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A thorough knowledge of factors important for loading efficiency and drug release is necessary to be able to develop optimal nano-carriers for the future. Other issues that are not fully understood is how small the carriers need to be, and which manufacturing procedures should be used.

Gene knockdown has emerged as an important tool for cancer gene therapy as well as for viral infections and dominantly inherited genetic disorders. The generation of suitable siRNA delivery systems poses some challenges, namely, to avoid nuclease degradation, to surpass the cytoplasmic membrane, and to release the nucleic acids into the cytosol. Aiming at evaluating the ability of thermoresponsive block copolymers formed by units of N-isopropylacrylamide and of (3-acrylamidopropyl)trimethylammonium chloride to efficiently deliver siRNAs, an extensive study was performed with four different copolymers using a human fibrosarcoma cell line as cell model. The silencing ability and cytotoxicity of the generated copolymer-based siRNA delivery systems were found to be dependent on the cloud point of the polymer, which corresponds to the transition temperature at which the aggregation or precipitation of the polymer molecules becomes thermodynamically more favorable than their solubilization. In the present study, a system capable of delivering siRNAs efficiently, specifically and without presenting relevant cytotoxicity, even in the presence of serum, was developed. Confocal fluorescence experiments showed that the ability of the generated systems to silence the target gene is related to some extent to nucleic acid internalization, being also dependent on polymer/siRNA dissociation at 37 °C. Thus, a delicate balance between nucleic acid internalization and intracellular release must be met in order to reach an ideal knockdown efficiency. The special features and potential for manipulation of the N-isopropylacrylamide-based copolymers make them suitable materials for the design and synthesis of new and promising siRNA delivery systems.

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Age-related macular degeneration (AMD) is a leading cause of blindness in developed countries, characterised by the degeneration of the retinal pigment epithelium (RPE), a pigmented cell monolayer that closely interacts with the photoreceptors. RPE transplantation is thus considered a very promising therapeutic option to treat this disease. In this work, porous honeycomb-like films are for the first time investigated as scaffold materials for human embryonic stem cell-derived retinal pigment epithelium (hESC-RPE). By changing the conditions during film preparation, it was possible to produce films with homogeneous pore distribution and adequate pore size (∼3-5 µm), that is large enough to ensure high permeability but small enough to enable cell adherence and spreading. A brief dip-coating procedure with collagen type IV enabled the homogeneous adsorption of the protein to the walls and bottom of pores, increasing the hydrophilicity of the surface. hESC-RPE adhered and proliferated on all the collagen-coated materials, regardless of small differences in pore size. The differentiation of hESC-RPE was confirmed by the detection of specific RPE protein markers. These results suggest that the porous honeycomb films can be promising candidates for hESC-RPE tissue engineering, importantly enabling the free flow of ions and molecules across the material. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 1646-1656, 2016.

Age-related macular degeneration (AMD) is the leading cause of vision loss in senior citizens in the developed world. The disease is characterised by the degeneration of a specific cell layer at the back of the eye -the retinal pigment epithelium (RPE), which is essential in retinal function. The most promising therapeutic option to restore the lost vision is considered to be RPE cell transplantation. This work focuses on the development of biodegradable biomaterials with similar properties to the native Bruch's membrane as carriers for RPE cells. In particular, the breath figure (BF) method was used to create semi-permeable microporous films, which were thereafter used as the substrate for the consecutive Langmuir-Schaefer (LS) deposition of highly organised layers of collagen type I and collagen type IV. The newly developed biomaterials were further characterised in terms of surface porosity, roughness, hydrophilicity, collagen distribution, diffusion properties and hydrolytic stability.Human embryonic stem cell-derived RPE cells (hESC-RPE) cultured on the biomaterials showed good adhesion, spreading and morphology, as well as the expression of specific protein markers. Cell function was additionally confirmed by the assessment of the phagocytic capacity of hESC-RPE.Throughout the study, microporous films consistently showed better results as cell culture materials for 2 hESC-RPE than dip-coated controls. This work demonstrates the potential of the BF-LS combined technologies to create biomimetic prosthetic Bruch's membranes for hESC-RPE transplantation.