Analisi struttura-funzione dei coloranti polimetinici per la terapia fotodinamica (PDT) attraverso gli approcci dell'experimental design e del Ca2+ imaging

Photodynamic therapy (PDT) is a minimally invasive treatment, clinically used to treat a wide range of medical conditions. It combines three non-toxic agents: visible or infrared light, molecular oxygen, and a light-absorbing molecule called photosensitizer (PS) to cause oxidative stress in the irradiated cells. The combination of these elements can induce a chain of reactions leading to cell death. One of the challenges in photodynamic therapy is to develop novel photosensitizers with enhanced photoactivity and low side effects. In this context, Polymethine Dyes (PMDs) have been proven to be promising candidates. On the other hand, experimental optimization and testing of different PMDs candidates is a time consuming and expensive process. For this reason, the purpose of this project was to test the possible use of the Design of Experiment (DoE), an efficient iterative method to attain maximum accuracy in the results, with minimal use of resources, to minimize the number of experiments and therefore, the time needed to characterize new PMDs. The main goal of my research was to investigate the relationship between different structures of PMDs and their different cellular response during PDT, using this statistical approach. In this regard, several empirical models have been developed, using the cell viability data of three different PMDs: a bromime-squaraine-C4 (BR-SQ-C4), a bromime-benzocyanine-C4 (Br-BCy-C4) and a carboxy-squaraine-C8 (COOH-SQ-C8). These models were then tested by comparing them with experimental data and used to identify the best PDT assay, featured by high efficacy and low side effects, for each different PMDs. Finally, the models were compared, in order to find out which PMDs is the most functional for PDT and which changes in the structure of a PMD may affect its function. The developed models showed a good predictive power and well described the photodynamic behaviour of the PMDs analyzed. The comparison then, made it possible to identify which PMDs responded best to the PDT, and therefore which structural characteristics were important to mediate photodynamic activity. The second goal of my research was to verify the role of intracellular calcium in PMDs-treated cells during PDT. Indeed, it is known that Ca2+ signalling play an important role in PDT-induced cell death processes. Therefore, I carried out Ca2+imaging experiments with cells treated with the studied PMDs, to verify possible Ca2+ responses. The results obtained showed the importance of Ca2+ signals in cells treated with the PMDs analyzed and confirmed the conclusions previously made on their photodynamic activity.