Browsing by Author "Fernandes, Joana Corrêa Mendes de Sousa"
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- Alginate nanoparticles for p53 encoding pDNA deliveryPublication . Fernandes, Joana Corrêa Mendes de Sousa; Valente, Joana Filipa Abreu Pereira; Chagas, Bárbara FerreiraIn developed countries with high life expectancies, colorectal cancer (CRC) ranks as the third most common cancer and the second deadliest globally. In 2022 alone, over 1.9 million cases were diagnosed, with CRC causing more than 900.000 deaths annually. Projections indicate a potential increase to 2.5 million cases per year by 2035. Adenocarcinoma, primarily originating from mucus-producing cells, constitutes over 90% of CRC cases. The prognosis of CRC significantly hinges on disease stage, treatment regimen, and patient conditions. The tumour suppressor protein p53 plays a pivotal role in regulating cell growth and preventing tumour formation and is often mutated, or inactivated in CRC cancer cells, making it a prime target for therapeutic intervention. Proper p53 function enables DNA repair or initiates programmed cell death (apoptosis) in cells with irreparable DNA damage, thwarting cancer cell proliferation. Low expression or loss of p53 function in CRC cells is common and can lead to increased genomic instability, impaired DNA repair, and resistance to apoptosis, thereby promoting tumour progression and aggressiveness. Additionally, low p53 expression correlates with chemotherapy resistance and poorer prognosis in CRC patients. Genetic therapy in CRC presents a tailored approach to fight against the disease at a genetic level. One promising strategy involves using plasmid DNA (pDNA) carrying the tp53 gene to restore or enhance p53 function within CRC cells. This targeted genetic intervention holds the potential to inhibit tumour growth, induce cancer cell death, and overcome resistance mechanisms encountered in conventional CRC therapies. However, pDNA's susceptibility to degradation leads to the need to use a delivery system to transport it to target cells. In this context, a growing interest in using natural polymers from renewable sources as biomaterials, such as alginate nanoparticles emerge as promising carriers due to their inert, biocompatible, and easily manipulable properties, with crosslinking achievable through divalent ions without adverse host effects. In this context, the present work focuses on the development of a simple and sustainable process for the extraction and purification of sodium alginate (SA) from brown Sargassum muticum and Saccorhiza polyschides aiming to develop DNA-loaded alginate nanoparticles. These nanoparticles were further characterized and tested in CRC cell lines (CaCo-2) and healthy cells (fibroblasts). Overall, the extraction and purification process parameters play a key role and significantly influence the quality of alginate and the extraction yield. Research efforts are focused on achieving simple and sustainable alginate extraction technologies to improve process efficiency, enhance extraction capacity, reduce cost and promote environmental friendliness. Alginate extraction from brown seaweed S. muticum and S. polyschides showed an optimized yield of SA extraction during 24 hours at 40 °C yielding 20% and 22%, respectively. Characterization wise, H-NMR, FT-IR, viscosity, molecular weight, and Thermal behaviour were studied. Throught this evaluation the efficiency and purity of the extraction were proved. Regarding the development of alginate nanoparticles, the optimized nanoparticle formulation showed a medium size of 167.9 ± 2.49 nm, a PDI of 0.1608 ± 0.009, and a stable zeta potential around 0 mV. Encapsulation efficiency reached 97%, with negligible changes in size when loaded with pDNA. Release tests indicated faster release at lower pH levels and a lower swelling behaviour for lower pH. At 72 hours CaCo-2 transfected with loaded nanoparticles exhibited a 30% diminish in cell viability, in healthy cells, the alginate-base nanocarrier didn’t show any cytotoxic effect.