Giovanna Esposito

Improved route for the visualization of stem cells labeled with a Gd‐/Eu‐Chelate as dual (MRI and fluorescence) agent

A simple labeling procedure of stem/progenitor cells based on the use of Gd‐HPDO3A and Eu‐HPDO3A, respectively, is described. The Gd‐chelate acts as T1‐agent for MRI visualization, whereas the corresponding Eu‐chelate acts as reporter in fluorescence microscopy. Owing to their substantial chemical equivalence, the two chelates are equally internalized in EPCs (endothelial progenitor cells), thus allowing their visualization by both techniques. The lanthanide chelates are entrapped in endosomic vesicles and the labeled cells retain biological activity with preservation of viability and pro‐angiogenesis capacity. Hyperintense spots in MR have been observed for Gd‐labeled EPCs injected under mice kidney capsule or grafted on a subcutaneous Matrigel plug up to 14 days after transplantation. Magn Reson Med 51:938–944, 2004.

Effect of the intracellular localization of a Gd-based imaging probe on the relaxation enhancement of water protons

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 × 1010 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. Magn Reson Med, 2006.

Magnetic Resonance Visualization of Tumor Angiogenesis by Targeting Neural Cell Adhesion Molecules with the Highly Sensitive Gadolinium-Loaded Apoferritin Probe

Tumor vessel imaging could be useful in identifying angiogenic blood vessels as well as being a potential predictive marker of antiangiogenic treatment response. We recently reported the expression of the neural cell adhesion molecule (NCAM) in the immature and tumor endothelial cell (TEC) lining vessels of human carcinomas. Exploiting an in vivo model of human tumor angiogenesis obtained by implantation of TEC in Matrigel in severe combined immunodeficiency mice, we aimed to image angiogenesis by detecting the expression of NCAM with magnetic resonance imaging. The imaging procedure consisted of (a) targeting NCAMs with a biotinylated derivative of C3d peptide that is known to have high affinity for these epitopes and (b) delivery of a streptavidin/gadolinium (Gd)-loaded apoferritin 1:1 adduct at the biotinylated target sites. The remarkable relaxation enhancement ability of the Gd-loaded apoferritin system allowed the visualization of TEC both in vitro and in vivo when organized in microvessels connected to the mouse vasculature. Gd-loaded apoferritin displayed good in vivo stability and tolerability. The procedure reported herein may be easily extended to the magnetic resonance visualization of other epitopes suitably targeted by proper biotinylated vectors.

Efficient Cellular Labeling by CD44 Receptor-Mediated Uptake of Cationic Liposomes Functionalized with Hyaluronic Acid and Loaded with MRI Contrast Agents

Cell tracking is now a well-established technique in magnetic resonance imaging (MRI) because of its three-dimensional capabilities, its good safety profile, and the superb anatomical resolution (near the cellular level, ~100 mm). For these applications, cells must be labeled with MRI contrast agents in order to decrease the relaxation times of the H atoms of water molecules. The most common contrast agents in current use are iron oxide particles (USPIO, SPIO, MPIO) because of their high sensitivity. They act as negative contrast agents because of their dominant effect on the magnetic susceptibility term. 6] Typically the R2 value of monocytes labeled with SPIO at a Fe concentration of 1.0 mg mL 1 is 13.1 s 1 mm . It has been shown that this leads to a detection limit of 58 labeled monocytes per voxel volume of 0.05 mL. These magnetic particles enable the visualization of labeled cells as dark spots in the MR images as a consequence of their largely dominant T2* effects. However, there is some concern about their long-term cellular toxicity related to the metabolic fate of the iron payload. 9] Other problems with superparamagnetic particles include difficulty in the quantification of cell number due to susceptibility artifacts and the potential transfer of contrast among cells (macrophages) after cell death, giving false-positive results. The use of positive contrast agents is a viable alternative to overcome these problems, but to attain sufficient contrast for the MRI of the labeled cells, it is necessary to entrap a number of Gd complexes on the order of 10–10 per cell. Various routes have been explored in pursuit of effecting the internalization of such large quantities of paramagnetic complexes. Pinocytosis is sometimes a useful route, but the cells must be kept in the presence of high concentrations (~50 mm) of highly stable, hydrophilic agents for many hours (12–24 h). 13] Rapid entrapment of the required Gd complexes may be attained by electroporation of the cell suspension; however, this treatment results in extensive cell death and serious damage to surviving cells. Finally, for cells endowed with phagocytic activity, it has been shown that cellular labeling can be achieved by using particles made of insoluble Gd complexes. 15] These particles are designed to be solubilized upon release of insolubilizing moieties effected by a specific enzyme present in the cellular compartment in which these particles have been entrapped. This procedure is particularly efficient, as it allows the entrapment of a large number of Gd complexes through a single internalization step. Herein we report a cellular labeling methodology that allows the rapid internalization of large amounts of soluble Gd complexes though exploitation of the receptor-mediated endocytotic route. The Gd payload is contained in the inner cavity of liposomes, which are appropriately functionalized at their external surface to bind a given receptor, thus starting the uptake process. As a proof of concept, hyaluronan (hyaluronic acid, HA) was used as a vector for the targeting procedure because HA receptors (CD44) are expressed in a variety of tumors such as breast, colon, intestinal, and brain, as well as melanoma, basal cell carcinoma, and stem cells. HA is a high-molecular-weight glycosaminoglycan polymer (Mr : 10 –10 Da) composed of repeating units of the disaccharide b1,3-N-acetylglucosaminyl-b1,4-glucuronide. Thus each diACHTUNGTRENNUNGsaccharide moiety contains a free carboxylate group, leading to highly negatively charged polymer. For the purpose of this application, HA can be bound to the surface of Gd-loaded liposomes in either a covalent or noncovalent manner. The formation of a noncovalent supramolecular adduct appeared a straightforward route, as cationic liposomes have been widely used in transfection experiments as carriers of negatively charged DNA macromolecules. 21] Cationic liposomes were prepared by using a mixture of phospholipids (POPC 52 % and DOTAP 25 %) and cholesterol (23 %). The lipid film was hydrated with an aqueous solution of Gd-HPDO3A (100 mm). The latter is a commercially available, highly hydrophilic, neutral MRI contrast agent largely used in clinical practice. The liposomes thus formed were extruded through progressively smaller pore sizes to select a population with an average diameter of 127 5 nm and a z potential of + 35 5 mV. The Gd complexes not entrapped in the liposomes were removed by dialysis. The supramolecular HA-functionalized Gd-liposome (HA–L) adducts were obtained by adding aliquots of a solution of HA to the liposome suspension. Interestingly, a dramatic increase in the supramolecular adduct size takes place (Figure 1 a) at the isoelectric point— the point at which the number of positive charges on the liposome equals the number of negative charges on the added HA polymer. Light scattering measurements showed that the particle diameter nearly triples (~350 nm) under these conditions, suggesting the formation of an aggregate that consists of three to four liposomes and several HA molecules (Scheme 1). At higher HA/liposome stoichiometric ratios, the prevalence of a monomeric liposome population was again detected. It appears that in the presence of an excess in positive [a] Dr. G. Esposito, Dr. S. Geninatti Crich, Prof. S. Aime Dipartimento di Chimica IFM and Molecular Imaging Center Via Nizza 52, 10125 Torino (Italy) Fax: (+ 39) 011-670-6487 E-mail : silvio.unito@unito.it

Visualization through magnetic resonance imaging of DNA internalized following "in vivo" electroporation.

The ability to visualize plasmid DNA entrapment in muscle cells undergoing an “in vivo” electroporation treatment was investigated on BALB/c mice using a 7-T magnetic resonance imaging (MRI) scanner using the paramagnetic Gd–DOTA–spd complex as imaging reporter. Gd–DOTA–spd bears a tripositively charged spermidine residue that yields a strong binding affinity toward the negatively charged DNA chain (6.4 kb, Ka = 2.2 × 103 M−1 for approximately 2500 ± 500 binding sites). Cellular colocalization of Gd-DOTA-spd and plasmid DNA has been validated by histological analysis of excised treated muscle. In vivo MRI visualization of Gd-DOTA-spd distribution provides an excellent route to access the cellular entrapment of plasmid DNA upon applying an electroporation pulse.

Synthesis and preliminary evaluation in tumor bearing mice of new 18F-labeled arylsulfone matrix metalloproteinase inhibitors as tracers for positron emission tomography

New fluorinated, arylsulfone-based matrix metalloproteinase (MMP) inhibitors containing carboxylate as the zinc binding group were synthesized as radiotracers for positron emission tomography. Inhibitors were characterized by Ki for MMP-2 in the nanomolar range and by a fair selectivity for MMP-2/9/12/13 over MMP-1/3/14. Two of these compounds were obtained in the (18)F-radiolabeled form, with radiochemical purity and yield suitable for preliminary studies in mice xenografted with a human U-87 MG glioblastoma. Target density in xenografts was assessed by Western blot, yielding Bmax/Kd = 14. The biodistribution of the tracer was dominated by liver uptake and hepatobiliary clearance. Tumor uptake of (18)F-labeled MMP inhibitors was about 30% that of [(18)F]fluorodeoxyglucose. Accumulation of radioactivity within the tumor periphery colocalized with MMP-2 activity (evaluated by in situ zimography). However, specific tumor uptake accounted for only 18% of total uptake. The aspecific uptake was ascribed to the high binding affinity between the radiotracer and serum albumin.

MRI and PET Compatible Bed for Direct Co-Registration in Small Animals

To obtain an accurate co-registration with stand-alone PET and MRI scanners, we developed a compatible bed system for mice and rats that enables both images to be acquired without repositioning the animals. MRI acquisitions were performed on a preclinical 7T scanner (Pharmascan, Bruker), whereas PET scans were acquired on a YAP-(S)PET (ISE s.r.l.). The bed performance was tested both on a phantom (NEMA Image Quality phantom) and in vivo (healthy rats and mice brain). Fiducial markers filled up with a drop of 18 F were visible in both modalities. Co-registration process was performed using the point-based registration technique. The reproducibility and accuracy of the co-registration were assessed using the phantom. The reproducibility of the translation distances was 0.2 mm along the z axis. On the other hand, the accuracy depended on the physical size of the phantom structures under investigation but was always lower than 4%. Regions of Interest (ROIs) drawn on the fused images were used for quantification purposes. PET and MRI intensity profiles on small structures of the phantom showed that the underestimation in activity concentration reached 90% in regions that were smaller than the PET spatial resolution, while the MRI allowed a good visualization of the 1 mm 0 rod. PET/MRI images of healthy mice and rats highlighted the expected superior capability of MRI to define brain structures. The simplicity of our developed MRI/PET compatible bed and the quality of the fused images obtained offers a promising opportunity for a future preclinical translation, particularly for neuroimaging studies.

Glucan particles loaded with a NIRF agent for imaging monocytes/macrophages recruitment in a mouse model of rheumatoid arthritis

Glucan Particles (GPs) are hollow pseudo-microspheres (average diameter 3–5 μm) obtained from common bakers yeast Saccharomyces cerevisiae, in which mannan, lipids and proteins are removed through a process of chemical extraction. GPs shell is mostly made of 1,3-β-D-glucan that allows for their prompt in vivo uptake by immune system cells. In this work, the inner cavity of the particles has been loaded with two amphiphilic fluorescent dyes (based on cyanine for in vivo imaging purposes and rhodamine for ex vivo microscopy experiments) through a sudden change in solvent polarity that allowed the entrapment of the molecules as microemulsion. The ability of fluorescent GPs to label immune cells in vivo and report on their recruitment in inflamed sites has been successfully demonstrated in a mouse model of rheumatoid arthritis (CIA) by NIRF imaging. Besides providing the visualization of the mononuclear cell infiltration in the lesion, the fluorescent signal well correlated with the clinical score associated with the disease. Very interestingly, the signal detected in lesions with the same clinical score allowed the assessment of the time evolution (progression or remission) of the pathology.

A stat3-mediated metabolic switch is involved in tumour transformation and stat3 addiction

Resumen del poster presentado al 36th FEBS Congress celebrado en Torino (Italia) del 25 al 30 de Junio de 2011.-- et al.