Nanoparticelle proteiche e polimeriche caricate con Cianina per l'applicazione nella terapia fotodinamica

Photodynamic therapy (PDT) has emerged as a novel therapeutic approach to treat cancer, offering a promising alternative to conventional treatments due to its high target selectivity for cancer cells and minimally invasive nature and side effects. The molecular mechanism of PDT involves three components, photosensitizer (PS), light, and molecular oxygen. When the PS is activated by light at the appropriate excitation wavelength, reacts with the molecular oxygen generating reactive oxygen species (ROS) responsible for cell death. The efficacy of PDT is highly dependent on the type of PS. Among the different classes of organic Near-Infrared (NIR) PSs, cyanine dyes (belonging to the polymethine dyes) have gained considerable attention in PDT. Specifically, heptamethine Cyanines (Cy7) can be considered promising PSs, thanks to their absorption and emission in the NIR (from 700 to 900 nm), perfectly matching the therapeutic window (600-900 nm), and resulting in a deeper tissue penetration, high fluorescence quantum yield, high selectivity, minimal toxicity in dark, and easy and cost-effective synthesis with a high degree of chemical purity. Despite these advantages, Cy7 showed poor stability in physiological environment with the formation of aggregates leading to lower production of ROS and thus lower activity. A promising solution to increase the stability and thus the bioavailability is the incorporation into nanoparticles (NPs). In this project, novel bromine indoline-based heptamethine-cyanine dyes (BrCy7) provided by the Department of Chemistry in the University of Turin, have been incorporated in both Human Serum Albumin Nanoparticles (HAS-NPs) and into Pegylated Poly(lactic-co-glycolic acid) Nanoparticles (PEG-PLGA NPs). The thesis project involved the characterization of the BrCy7 and NPs, followed by the assessment of NPs+BrCy7 complexes to evaluate the stability, ROS production, and efficacy compared to the free BrCy7. For the synthesis of HSA-NPs, a statistical multivariate design such as the Design of Experiment (DoE), based on different desolvation method synthesis reported in the literature, was set up to investigate how different variables can affect the size, Entrapment Efficiency (EE%) and yield of the dye-loaded HSA-NPs. However, only the evaluation of Entrapment Efficiency (EE%) was possible (due to the insolubility of the nanosystem), with an increase in EE% up to 99% in some chemical syntheses. On the other hand, the incorporation of BrCy7 into PEG-PLGA NPs showed an improvement of the dye stability in aqueous medias, as well as a high ROS production. This nanosystem was also tested in vitro, to evaluate the cytotoxicity and photoactivity against pancreatic cancer (PANC-1) cell line equilibrated in an acidic pH (6.6) environment. Bare PEG-PLGA NPs showed good cytocompatibility, however, the presence of BrCy7 seems to decrease the cytocompatibility in the dark and after irradiation, even if it was not possible to clearly highlight a difference between the untreated and treated cells. This research contributes to providing a basis for further investigation into the effect of NPs on the stability of NIR-PSs, aiming to obtain stable PSs in aqueous environments for future application in PDT. However, further investigations are needed to understand how to optimize the production of HSA NPs and for the selection of an optimal concentration of PEG-PLGA-BrCy7 that does not induce cytotoxic effects in dark conditions.