Development and characterization of lipid-based nanosystems for controlled release of bioactive compounds

Tese de Doutoramento em Engenharia Química e Biológica.

A novel hanging spherical drop system for the generation of cellular spheroids and high throughput combinatorial drug screening

We propose a novel hanging spherical drop system for anchoring arrays of droplets of cell suspension based on the use of biomimetic superhydrophobic flat substrates, with controlled positional adhesion and minimum contact with a solid substrate. By facing down the platform, it was possible to generate independent spheroid bodies in a high throughput manner, in order to mimic in vivo tumour models on the lab-on-chip scale. To validate this system for drug screening purposes, the toxicity of the anti-cancer drug doxorubicin in cell spheroids was tested and compared to cells in 2D culture. The advantages presented by this platform, such as feasibility of the system and the ability to control the size uniformity of the spheroid, emphasize its potential to be used as a new low cost toolbox for high-throughput drug screening and in cell or tissue engineering.

In vitro digestion and stability assessment of b-lactoglobulin/riboflavin nanostructures

B-Lactoglobulin (b-Lg) is the major protein fraction of bovine whey serum and a primary gelling agent. b-Lg has a high nutritional value, is stable at low pH being highly resistant to proteolytic degradation in the stomach, besides, it has the ability of acting as an encapsulating agent. This study aims at assessing the ability of b-Lg nanostructures to associate a nutraceutical - i.e. riboflavin - and release it in a controlled manner throughout an in vitro gastrointestinal (GI) system. For this reason b-Lg nanostructures loaded with riboflavin were critically characterized in terms of their morphology (i.e. size, polydispersity, -potential and shape) by dynamic light scattering (DLS) and transmission electron microscopy (TEM), and efficiency to associate to riboflavin through spectrofluorimetry. Furthermore, these nanocomplexes were evaluated in an in vitro GI model, simulating the physiological conditions. Stable b-Lg nanostructures were obtained at pH 6, of spherical shape, characterized by particle size of 172±1 nm, low polydispersity (i.e. PDI of 0.06±0.02), -potential of -32±3 mV and association efficiency (AE) of 26±1 %. b-Lg nanostructures showed to be stable upon their passage throughout stomach (i.e. particle size, PDI and potential of 248±10 nm, 0.18±0.03 and 18±3 mV, respectively). Concerning their passage throughout the intestine, such nanostructures were mostly degraded in the duodenum. Regarding riboflavin, a release of about 11 % was observed after their passage through stomach, while 35 %, 38 % and 5 % were the released percentages of the total riboflavin associated observed after passage through duodenum, jejunum and ileum, respectively. Hence,b-Lg nanostructures showed to be suitable carriers for riboflavin until the intestine, where their degradation occurs. b-Lg also showed to be structurally stable, under food simulant conditions (yoghurt simulant, composed of 3 % acetic acid), over 14 days, with a protective effect upon riboflavin activity, releasing it in a 7 day period.

Evaluation of zearalenone in vitro removal by Saccharomyces cerevisiae strains isolated from bovine forage

Zearalenone (ZEN) is a mycotoxin that has relatively low acute toxicity. However, it is a potent oestrogen, interfering with the reproductive tract of animals. Among other effects, ZEN decreases animals fertility, and induces fibrosis in the uterus, breast cancer and endometrial carcinoma (Zinedine et al., 2007). Anti-mycotoxin additives (AMA) are defined as a group of products that, when added to animal feed, are capable of adsorbing, inactivating, or neutralizing mycotoxins in the gastrointestinal tract of animals. One example of these products are adsorbents based on yeast cell walls, a safe and beneficial animal feed additive (Abreu et al., 2008). When based on active cells, yeast based products also act as a probiotic, contributing to improve the general animal health because it stimulates their immune system and promotes the integrity of intestinal mucosa (Albino et al., 2006). Strains of Saccharomyces cerevisiae isolated from silage were tested for their ZEN removal capability. Their effect on - and b-zearalenol (-ZOL and b-ZOL) was also tested. Strains were grown on YPD separately supplemented with ZEN, -ZOL and b-ZOL, and their elimination from culture media was quantified over time by HPLC-FL.

Zebrafish embryos as in vivo model for toxicity evaluation: screening of nanoparticle formulations DODAB:MO and DODAC:MO

Dissertação de mestrado em Molecular Genetics

Síntese de novas tieno[3,2-b]piridinas com potencial atividade biológica contendo 1,2,3-triazoles 1,4-dissubstituídos obtidos por reação "click" de ciclioadição azida-alquino catalisada por Cu(I)-CuAAC

Dissertação de mestrado em Química Medicinal

Toxicity induced by a metal mixture (Cd, Pb and Zn) in the yeast Pichia kudriavzevii: The role of oxidative stress

The present work aims to contribute for the elucidation of the role of oxidative stress in the toxicity associated with the exposure of Pichia kudriavzevii to multi-metals (Cd, Pb and Zn). Cells of the non-conventional yeast P. kudriavzevii exposed for 6 h to the action of multi-metals accumulated intracellular reactive oxygen species (ROS), evaluated through the oxidation of the probe 2,7-dichlorodihydrofluorescein diacetate. A progressive loss of membrane integrity (monitored using propidium iodide) was observed in multi-metal-treated cells. The triggering of intracellular ROS accumulation preceded the loss of membrane integrity. These results suggest that the disruption of membrane integrity can be attributed to the oxidative stress. The exposure of yeast cells to single metal showed that, under the concentrations tested, Pb was the metal responsible for the induction of the oxidative stress. Yeast cells coexposed to an antioxidant (ascorbic acid) and multi-metals did not accumulate intracellular ROS, but loss proliferation capacity. Together, the data obtained indicated that intracellular ROS accumulation contributed to metal toxicity, namely for the disruption of membrane integrity of the yeast P. kudriavzevii. It was proposed that Pb toxicity (the metal responsible for the toxic symptoms under the conditions tested) result from the combination of an ionic mechanism and the intracellular ROS accumulation.