Guided nanoparticles for tumor imaging and therapy

The passive immunotherapy based on the use of monoclonal antibodies has recently improved the therapeutic response and survival in hematologic and solid tumor. However, some patients show no response to these treatments, moreover a resistance mechanism can rise up. For these reasons many conjugates of antibodies and radionuclides or cytotoxic molecules are currently in clinical trials. As in the cancer treatment, the prognosis is strongly influenced by the possibility of an early diagnosis; there is the neccesity of diagnostic methods with high sensitivity and specificity. Therefore in the last years new protocols based on immuno-imaging are object of interest. In order to increase the transport of a large amount of drug and tracker molecules at tumor lesions site, nanotechnology can offer effective solutions. The nanosystems have the characteristic to focus in neoplastic lesions thanks to the EPR effect; the accumulation and the retention in the injury sites can be implemented by conjugating nanoparticles with targeting moieties such as antibodies or growth factors. Combining nanomedicine together with recombinant antibody technology, it is possible to increase the specificity of cancer treatments greatly, thus allowing the decrease of therapeutic doses and increasing the sensitivity of diagnostic tools currently used (MRI). This thesis work was done in collaboration with the reaserch group of Prof. Meneghetti (University of Padua) and the groups of Prof. Mancin and Prof. Papini (University of Padua) in the context of the European project “Nanophoto”. The work describes the production and characterization of different types of nanoconjugates and their potential use in imaging and cancer therapy. The first part of the study was focused on the synthesis and characterization of metal nanoparticles, gold and iron oxide, not targeted. Gold is a material used in medicine thanks to its biocompatibility and its unique chemical and optical properties. Among these, the ability to produce Raman signal is interesting, this can be amplified more than 108 times by using SERS dies. We have shown that by Raman spectroscopy it is possible to quantify the amount of NPs uptaken by the cells. This result provides the basis for an ultrasensitive method that can detect the amount of nanocompound conveyed at neoplatic lesions. By applying FeOX-MNPs (magnetic nanoparticles) we have shown that: a) magnetic NPs can be used to sort modified cell populations (see cells loaded with FeOX-MNPs coated with siRNA or cDNA) b) it is possible to direct into the site of interest specific cell populations by external magnetic field (see anti tumor T cells or macrophage, loaded with FeOX MNPs). The second part of the study was focused on the analysis of the targeting of gold and silica nanoparticles, conjugated to monoclonal antibodies specific for TAAs, in order to increase the accumulation in the site of interest. The binding specificity and internalization of these nanocompounds has been demonstrated “in vitro” and “ex vivo” using antigen positive and negative cells and tissue harvested from syngenic mouse models (C57/B16 mice injected sc with B16 cells trasfected with the Ags of interest). It was also assessed “in vitro” the therapeutic potential of both gold nanoparticles, loaded with a chemoterapeutic agent, doxorubicin, and silica nanoparticles loaded with the photosensitiser Foscan®. In both cases the results are promising: it is highlighted a toxicity of 60% and 40% greater than not loaded NPs, using NPs loaded with doxorubicin and Foscan® respectively. We have also proved that it is possible to detect single antigen positive cells and develope multi-target methods, by using Raman spectroscopy and gold nanoparticles conjugated with different mAbs and loaded with SERS dies.