Sanità, Gennaro (2020) Uptake SPARC-mediated of HSA-modified nanoparticles for photoacoustic imaging in breast cancer. [Tesi di dottorato]

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In this work, an innovative type of hybrid organic-metal HSA modified nanoparticles (MelaSil_Ag-HSA NPs) is presented. MelaSil_Ag NPs are constituted by an external shell of silicon and a melanin-like compound (5,6- dihydroxyindole-2-carboxylic acid, DHICA), and an internal core of metallic silver. Silicon has been selected for its biological compatibility, low costs and physical properties, while DHICA has been chosen for its photoacoustic (PA) properties and very high biocompatibility. Metallic silver core improves the photoacoustic signal produced by DHICA, working like an amplifier antenna. Preliminary biological assessments were performed using two pancreas cancer cell lines (BxPC-3 and PANC-1), to evaluate cytotoxicity and internalization of MelaSil_Ag NPs. Results have shown good NPs internalization and cytocompatibility in both cell lines up to 50 μg/mL for 72 h, while a high level of cytotoxicity was observed when a concentration of 100 μg/mL was tested. In addition, to evaluate the effects of MelaSil_Ag on blood, NPs were tested in hemotoxicity assays, assessing the %-lysed Red Blood Cells (RBCs). Data showed 100% cytotoxicity already at 1 h of incubation at 50 μg/ml. The photoacoustic signal (PAS) of MelaSil_Ag NPs was tested into polyethylene (PE) tubes and in ex vivo experiments, results showed very high increase of PAS of MelaSil_Ag compared to MelaSil NPs (NPs without silver core). The conjugation of recombinant human serum albumin (rHSA), by EDC/NHS chemistry strategy, on MelaSil_Ag NPs surface totally removed the hemotoxicity previously observed. Furthermore, viability increase was observed in breast cancer cells (HS578T) and normal breast cells (MCF10a) incubated with MelaSil_Ag-HSA NPs compared to bare NPs, up to 100 μg/mL for 72h of incubation. This data confirms the increase of NPs biocompatibility due to HSA surface functionalization. Further studies were performed to verify the interaction of MelaSil_Ag NPs with corona proteins. Data showed high diminution of interacting proteins in HSA conjugated NPs compared to bare NPs. HSA was farther exploited to perform selective uptake of MelaSil_Ag-HSA through the interaction with SPARC. This protein, highly expressed in tumor cells (like HS578T), can bind HSA and to promote NPs internalization in cancer cells. The binding of SPARC to HSA was confirmed by Co-IP and Dot-blot assays. Furthermore, uptake experiments with MelaSil_Ag- HSA NPS in HS578T (SPARC positive cells) and MCF10a (SPARC negative cells) were performed. Results showed very high levels of MelaSil_Ag-HSA uptake only by HS578T cells. The involvement of SPARC in the internalization of the MelaSil_Ag-HSA NPs was evaluated by using a clathrin inhibitor. Finally, the MelaSil_Ag-HSA NPs showed very low decrease of PAS (about 10% compared to bare NPs) after HSA bioconjugation, but the signal stability in continuous irradiation, showed a very strong increase. In conclusion, this innovative hybrid NP showed high biocompatibility and hemocompatibility level, good stability and PA properties and selective internalization into cancer cells by SPARC protein.

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