experiments show that agmatine, besides all the known biological effects, has also part, at least in hepatocytes, in the modulation of programmed cell death.

Hyperpolarized 1-13C-2-oxoglutarate as contrast agent in13C Magnetic Resonance diagnostic technique (13C-MRI) for use in the diagnosis of cancer. In particular, upon administration of said 1-13C-2-oxoglutarate, signals of 1-13C-glutamate are detected. More in particular, different MR signals from13C nuclei are detected and compared, said comparison being useful to determine a difference between tumor and non-tumor tissues, to determine the aggressiveness of a tumor or the efficacy of an anti-tumor therapy

1-13C-1,1-Bis(acetoxy(methyl))-2,2'-cyclopropane of formula (I): The compound can be hyperpolarized and used as a contrast agent in 13C Magnetic Resonance diagnostic technique (13C-MR) for the diagnosis of tumor.