Enza Di Gregorio

Probing Protein Conformation in Cells by EPR Distance Measurements using Gd3+ Spin Labeling

Protein structure investigations are usually carried out in vitro under conditions far from their native environment in the cell. Differences between in-cell and in vitro structures of proteins can be generated by crowding effects, local pH changes, specific and nonspecific protein and ligand binding events, and chemical modifications. Double electron-electron resonance (DEER), in conjunction with site-directed spin-labeling, has emerged in the past decade as a powerful technique for exploring protein conformations in frozen solutions. The major challenges facing the application of this methodology to in-cell measurements are the instabilities of the standard nitroxide spin labels in the cell environment and the limited sensitivity at conventional X-band frequencies. We present a new approach for in-cell DEER distance measurement in human cells, based on the use of: (i) reduction resistant Gd(3+) chelates as spin labels, (ii) high frequency (94.9 GHz) for sensitivity enhancement, and (iii) hypo-osmotic shock for efficient delivery of the labeled protein into the cell. The proof of concept is demonstrated on doubly labeled ubiquitin in HeLa cells.

On the Fate of MRI Gd-Based Contrast Agents in Cells. Evidence for Extensive Degradation of Linear Complexes upon Endosomal Internalization

Commercial Gd-containing complexes are often used as MRI reporters in cellular labeling procedures as they are internalized into endosomes by pinocytosis. A methodology has been applied to assess the relative stability of three commercial Gd contrast agents following cellular uptake in fibroblasts and macrophages. It has been found that the acyclic series of Gd MRI contrast agents are degraded much more rapidly than their macrocyclic analogues, following endosomal internalization into living cells. This helps to explain their causal role in the development of nephrogenic systemic fibrosis in renally impaired patients. The methodology has also been applied to assess the fate of Gd-DTPA-BMA-loaded liposomes upon their endosomal internalization. Resistant liposomes prevent the degradation of the complex, whereas liposomes designed to release their payload in the acidic environments show a loss of integrity of Gd-DTPA-BMA analogous to the one observed upon internalization of the free complex.

An MRI Method To Map Tumor Hypoxia Using Red Blood Cells Loaded with a pO2-Responsive Gd-Agent

Hypoxia is a typical hallmark of many solid tumors and often leads to therapy resistance and the development of a more aggressive cancer phenotype. Oxygen content in tissues has been evaluated using numerous different methods for several imaging modalities, but none has yet reached the required standard of spatial and temporal resolution. Magnetic Resonance Imaging (MRI) appears to be the technique of choice and several pO2-responsive probes have been designed for it over the years. In vivo translation is often hampered in Gd-relaxation agents as it is not possible to separate effects that arise from changes in local concentration from those associated with responsive properties. A novel procedure for the MRI based assessment of hypoxia is reported herein. The method relies on the combined use of Gd-DOTP- and Gd-HPDO3A-labeled red blood cells (RBCs) where the first probe acts as a vascular oxygenation-responsive agent, while the second reports the local labeled RBC concentration in a transplanted breast tumor mouse model. The MRI assessment of oxygenation state has been validated by photoacoustic imaging and ex vivo immunofluorescence. The method refines tumor staging in preclinical models and makes possible an accurate monitoring of the relationship between oxygenation and tumor growth.

Frequency-Encoded MRI-CEST Agents Based on Paramagnetic Liposomes/RBC Aggregates

Paramagnetic liposomes containing Dy-HPDO3A in their inner water compartment and carrying a residual positive charge on their outer surface have been electrostatically bound to the membrane of red blood cells (RBCs). These aggregates yield two chemical exchange saturation transfer (CEST) pools represented by liposomal water protons (LipoCEST) and cytoplasmatic water protons (ErythroCEST), respectively. The absorption frequencies of the two pools fall at the negative and positive side of the solvent water resonance as expected from the dipolar (LipoCEST) and BMS (bulk magnetic susceptibility) (ErythroCEST) origin of the paramagnetic induced shift of their water protons resonances, respectively. In vivo magnetic resonance imaging (MRI) shows that the liposomes/RBC aggregates report about the vascular volume whereas the residual LipoCEST effect informs about the presence of released liposomes in the region of interest (ROI). Besides being an innovative blood cell labeling for MRI, the LipoCEST/RBC aggregates provide a route to improve the circulation lifetime of the liposomes and the CEST procedure allows assessing the deassembly of the aggregates and accumulation of the liposomes in the ROI.

Gd-loaded-RBCs for the assessment of tumor vascular volume by contrast-enhanced-MRI

The assessment of the fractional vascular volume (vV) in the tumor area is of great interest in the characterization of tumor and it can be useful to monitor the outcome of anti-angiogenetic therapies. The high spatial and temporal resolution of Magnetic Resonance Imaging makes it the election imaging modality to monitor in vivo the vascular volume changes. Commonly used MRI methods to obtain this information rely on the administration of contrast agents that modify the bulk water relaxation times but, unfortunately, they can provide only an estimate of vV since they are not fully retained in the vascular space. Herein, Gd-loaded Red Blood Cells (Gd-RBCs) are proposed as a contrast agent able to provide quantitative information on tumor vascularization. Being Gd-RBCs fully retained in the vascular space, the proposed method does not suffer for the limitations associated to the use of extracellular Gd-agents that quickly extravasate in the leaky tumor vasculature. Furthermore, the long half-life and biocompatibility of Gd-RBCs allows repeating the measurement many times upon their administration; this ensures the possibility to in vivo evaluate the change of vascular volume during tumor growth. For these reasons, Gd-RBCs may represent a highly biocompatible imaging reporter of vasculature, able to quantitatively assess changes in the vascular volume in the ROI.

Gadolinium Retention in the Rat Brain: Assessment of the Amounts of Insoluble Gadolinium-containing Species and Intact Gadolinium Complexes after Repeated Administration of Gadolinium-based Contrast Agents

Purpose To evaluate the speciation of gadolinium-containing species after multiple administrations of the gadolinium-based contrast agents (GBCAs) gadodiamide and gadoteridol and to quantify the amount of intact gadolinium complexes and insoluble gadolinium-containing species. Materials and Methods A total dose of 13.2 mmol per kilogram of body weight of each GBCA was administered in healthy Wistar rats over a period of 8 weeks. Three days after the final administration, rats were sacrificed, and the brains were excised and divided into three portions. Each portion of brain homogenate was divided into two parts, one for determination of the total gadolinium concentration with inductively coupled plasma mass spectrometry and one for determination of the amount of intact GBCA and gadolinium-containing insoluble species. Relaxometric measurements of gadodiamide and gadolinium trichloride in the presence of polysialic acid were also performed. Results The mean total gadolinium concentrations for gadodiamide and gadoteridol, respectively, were 0.317 μg/g ± 0.060 (standard deviation) and 0.048 μg/g ± 0.004 in the cortex, 0.418 μg/g ± 0.078 and 0.051 μg/g ± 0.009 in the subcortical brain, and 0.781 μg/g ± 0.079 and 0.061 μg/g ± 0.012 in the cerebellum. Gadoteridol comprised 100% of the gadolinium species found in rats treated with gadoteridol. In rats treated with gadodiamide, the largest part of gadolinium retained in brain tissue was insoluble species. In the cerebellum, the amount of intact gadodiamide accounts for 18.2% ± 10.6 of the total gadolinium found therein. The mass balance found for gadolinium implies the occurrence of other soluble gadolinium-containing species (approximately 30%). The relaxivity of the gadolinium polysialic acid species formed in vitro was 97.8 mM/sec at 1.5 T and 298 K. Conclusion Gadoteridol was far less retained, and the entire detected gadolinium was intact soluble GBCA, while gadodiamide yielded both soluble and insoluble gadolinium-containing species, with insoluble species dominating.

Sensitive MRI detection of internalized T1 contrast agents using magnetization transfer contrast.

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

Enzyme‐Responsive LipoCEST Agents: Assessment of MMP‐2 Activity by Measuring the Intra‐liposomal Water 1H NMR Shift

Mobile proton-containing solutes can be detected by MRI by the chemical exchange saturation transfer (CEST) method. CEST sensitivity is dramatically enhanced by using, as exchanging protons, the water molecules confined inside liposomes, shifted by a paramagnetic shift reagent. The chemical shift of the intraliposomal water resonance (δIL ) is affected by the overall shape of the supramolecular system. δIL of a spherical LipoCEST acts as a sensitive reporter of the distribution of streptavidin proteins anchored at the liposome surface by biotinylated phospholipids. This finding prompted the design of a MMP-2 responsive LipoCEST agent as the streptavidin moieties can be released from the liposome surfaces when a properly tailored enzyme-cleavable peptide is inserted on the phospholipids before the terminal biotin residues. δIL reports on the overall changes in the supramolecular architecture associated to the cleavage carried out by MMP-2.

MEMRI and tumors: a method for the evaluation of the contribution of Mn(II) ions in the extracellular compartment

The purpose of the work was to set‐up a simple method to evaluate the contribution of Mn2+ ions in the intra‐ and extracellular tumor compartments in a MEMRI experiment. This task has been tackled by “silencing” the relaxation enhancement arising from Mn2+ ions in the extracellular space. In vitro relaxometric measurements allowed assessment of the sequestering activity of DO2A (1,4,7,10‐tetraazacyclododecane‐1,7‐diacetic acid) towards Mn2+ ions, as the addition of Ca‐DO2A to a solution of MnCl2 causes a drop of relaxivity upon the formation of the highly stable and low‐relaxivity Mn‐DO2A. It has been proved that the sequestering ability of DO2A towards Mn2+ ions is also fully effective in the presence of serum albumin. Moreover, it has been shown that Mn‐DO2A does not enter cell membranes, nor does the presence of Ca‐DO2A in the extracellular space prompt migration of Mn ions from the intracellular compartment.

CEST‐MRI studies of cells loaded with lanthanide shift reagents

Magnetic resonance imaging has been used extensively to track in vivo implanted cells that have been previously labeled with relaxation enhancers. However, this approach is not suitable to track multiple cell populations, as it may lead to confounding results in case the contrast agent is released from the labeled cells. This paper demonstrates how the use of CEST agents can overcome these issues. After encapsulating paramagnetic lanthanide shift reagents, we may shift the absorption frequency of the intracellular water resonance (δIn), thus generating frequency‐encoding CEST responsive cells that can be visualized in the MR image by applying the proper RF irradiation.