Corvo, Maria Luísa Teixeira de Azevedo Rodrigues, 1960-Almeida, Rodrigo Freire Martins deMendes, Isidro Alexandre Calheiros2025-04-0420252025http://hdl.handle.net/10400.5/100021Tese de mestrado, Bioquímica e Biomedicina, 2025, Universidade de Lisboa, Faculdade de CiênciasTriple-negative breast cancer (TNBC) has no receptors and high Ki-67 levels, being a highly invasive subtype, prone to develop metastasis, with limited treatment options due to most therapeutic agents’ inability to effectively cross the blood-brain barrier (BBB), posing a significant global health challenge. This study aimed to develop a targeted drug delivery system capable of bypassing the BBB to deliver a promising compound (Mol. X) to metastatic cells in the brain. Two long-circulating liposomal formulations coated with polyethylene glycol (PEG) were developed: a non-targeted [Mol. X-Lip], and a TNBC-targeted formulation relying on the use of chlorotoxin (CTX) [Mol. X-Lip-CTX]. CTX was covalently coupled to the distal end of PEG using the direct coupling method. Both formulations were characterized in terms of mean size, polydispersity index, zeta potential, and total lipid and Mol. X contents allowing the determination of incorporation efficiency (IE). For the Mol. XLip-CTX, the covalently linked CTX was quantified, and the attachment capacity (AC) was calculated. Mol. X-Lip exhibited an IE of 85 %, a mean size of 128 nm, a polydispersity index of 0.049, and a zeta potential of -3 mV, as expected for pegylated liposomes. The Mol. X-Lip-CTX showed an IE of 69 %, a CTX AC of 4.5 nmol per µmol of lipids, a mean size of 247 nm, a polydispersity index of 0.288, and a zeta potential of -5 mV. In cell viability assay using the TNBC brain-tropism cell line (MDA-MB-231 Br4), Mol. X-Lip showed no toxicity at any concentration. Additionally, immunocytochemistry analysis targeting the biomarker Ki-67 revealed inconclusive results. Finally, a cell viability assay was conducted using the Mol. X-Lip-CTX, where no cytotoxicity was observed. In conclusion, successful incorporation of the hydrophobic compound Mol. X, and anchoring CTX into liposomes were achieved, without detect toxicity on the cells at any Mol. X concentration.engMetástases encefálicas de cancro da mamabarreira hematoencefálicalipossomaclorotoxinasistema de entrega de medicamentos direcionadoTeses de mestrado - 2025master thesis