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.

Technology market transfer plan launch of DELIVES to E.U. Market©

Dissertação apresentada na Faculdade de Ciências e Tecnologia da Universidade Nova de Lisboa para obtenção do grau de Mestre em Biotecnologia

Tauroursodeoxycholic acid prevents Bax-induced membrane perturbation and cytochrome C release in isolated mitochondria

Biochemistry, 2003, 42 (10), pp 3070–3080 DOI: 10.1021/bi026979d

Development of chitosan-based microparticles for pulmonary drug delivery

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

Development of composite particles for pulmonary delivery

In this work, biocompatible and biodegradable poly(D-L-lactide-co-glycolide) (PLGA) microparticles with the potential for use as a controlled release system of vaccines and other drugs to the lung were manufactured using supercritical CO2, through the Supercritical Assisted Atomization (SAA) technique. After performing a controlled variance in production parameters (temperature, pressure, CO2/solution flow ratio) PLGA microparticles were characterized and later used to encapsulate active pharmaceutical ingredients (API). Bovine serum albumin (BSA) was chosen as model protein and vaccine, while sildenafil was the chosen drug to treat pulmonary artery hypertension and their effect on the particles characteristics was evaluated. All the produced formulations were characterized in relation to their morphology (Morphologi G3 and scanning electronic microscopy (SEM)), to their physical-chemical properties (X-ray diffraction (XRD, differential scanning calorimetry (DSC), Fourier transform infrared (FTIR)) and aerodynamic performance using an in vitro aerosolization study – Andersen cascade impactor (ACI) - to obtain data such as the fine particle fraction (FPF) and the mass median aerodynamic diameter (MMAD). Furthermore, pharmacokinetic, biodegradability and biocompatibility tests were performed in order to verify the particle suitability for inhalation. The resulting particles showed aerodynamic diameters between the 3 and 5 μm, yields up to 58% and FPF percentages rounding the 30%. Taken as a whole, the produced microparticles do present the necessary requests to make them appropriate for pulmonary delivery.

Double-walled PLLA/PLGA particles for the sustained release of Meloxicam: preparation, characterisation and contolled release studies

There were two main objectives in this thesis investigation, first, the production, characterisation, in vitro degradation and release studies of double walled microspheres for drug release control. The second one, and the most challenging, was the production of double walled nanospheres, also for drug control delivery. The spheres were produced using two polymers, the Poly(L-lactide)Acid, PLLA, and the Poly(L-lactide-co-glycolic)Acid, PLGA.Afterwards, a model drug, Meloxicam, which is an antiinflammatory drug, was encapsulated into the particles. Micro and nanospheres were produced by the solvent extraction/evaporation method, where perfect spherical particles were obtained. By varying the polymers PLLA/PLGA mass ratio, different core and shell composition, as well as several shell and core thickness were observed. In the particles with a PLLA/PLGA mass ratio 1:1, the shell is composed by PLLA and the core by PLGA. It was also verified that the Meloxicam has a tendency to be distributed in the PLGA layer. Micro and nanoparticles were characterised in morphology, size, polymer cristalinity properties and drug distribution. Particles degradation studies was performed, where the particles in a PVA solution of pH 7,4 where placed in an incubator, during approximately 40 days, at 120rpm, and 37ºC, simulating, as much as possible, the human body environment. From these studies, the conclusion was that particles containing a PLGA shell and a PLLA core degrade more rapidly, due to the fact that PLLA is more hydrophobic than the PLGA. Concerning the drug release controlled results, done also for 40 and 50 days, they showed that the microspheres containing a shell of PLLA release more slowly than when the shell is composed of PLGA. This result was predictable, since the drug is solubilised in the PLGA polymer and so, in that case, the PLLA shell works like a barrier between the drug and the outer medium. Another positive aspect presented by this study is the lower initial burst effect, obtained when using double walled particles, which is one of the advantages of the same. In a second part of this investigation, the production of the nanospheres was the main goal, since it was not yet accomplished by other authors or investigators. After several studies, referring to the speed, time and type of agitation, as well as, the concentration and volume of the first aqueous solution of poly-vinyl-alcohol (PVA) during the process of solvent extraction/evaporation it was possible to obtain double walled nanospheres.(...)

Development of principle culture medium formulations for Saccharomyces cerevisiae

Saccharomyces cerevisiae as well as other microorganisms are frequently used in industry with the purpose of obtain different kind of products that can be applied in several areas (research investigation, pharmaceutical compounds, etc.). In order to obtain high yields for the desired product, it is necessary to make an adequate medium supplementation during the growth of the microorganisms. The higher yields are typically reached by using complex media, however the exact formulation of these media is not known. Moreover, it is difficult to control the exact composition of complex media, leading to batch-to-batch variations. So, to overcome this problem, some industries choose to use defined media, with a defined and known chemical composition. However these kind of media, many times, do not reach the same high yields that are obtained by using complex media. In order to obtain similar yield with defined media the addition of many different compounds has to be tested experimentally. Therefore, the industries use a set of empirical methods with which it is tried to formulate defined media that can reach the same high yields as complex media. In this thesis, a defined medium for Saccharomyces cerevisiae was developed using a rational design approach. In this approach a given metabolic network of Saccharomyces cerevisiae is divided into a several unique and not further decomposable sub networks of metabolic reactions that work coherently in steady state, so called elementary flux modes. The EFMtool algorithm was used in order to calculate the EFM’s for two Saccharomyces cerevisiae metabolic networks (amino acids supplemented metabolic network; amino acids non-supplemented metabolic network). For the supplemented metabolic network 1352172 EFM’s were calculated and then divided into: 1306854 EFM’s producing biomass, and 18582 EFM’s exclusively producing CO2 (cellular respiration). For the non-supplemented network 635 EFM’s were calculated and then divided into: 215 EFM’s producing biomass; 420 EFM’s producing exclusively CO2. The EFM’s of each group were normalized by the respective glucose consumption value. After that, the EFMs’ of the supplemented network were grouped again into: 30 clusters for the 1306854 EFMs producing biomass and, 20 clusters for the 18582 EFM’s producing CO2. For the non-supplemented metabolic network the respective EFM’s of each metabolic function were grouped into 10 clusters. After the clustering step, the concentrations of the other medium compounds were calculated by considering a reasonable glucose amount and by accounting for the proportionality between the compounds concentrations and the glucose ratios. The approach adopted/developed in this thesis may allow a faster and more economical way for media development.

Da assessoria ao jornalismo : análise do processo de produção de press release na era digital em Portugal

A presente dissertação pretende fazer a análise do processo de produção do press release na assessoria em Portugal, a eficácia dessa ferramenta de comunicação junto dos jornalistas e, por inerência, a evolução da figura do assessor enquanto profissional reconhecido junto da comunidade jornalística. São também objetivos, compreender a relevância de um press release, perceber se gera efeito, analisar a possível forma de melhorar esta ferramenta e, ainda, perceber se esta ferramenta sofreu algum tipo de adaptação à era digital. A investigação inicia-se com a incursão pelos contextos e história das áreas profissionais em estudo, a assessoria de comunicação em agências e o jornalismo em Portugal, no quadro da crise económica e financeira de 2008 a 2013. O enfoque deste estudo será o procedimento e a eficácia de um press release, no período considerado. A pós-produção desta ferramenta implica o contacto entre dois interlocutores, os profissionais de assessoria e os profissionais do jornalismo. Finda esta investigação com análise baseada em seis entrevistas semiestruturadas, divididas em categorias profissionais e setores de atividade: jornalistas, assessores, assessores ex-jornalistas, nas áreas de saúde e consumo. Deste estudo resulta que o press release, privilegiando-se a sua estrutura e conteúdo, é, como foi, uma ferramenta fundamental muitas vezes, e nos dias de hoje, no auxílio às negociações one to one.

Blocking tumor exosome release using nanovectorization systems

Exosomes are small membrane vesicles secreted by most cell types, either normal or malignant and are found in most body fluids such as saliva, plasma and breast milk. In the past decade, the interest in these vesicles has been growing more and more since it was found that besides their beneficial functions such as the removal of cellular debris and unnecessary proteins during cell maturation process, they can also interact with other cells and transfer information between them, thus helping diseases like cancer to progress. The present work intended to use gold nanoparticles as vehicles for gene silencing in an attempt to reduce the tumor-derived exosome secretion, regulated by Rab27a protein, and also aimed to compare the exosome secretion between two breast cell lines, MCF7 and MDA. Changes in RAB27A gene expression were measured by Real-time Quantitative PCR and it was revealed a decreased in RAB27A gene expression, as expected. Exosomes were isolated and purified by two different methods, ultracentrifugation and the commercial kit ExoQuick™ Solution, and further characterized using Western Blot analysis. ExoQuick™ Solution was proven to be the most efficient method for exosome isolation and it was revealed that MDA cells secrete more exosomes. Furthermore, the isolated MCF7-derived exosomes were placed together with a normal bronchial/tracheal epithelial cell line (BTEC) for an additional assay, which aimed to observe the uptake of exosomes by other cells and the exosomes’ capability of promoting cell-cell communication. This observation was made based on alterations in the expression levels of c-Myc and miR-21 genes and the fact that they both have an increased expression in BTEC cells incubated with tumor-derived exosomes when compared to control cells (without incubation with the exosomes) lead us to the conclusion that the exosome uptake and exchange of information between the exosomes and the normal cells did occurred.