Cytotoxicity of temperature-responsive cationic diblock copolymers in human cancer and non-cancer cells lines

Abstract in proceedings of the Fourth International Congress of CiiEM: Health, Well-Being and Ageing in the 21st Century, held at Egas Moniz’ University Campus in Monte de Caparica, Almada, from 3–5 June 2019.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.info:eu-repo/semantics/publishedVersio

Co-culture of human induced pluripotent stem cell-derived retinal pigment epithelial cells and endothelial cells on double collagen-coated honeycomb films

In vitro cell culture models representing the physiological and pathological features of the outer retina are urgently needed. Artificial tissue replacements for patients suffering from degenerative retinal diseases are similarly in great demand. Here, we developed a co-culture system based solely on the use of human induced pluripotent stem cell (hiPSC)-derived cells. For the first time, hiPSC-derived retinal pigment epithelium (RPE) and endothelial cells (EC) were cultured on opposite sides of porous polylactide substrates prepared by breath figures (BF), where both surfaces had been collagen-coated by Langmuir–Schaefer (LS) technology. Small modifications of casting conditions during material preparation allowed the production of free-standing materials with distinct porosity, wettability and ion diffusion capacity. Complete pore coverage was achieved by the collagen coating procedure, resulting in a detectable nanoscale topography. Primary retinal endothelial cells (ACBRI181) and umbilical cord vein endothelial cells (hUVEC) were utilised as EC references. Mono-cultures of all ECs were prepared for comparison. All tested materials supported cell attachment and growth. In mono-culture, properties of the materials had a major effect on the growth of all ECs. In co-culture, the presence of hiPSC-RPE affected the primary ECs more significantly than hiPSC-EC. In consistency, hiPSC-RPE were also less affected by hiPSC-EC than by the primary ECs. Finally, our results show that the modulation of the porosity of the materials can promote or prevent EC migration. In short, we showed that the behaviour of the cells is highly dependent on the three main variables of the study: the presence of a second cell type in co-culture, the source of endothelial cells and the biomaterial properties. The combination of BF and LS methodologies is a powerful strategy to develop thin but stable materials enabling cell growth and modulation of cell-cell contact. Statement of significance: Artificial blood-retinal barriers (BRB), mimicking the interface at the back of the eye, are urgently needed as physiological and disease models, and for tissue transplantation targeting patients suffering from degenerative retinal diseases. Here, we developed a new co-culture model based on thin, biodegradable porous films, coated on both sides with collagen, one of the main components of the natural BRB, and cultivated endothelial and retinal pigment epithelial cells on opposite sides of the films, forming a three-layer structure. Importantly, our hiPSC-EC and hiPSC-RPE co-culture model is the first to exclusively use human induced pluripotent stem cells as cell source, which have been widely regarded as an practical candidate for therapeutic applications in regenerative medicine.publishedVersionPeer reviewe

Impact of Optimized Breastfeeding on the Costs of Necrotizing Enterocolitis in Extremely Low Birthweight Infants

To estimate risk of NEC for ELBW infants as a function of preterm formula and maternal milk (MM) intake and calculate the impact of suboptimal feeding on NEC incidence and costs

Thermoresponsive polymers in controlled drug delivery and gene delivery

In this thesis, emphasis is given to the study of temperature-responsive systems and their use in drug and gene delivery applications. Studies on polymer-surfactant systems were conducted, using amino acid-based surfactants. In these studies, ethyl (hydroxyethyl) cellulose (EHEC) was the investigated polymer, while the interactions with six different surfactants were analyzed. Three of these were anionic and lysine-based, having a gemini-like structure and different alkyl chain length. In addition, three cationic arginine-based surfactants were investigated. Of these, two had a gemini structure, while the third was monomeric. The polymer-surfactant systems were characterized in terms of their rheological properties, i.e. complex viscosity, gel points, gel properties and thermodynamic features in form of cloud points. It was shown that the mixtures were generally low viscous at room temperature, and that the viscosity increased at higher temperatures, in agreement with the formation of a polymer network stabilized by hydrophobic associations and electrostatic repulsions. Sol-gel transitions were also observed, suggesting that the gels could be employed as pharmaceutical formulations undergoing gelation in situ, once injected. Importantly, surfactants with long alkyl chains and gemini-like structure were the most efficient, which implies that very low amounts are needed in order to induce the sol-gel transition at elevated temperatures. The biocompatibility of the EHEC-surfactant systems was evaluated by performing in vitro experiments on a human cell line (HeLa cells). These studies revealed that the higher toxicity of the long-chain/gemini-like surfactants was significantly compensated by their superior efficiency at low concentrations. Experiments were further carried out with the intention of developing a microparticulate system for controlled drug delivery. An emulsification-solvent evaporation method was employed according to a planned experimental design that aimed at evaluating the process parameters on the size properties of the microparticles. Chitosan, a hydrophobicallymodified chitosan derivative (HM-chitosan) and poly lactic-co-glycolic acid (PLGA) were investigated as the polymeric matrix. The blank microparticles were characterized in terms of size and size distribution. Following the multivariate analysis, specific parameters were chosen to produce the drug-loaded microparticles. Naltrexone, an opioid antagonist, was used as the model drug. The microparticles were evaluated in terms of their morphological properties (scanning electron microscopy), and the drug encapsulation efficiency and loading capacity were determined. In vitro drug release experiments were carried out. The results from this study showed that HM-chitosan microparticles were recovered in a high yield and that the encapsulation efficiency was higher as compared to the use of the other polymers. Importantly, this polymer also demonstrated the higher capacity to retain the drug, which was slowly released for at least 50 days. Finally, four cationic temperature-responsive block copolymers were evaluated as gene carriers. These polymers had a structure based on poly (N-isopropylacrylamide)-blockpoly(( 3-acrylamidopropyl) trimethylammonium chloride) (PNIPAAM-b-PAMPTMA(+)), and were distinct in terms of the length of the blocks. Complexes between polymer and plasmid DNA were prepared at different polymer/DNA ratios, and used to transfect HeLa cells. Because the plasmid DNA coded for green fluorescent protein (GFP), the expression of this protein was followed by flow cytometry. Cell viability studies were simultaneously carried out in order to evaluate the cytotoxic effects of the complexes. The results showed that polymers with longer PNIPAAM or shorter PAMPTMA(+) were the most effective carriers. A series of physicochemical experiments were carried out (dynamic light scattering, zeta potential and turbidity), so as to gain insights into copolymer structureactivity relationships. It was shown that the most effective carriers had a compact PNIPAAM core that collapsed at 37 °C surrounded by a positively charged corona

Colagénio da Catostylus tagi como matriz polimérica destinada à veiculação de fármacos proteicos

Tese de mestrado, Farmacotecnia Avançada, Universidade de Lisboa, 2009Despite the great potential of protein pharmaceuticals in the pharmaceutical industry, proteins are rather fragile molecules and their administration often requires encapsulation in protective systems. In this work, a microparticulate protein delivery system was developed using collagen from the medusa Catostylus tagi as polymeric matrix. Collagen microparticles (CMPs) were produced by an emulsification-gelationsolvent extraction method, and a high loading efficiency was found for the entrapment of two model proteins – lysozyme and α-lactalbumin. A fraction of CMPs was separated and chemically treated with genipin (GP) and another with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), in order to strengthen the collagenous matrix and reduce pore size. The non-crosslinked CMPs were spherical, rough-surfaced and had an average size of 5-15 μm. Slight size decrease, along with the reduction in pore size and number was found to occur upon treatment with EDC. When the particles were treated with GP, in contrast, no reticulation could be measured under the conditions used, and the particles suffered shape modification and diameter enlargement. These results pointed out to the unsuitability of GP as a crosslinking agent for this purpose and its use was hence abandoned. The EDC-crosslinked microparticles showed a moderate hydrophobic behavior. A positive surface charge was assessed by zeta potential determination, which suggested a higher affinity towards negatively charged molecules and cellular membranes.Untreated collagen matrixes, due to their weakness and large pore size, seemed unable of significantly retaining entrapped molecules; chemical treatment with EDC, on the other hand, produced microparticles that were significantly resistant to water uptake, allowing a slow and sustained release of the proteins, as shown by in vitro experiments. Finally, the assessment of lysozyme’s biological activity showed that the protein remained active after both the encapsulation and reticulation processes. In summary, the work here described shows the potential use of a marine collagen in the production of microparticles for the controlled release of therapeutic proteins