Cristina Anselmi

Magneto‐Plasmonic Au‐Fe Alloy Nanoparticles Designed for Multimodal SERS‐MRI‐CT Imaging

Diagnostic approaches based on multimodal imaging are needed for accurate selection of the therapeutic regimens in several diseases, although the dose of administered contrast drugs must be reduced to minimize side effects. Therefore, large efforts are deployed in the development of multimodal contrast agents (MCAs) that permit the complementary visualization of the same diseased area with different sensitivity and different spatial resolution by applying multiple diagnostic techniques. Ideally, MCAs should also allow imaging of diseased tissues with high spatial resolution during surgical interventions. Here a new system based on multifunctional Au-Fe alloy nanoparticles designed to satisfy the main requirements of an ideal MCA is reported and their biocompatibility and imaging capability are described. The MCAs show easy and versatile surface conjugation with thiolated molecules, magnetic resonance imaging (MRI) and computed X-ray tomography (CT) signals for anatomical and physiological information (i.e., diagnostic and prognostic imaging), large Raman signals amplified by surface enhanced Raman scattering (SERS) for high sensitivity and high resolution intrasurgical imaging, biocompatibility, exploitability for in vivo use and capability of selective accumulation in tumors by enhanced permeability and retention effect. Taken together, these results show that Au-Fe nanoalloys are excellent candidates as multimodal MRI-CT-SERS imaging agents.

Top-down synthesis of multifunctional iron oxide nanoparticles for macrophage labelling and manipulation

Multifunctional iron oxide (FeOx) magnetic nanoparticles (MNPs) are promising items for biomedical applications. They are studied as theranostic agents for cancer treatment, selective probes for bioanalytical assays, controllable carriers for drug delivery and biocompatible tools for cell sorting or tissue repair. Here we report a new method for the synthesis in water of FeOx–MNPsvia a top-down physical technique consisting in Laser Ablation Synthesis in Solution (LASiS). LASiS is a green method that does not require chemicals or stabilizers, because nanoparticles are directly obtained in water as a stable colloidal system. A gamut of characterization techniques was used for investigating the structure of FeOx–MNPs that have a polycrystalline structure prevalently composed of magnetite (ca. 75%) and hematite (ca. 22%). The FeOx–MNPs exhibit very good magnetic properties if compared to what is usually reported for iron oxide nanoparticles, with saturation magnetization close to the bulk value (ca. 80 emu g−1) and typical signatures of the coexistence of ferrimagnetic and antiferromagnetic phases in the same particle. The functionalization of FeOx–MNPs after the synthesis was possible with a variety of ligands. In particular, we succeeded in the functionalization of FeOx–MNPs with carboxylated phosphonates, fluorescent alkylamines, fluorescent isothiocyanates and bovine serum albumin. Our FeOx–MNPs showed excellent biocompatibility. Multifunctional FeOx–MNPs were exploited for macrophage cell labelling with fluorescent probes as well as for cell sorting and manipulation by external magnetic fields.

Self-potentiation of ligand-toxin conjugates containing ricin A chain fused with viral structures.

A chimeric protein was obtained by fusing together the ricin toxin A chain (RTA) gene and a DNA fragment encoding the N terminus of protein G of the vesicular stomatitis virus. Chimeric RTA (cRTA) retained full enzymic activity in a cell-free assay, but was 10-fold less toxic against human leukemic cells than either native RTA (nRTA) or unmodified recombinant RTA (rRTA). However, conjugates made with cRTA and human transferrin (Tfn) showed 10-20-fold greater cell killing efficacy than Tfn-nRTA or Tfn-rRTA conjugates despite equivalent binding of the three conjugates to target tumor cells. As a consequence, by fusion of the KFT25 peptide to the RTA sequence, the specificity factor (i.e. the ratio between nonspecific and specific cytotoxicity) of Tfn-cRTA was increased 90-240 times with respect to those of Tfn-nRTA and Tfn-rRTA. cRTA interacted with phospholipid vesicles with 15-fold faster kinetics than nRTA at acidic pH. Taken together, our results suggest that the ability of vesicular stomatitis virus protein G to interact with cell membranes can be transferred to RTA to facilitate its translocation to the cell cytosol. Our strategy may serve as a general approach for potentiating the cytotoxic efficacy of antitumor immunotoxins.

Reductive Activation of Type 2 Ribosome-inactivating Proteins Is Promoted by Transmembrane Thioredoxin-related Protein

Background: PDI is implicated in the intracellular reduction of ricin; other oxidoreductases also have a role in this process. Results: Overexpression and silencing of TMX affect ricin cytotoxicity. Conclusion: TMX participates with PDI and/or other reductases in the reduction of ricin and other proteins. Significance: These findings contribute to the understanding of disulfide reduction in cell intoxication by toxins and virus assembly and entry. Members of the type 2 ribosome-inactivating proteins (RIPs) family (e.g. ricin, abrin) are potent cytotoxins showing a strong lethal activity toward eukaryotic cells. Type 2 RIPs contain two polypeptide chains (usually named A, for “activity”, and B, for “binding”) linked by a disulfide bond. The intoxication of the cell is a consequence of a reductive process in which the toxic domain is cleaved from the binding domain by oxidoreductases located in the lumen of the endoplasmic reticulum (ER). The best known example of type 2 RIPs is ricin. Protein disulfide isomerase (PDI) was demonstrated to be involved in the process of ricin reduction; however, when PDI is depleted from cell fraction preparations ricin reduction can still take place, indicating that also other oxidoreductases might be implicated in this process. We have investigated the role of TMX, a transmembrane thioredoxin-related protein member of the PDI family, in the cell intoxication operated by type 2 RIPs ricin and abrin. Overexpressing TMX in A549 cells resulted in a dramatic increase of ricin or abrin cytotoxicity compared with control mock-treated cells. Conversely, no difference in cytotoxicity was observed after treatment of A549 cells or control cells with saporin or Pseudomonas exotoxin A whose intracellular mechanism of activation is not dependent upon reduction (saporin) or only partially dependent upon it (Pseudomonas exotoxin A). Moreover, the silencing of TMX in the prostatic cell line DU145 reduced the sensitivity of the cells to ricin intoxication further confirming a role for this enzyme in intracellular ricin activation.

Chemical conjugation of ΔF508-CFTR corrector deoxyspergualin to transporter human serum albumin enhances its ability to rescue Cl− channel functions

The most common mutation of the cystic fibrosis (CF) gene, the deletion of Phe508, encodes a protein (DeltaF508-CFTR) that fails to fold properly, thus mutated DeltaF508-cystic fibrosis transmembrane conductance regulator (CFTR) is recognized and degraded via the ubiquitin-proteasome endoplasmic reticulum-associated degradation pathway. Chemical and pharmacological chaperones and ligand-induced transport open options for designing specific drugs to control protein (mis)folding or transport. A class of compounds that has been proposed as having potential utility in DeltaF508-CFTR is that which targets the molecular chaperone and proteasome systems. In this study, we have selected deoxyspergualin (DSG) as a reference molecule for this class of compounds and for ease of cross-linking to human serum albumin (HSA) as a protein transporter. Chemical cross-linking of DSG to HSA via a disulfide-based cross-linker and its administration to cells carrying DeltaF508-CFTR resulted in a greater enhancement of DeltaF508-CFTR function than when free DSG was used. Function of the selenium-dependent oxidoreductase system was required to allow intracellular activation of HSA-DSG conjugates. The principle that carrier proteins can deliver pharmacological chaperones to cells leading to correction of defective CFTR functions is therefore proven and warrants further investigations.

A non-parametric method for the analysis of experimental tumour growth data

Analysis of tumour growth is required to investigate the biology of tumours and to determine the effects of new anti-tumour therapies. A non-parametric mathematical method for the analysis of a set of experimental tumour growth data is described. The method is based on the similarity between time series of tumour size measurements (e.g. tumour volume), similarity being defined as the Euclidean distance between data measured for each tumour at the same time. Subsets of similar time series are found for a given population of tumours. A biologically meaningful parameter H has been derived which is a measure of the scattering of experimental volume samples. The method has been applied to the analysis of the growth of (i) untreated multicellular tumour spheroids obtained with different cell lines and (ii) spheroids, treated with cytotoxic drugs (immunotoxins). Results are compared with those previously obtained by applying the classical Gompertz growth model to the analysis of treated and untreated spheroids.