Development of Chitosan and Poly (Vinyl Alcohol) Blended scaffolds for cell culture using supercritical fluids technology

Dissertação apresentada na Faculdade de Ciências e Tecnologia da Universidade Nova de Lisboa para obtenção do grau de Mestre em Mestrado Integrado em Engenharia Química e Bioquímica

Edible coatings based on chitosan-beeswax emulsions

Dissertação para obtenção do Grau de Mestre em Engenharia Química e Bioquímica

Development of chitosan-based microparticles for pulmonary drug delivery

Dissertação para obtenção do Grau de Mestre em Engenharia Química e Bioquímica

Influence of the molecular weight in mechanical properties and degradation kinetics of chitosan inverted colloidal crystals

Tissue engineering arises from the need to regenerate organs and tissues, requiring the development of scaffolds, which can provide an optimum environment for tissue growth. In this work, chitosan with different molecular weights was used to develop biodegradable 3D inverted colloidal crystals (ICC) structures for bone regeneration, exhibiting uniform pore size and interconnected network. Moreover, in vitro tests were conducted by studying the influence of the molecular weight in the degradation kinetics and mechanical properties. The production of ICC included four major stages: fabrication of microspheres; assembly into a cohesive structure, polymeric solution infiltration and microsphere removal. Chitosan’s degree of deacetylation was determined by infrared spectroscopy and molecular weight was obtained via capillary viscometry. In order to understand the effect of the molecular weight in ICC structures, the mass loss and mechanical properties were analyzed after degradation with lysozyme. Structure morphology observation before and after degradation was performed by scanning electron microscopy. Cellular adhesion and proliferation tests were carried out to evaluate ICC in vitro response. Overall, medium molecular weight ICC revealed the best balance in terms of mechanical properties, degradation rate, morphology and biological behaviour.

Chitosan-based magnetic nanoparticles for osteosarcoma theranostic

Cancer is a well-known disease with a significant impact in society not only due to its incidence, more evident in more developed countries, but also due to the expenses related to medical treat-ments. Cancer research is considered an increasingly logical science with great potential for the development of new treatment options. Advances in nanomedicine have resulted in rapid devel-opment of nanomaterials with considerable potential in cancer diagnostics and treatment. The combination of diagnosis and treatment in a single nano-platform is named theranostic. In this PhD thesis a theranostic system for osteosarcoma was proposed, composed by a magnetic core, a polymeric coating, and a chemotherapeutic drug. The presence of a specific targeting agent, in this case a monoclonal antibody, provides high specificity to the proposed theranostic system. For the core of the proposed theranostic system, stable aqueous suspensions of superparamagnetic iron oxide nanoparticles with an average diameter of 9 nm were produced. Chitosan-based poly-meric nanoparticles with a hydrodynamic diameter around 150 nm were successfully produced. Incorporation of iron oxide nanoparticles into the polymeric ones increased their hydrodynamic diameter to at least 250 nm. A monoclonal antibody specific for a transmembranar protein (car-bonic anhydrase IX) present in solid tumors was developed by hybridoma technology. Functional hybridomas producing the desired monoclonal antibodies were obtained. The proposed theranostic system functionality was evaluated in separated parts of its components. Uncoated and coated iron oxide nanoparticles with chitosan-based polymers generated heat under the application of an external alternating magnetic field. Uncoated iron oxide nanoparticles sta-bilized with oleic acid were able to enhance contrast in magnetic resonance imaging. Drug deliv-ery studies were conducted in chitosan-based polymeric nanoparticles without and with the in-corporation of iron oxide nanoparticles, demonstrating to be an effective drug delivery platform for doxorubicin. The theranostic system proposed in this PhD thesis is very promising for cancer theranostic, demonstrating to be applicable in solid tumors such as osteosarcoma.

Development of biocompatible and “smart” porous structures using CO2-assisted processes

Dissertação apresentada para a obtenção do grau de Doutor em Engenharia Química, especialidade Engenharia da Reacção Química, pela Universidade Nova de Lisboa, Faculdade de Ciências e Tecnologia

Evaluation of a new vaccine based on pDNA and recombinant protein against Helicobacter pylori

Dissertação para obtenção do Grau de Mestre em Genética Molecular e Biomedicina

Production of chitin-glucan complex by Pichia pastoris

Dissertation for the Degree of Master in Biotechnology

Porous structures for the purification of biopharmaceuticals

This work aimed at the development of a (bio)polymeric monolithic support for biopharmaceuticals purification and/or capture. For that, it was assured that functional groups on its surface were ready to be involved in a plethora of chemical reactions for incorporation of the desired and most suitable ligand. Using cryogelation as preparation method a screening on multiple combinations of materials was performed in order to create a potentially efficient support with the minimal footprint, i.e. a monolithic support with reasonable mechanical properties, highly permeable, biocompatible, ready to use, with gravitational performance and minimal unspecific interactions towards the target molecules, but also biodegradable and produced from renewable materials. For the pre-selection all monoliths were characterized physico-chemically and morphologically; one agarose-based and two chitosan-based monoliths were then subjected to further characterizations before and after their modification with magnetic nanoparticles. These three specimens were finally tested towards adenovirus and the recovery reached 84% for the chitosan-GMA plain monolith prepared at -80°C. Monoliths based on chitosan and PVA were prepared in the presence and absence of magnetic particles, and tested for the isolation of GFP directly from crude cellular extracts. The affinity ligand A4C7 previously selected for GFP purification was synthesized on the monolith. The results indicated that the solid-phase synthesis of the ligand directly onto the monolith might require optimization and that the large pores of the monoliths are unsuitable for the purification of small proteins, such as GFP.

Smart macroporous structures for the purification of viral particles

Dissertação para obtenção do Grau de Mestre em Engenharia Química e Bioquímica

Studies towards modified chitooligosaccharides: a new approach to NAG-NAM moiety

Dissertação para obtenção do Grau de Mestre em Bioorgânica

Soft biomaterials as a substitute for the Nucleus Pulposus

One of the largest health problems faced worldwide, when evaluated by direct (clinical) as well indirect cost (absenteeism), is the degeneration of the intervertebral disc (IVD) that leads to back pain and, potentially disability and individual´s quality of life decreasing. The intervertebral disc is a mechanical and biological complex structure, formed by a tough outer layer of fibrocartilage called Annulus Fibrosus (AF),which surrounds a soft, elastic and gelatinous core called Nucleus Pulposus (NP). These two structures are completed by two upper and lower encasing layer called Vertebral Endplates (VEP). The degeneration of the IVD is marked by the dehydration of the Nucleus Pulposus, reducing the hydrostatic pressure inside the nucleus, resulting in a loss of capability to support compressive forces, during the active period, and to regain height during the resting period. This situation will compromise the role of shock absorber by the NP and transfers these forces to the AF. This transfer will result in cracks on the AF, deteriorating the IVD, allowing the ingrowth of vessels and nerves. This project was based on the developing a protocol to test suitable NP replacements, in hope to future assessment of discrete mechanical values and characteristics for an NP replacement. For this, Nucleus pulposus samples from goat, encapsulated Hydromed gel denominated “Raviolis” and Chitosan gels, produced via wet route using an ammonium environment, were confined compressed. Chitosan was rheologically tested and swelling capability of all the three type of materials was assessed. Results showed that the Nucleus Pulposus and “Raviolis” have similar mechanical behavior, being able to swell and “build up” hydrostatic pressure after a compression stage, while the Chitosan gel did not showed that ability. Therefore, “Raviolis” are a more suitable candidate to replace the NP than Chitosan gels. It was also observed that confined compression is the key test to perform on any possible candidate to replace the NP.

Novel antibiotic-loaded orthopaedic bone cements: insights on drug release profiles and biocompatibility

Acrylic bone cement (BC) is widely used as an anchor of artificial joints. Bacterial infection due to biofilm formation and inflammation are common and difficult to treat problems associated with commercial available BC formulations. Research on novel BC compositions is urgently needed. The main objective of this thesis was to develop a new biocompatible antibiotic-loaded BC with improved release profile. To achieve that aim several additives were incorporated, as an antibiotic (levofloxacin) to combat bacterial growth, an anti-inflammatory drug (diclofenac) to decrease the inflammatory process and two well-known and broadly used biopolymers, alginate and chitosan in order to increase matrix porosity, and in this way to intensify the amount of released drug. Novel BC formulations were tested in order to find the most suitable one that had potential to proceed to clinical application. Numerous tests were conducted as: a) evaluation of drug release profiles in different biomimetic media, b) mechanical and surface studies, c) microbiological activity testing against Staphylococcus aureus and d) in vitro biocompatibility assays (fibroblasts and osteoblasts). In general, the addition of biopolymers increased drug release, didn’t compromised BC mechanical properties and increased BC hydrophilicity. Microbiological testing revealed that Lev[BC]Chi was the only matrix that reduced significantly biofilm formation. On the contrary, alginate and diclofenac loading into BC seemed to increase biofilm growth. Biocompatibility studies showed some decrease in cell viability, in particularly on osteoblasts, mainly due to the high amounts of released drugs. In conclusion, the present work has shown that the matrix with more potential to proceed in further investigations was Lev[BC]Chi. Other conditions (namely additives and drugs concentrations) should be evaluated with the other tested BC matrices before being discharged.

Production and characterization of electrospun composite fibers: confinement of thermosensitive microgels

Materials engineering focuses on the assembly of materials´ properties to design new products with the best performance. By using sub-micrometer size materials in the production of composites, it is possible to obtain objects with properties that none of their compounds show individually. Once three-dimensional materials can be easily customized to obtain desired properties, much interest has been paid to nanostructured poly-mers in order to build biocompatible devices. Over the past years, the thermosensitive microgels have become more common in the framework of bio-materials with potential applicability in therapy and/or diagnostics. In addition, high aspect ratio biopolymers fibers have been produced using the cost-effective method called electrospinning. Taking advantage of both microgels and electrospun fibers, surfaces with enhanced functionalities can be obtained and, therefore employed in a wide range of applications. This dissertation reports on the confinement of stimuli-responsive microgels through the colloidal electro-spinning process. The process mainly depends on the composition, properties and patterning of the precur-sor materials within the polymer jet. Microgels as well as the electrospun non-woven mats were investigated to correlate the starting materials with the final morphology of the composite fibers. PNIPAAm and PNIPAAm/Chitosan thermosensitive microgels with different compositions were obtained via surfactant free emulsion polymerization (SFEP) and characterized in terms of chemical structure, morphology, thermal sta-bility, swelling properties and thermosensitivity. Finally, the colloidal electrospinning method was carried out from spinning solutions composed of the stable microgel dispersions (up to a concentration of about 35 wt. % microgels) and a polymer solution of PEO/water/ethanol mixture acting as fiber template solution. The confinement of microgels was confirmed by Scanning Electron Microscopy (SEM). The electrospinning process was statistically analysed providing the optimum set of parameters aimed to minimize the fiber diameter, which give rise to electrospun nanofibers of PNIPAAm microgels/PEO with a mean fiber diameter of 63 ± 25 nm.

Improving silicon probe performance through layer-by-layer coating

Fully comprehending brain function, as the scale of neural networks, will only be possi-ble with the development of tools by micro and nanofabrication. Regarding specifically silicon microelectrodes arrays, a significant improvement in long-term performance of these implants is essential. This project aims to create a silicon microelectrode coating that provides high-quality electrical recordings, while limiting the inflammatory response of chronic implants. To this purpose, a combined chitosan and gold nanoparticles coating was produced allied with electrodes modification by electrodeposition with PEDOT/PSS in order to reduce the im-pedance at 1kHz. Using a dip-coating mechanism, the silicon probe was coated and then charac-terized both morphologically and electrochemically, with focus on the stability of post-surgery performance in anesthetized rodents. Since not only the inflammatory response analysis is vital, the electrodes recording degradation over time was also studied. The produced film presented a thickness of approximately 50 μm that led to an increase of impedance of less than 20 kΩ in average. On a 3 week chronic implant, the impedance in-crease on the coated probe was of 641 kΩ, compared with 2.4 MΩ obtained for the uncoated probe. The inflammatory response was also significantly reduced due to the biocompatible film as proved by histological tests.