Brito, Maria Alexandra de Oliveira Silva Braga Pedreira de, 1960-Malhó, Rui, 1967-Romão, Joana Raquel Aniceto2023-05-2920232022http://hdl.handle.net/10451/57650Tese de mestrado, Biologia Molecular e Genética , 2022, Universidade de Lisboa, Faculdade de Ciências15-25 % of breast cancer (BC) patients develop brain metastases (BM), a poor prognosis condition due to the restricted blood-brain barrier (BBB) permeability. Platelet-derived growth factor subunit B (PDGF-B) was associated with BC cells (BCCs) proliferation. Furthermore, salinomycin (SAL) was effective in the eradication of BCCs. Nanoformulations coupled to chlorotoxin (CTX), appear as a strategy to overcome the BBB and achieve target-specific delivery. This prompted us to develop a new nanomedicines platform decorated with CTX for combined drug (SAL) and genetic (siRNA) therapeutic approach to abrogate BC brain metastases (BCBM). Liposomes with siPDGF-B were developed and their biological activity towards triple negative BCCs (4T1 cells) was determined based on cell viability and PDGF-B silencing. SAL incorporation was optimized, and characterized, and SAL encapsulated median lethal dose (LD50) was determined. The efficiency of co-administration was studied based on cell viability, PDGF-B silencing, and proliferation. The safety of both formulations for brain microvascular endothelial cells (b.End5 cells) was studied. BBB transposition efficiency, and efficacy to abrogate BCCs were evaluated in a co-culture model. Finally, the BBB integrity was ensured by transendothelial electrical resistance (TEER) and the βcatenin labeling. siPDGF-B presented no effect on 4T1 cells’ viability, while PDGF-B silencing efficacy achieved 31%. SAL showed around 33% SAL incorporation efficiency, achieving a LD50 of 24.76 µM. Moreover, coadministration treatment modulated 4T1 cells’ proliferation. Importantly, both formulations showed no effect on b.End5 cells’ viability. Using the co-culture model, liposomes’ ability to act on BCCs, decreasing PDGF-B expression was demonstrated. Additionally, liposomes individually lead to cell senescence. Lastly, TEER and β-catenin labeling revealed b.End5 monolayer disruption by SAL. Overall, the delivery of SAL appears to impair the endothelium, while siPDGF-B delivery in a targeted and BBB-permeant platform emerges as a new approach for BCBM treatment.engmetástases encefálicas do cancro da mamabarreira hematoencefálicalipossomafator de crescimento derivado de plaquetassalinomicinaTeses de mestrado - 2023master thesis203486781