99MTC-RADIOLABELED NANOPARTICLES FOR TARGETED DETECTION AND TREATMENT OF HER2-POSITIVE BREAST CANCER

Introduction: The HER2 receptor overexpression is normally associated to aggressive and infiltrating breast cancer (BC) phenotype with propensity to spread into metastases. Nowadays, the detection of HER2 in primary tumor lesions and in their metastases is based on invasive methods as well therapy clinical outcomes are not satisfactory yet. Recent advances in nanotechnology have led to the development of nanoparticles able to host various functionalities for specific targeting and to be loaded with therapeutic molecules, making possible the simultaneous diagnosis and treatment of human cancers (theranostic). In the present Thesis study, was evaluated the potential use of targeted silica nanoparticles (SiNPs) as theranostic agent for HER2+ breast cancer. Methods: SiNPs were engineered with anti-HER2 monoclonal antibody Trastuzumab, in the form of half-chain (Hc-TZ), and radiolabeled employing 99mTc for in vivo SPECT imaging detection of HER2+ BC lesions. Subsequently, SiNPs were loaded with doxorubicin (DOX) for treatment evaluation. Experimental design was divided in three main tasks. First, we evaluated the contribution offered by active targeting (Hc-TZ) to the selective distribution of SiNPs in solid HER2 positive BC lesions. To this aim, both Hc-TZ conjugated (SiNP-TZ) and non-conjugated (SiNP) nanosilica shells were radiolabeled with 99mTc-Tricarbonyl complex, through nitrilotriacetic acid (NTA) linker procedure, and their distribution kinetics evaluated in vitro and ex vivo in ad hoc cancer models. Nanoparticles were simultaneous filled with a fluorescent dye and their uptake were also assessed by FACS analysis and fluorescence microscopy. In the second experimental step, nanoparticles were also engineered with several amount of Hc-TZ (SiNPs to Hc-TZ ratio, 1:2 and 1:8 respectively), and were 99mTc-labeled at histidine residues of the antibody chain for ex vivo/in vivo biodistribution evaluation. Finally, SiNP-TZ were loaded with DOX and in vitro/in vivo DOX distribution in HER2 positive models was evaluated using confocal microscopy and Optical Imaging, in comparison to liposomal doxorubicin (Caelyx). The treatment efficacy of DOX-SiNP-TZ (1:8 Hc-TZ) versus Caelyx was evaluated in vivo for six weeks of treatment, also using PET molecular imaging ([18F]FDG) approach. Results: In vitro assays showed a higher fluorescence signal (FICT) in SK-BR-3 compared to MDA-MB-468 cells, exclusively for targeted SiNP-NTA-TZ/SiNP-TZ with an increase over time. Ex vivo biodistribution of 99mTc-labelled nanoparticles via NTA, at 4 h post-injection of SiNP-NTA-TZ and/or non-targeted (SiNP-NTA), exhibited values of 3.53 and 1.69 in tumor (tumor to muscle ratio) respectively, with a rapid reduction over time for targeted nanoparticles. These results indicated the presence of an antibody-receptor mediated tumor uptake of SiNP-NTA-TZ, with a faster washout of nanoparticles radiolabeled shell. In the second set of experiments, performed with 99mTc-SiNP-TZ labelled on TZ half chain, uptake was confirmed at 4 h p.i. for SiNP-TZ (1:8 Hc-TZ) with similar results to SiNP-TZ (1:2 Hc-TZ). Instead, was showed a progressive retention of radioactivity until 24 h p.i., confirming the presence of radiolabeled Hc-TZ to the tumor also at latter times, with improved results for SiNP-TZ (1:8 Hc-TZ), also in terms of radiochemical yield. Doxorubicin loaded SiNP-TZ (1:8 Hc-TZ) showed in vivo similar delivery results in comparison to Caelyx at 6 h p.i., meanwhile at the end of treatment tumor volume reduction resulted significant improved by targeted nanoparticles administration. Conclusion: Results of this Thesis study, demonstrated a promising specificity and treatment efficacy of the silica nanoparticles-based system SiNP-TZ, encouraging its potential use as theranostic agent for HER2+ breast cancer lesions.