This research focused on investigating and comparing the cytotoxicity and cellular uptake of RRR-alpha-tocopheryl polyethylene glycol succinate (TPGS, with that of alpha-tocopheryl succinate (α-TS). Both TPGS and α-TS are water-soluble forms of vitamin E with important clinical applications. Cytotoxicity assays with RAW 264.7 and LNCaP cells incubated overnight with TPGS or α-TS at concentrations ≥ 12.4 μM suggest that α-TS is more cytotoxic than TPGS. Macrophages were found to be more sensitive than LNCaP cells when treated with similar concentrations of α-TS. For both cell lines, most of the TPGS or α-TS taken up remained esterified after 24 hours. Our results suggest that cell death was due to TPGS and/or α-TS and not alpha-tocopherol. A para-hydroxyanilide of α-TS (p-HATS) that could be used to distinguish between cellular TPGS and α-TS was studied. It was found that p-HATS can be detected electrochemically and that it is hydrolyzed to α-TOH.

Dans le contexte de développement durable actuel, les matériaux biosourcés et biodégradables commencent à prendre une place importante d’un point de vue économique et écologique. L’objectif de remplacer les polyoléfines utilisées actuellement dans des domaines clés tels que l’emballage et l’automobile est cependant difficile car les matériaux « verts » sont rarement aussi performants. Ils présentent en général des lacunes au niveau de leurs propriétés mécaniques et sont sensibles à des dégradations qui limitent leur durée de vie. Le poly(butylène succinate) est un polyester connu depuis longtemps mais qui regagne de l’intérêt grâce à son potentiel biosourcé et biodégradable ainsi que ses propriétés mécaniques proches de celles des polyoléfines. Néanmoins, sa rigidité est encore trop faible et sa sensibilité importante à l’hydrolyse limite son utilisation dans le temps, même dans des conditions standards de température et d’humidité. Des améliorations de la stabilité du PBS sont possibles et plusieurs solutions ont été envisagées. L’intérêt s’est porté sur la modification de la balance hydrophile/hydrophobe du PBS par l’ajout d’un comonomère ramifié très hydrophobe, le Pripol 1009 ou possédant un cycle aromatique, l’acide téréphtalique. L’addition de charges dans le matériau peut également s’avérer efficace pour neutraliser les fins de chaines acides catalysant la dégradation, en utilisant du carbonate de calcium, ou pour limiter la perméabilité du matériau, par incorporation de talc. Dans l’optique de moduler les propriétés mécaniques du PBS, la voie de modification la plus fructueuse est le mélange par incorporation de charges minérales, comme le carbonate de calcium ou le talc, ou le mélange avec d’autres polyesters possédant une rigidité plus importante, tels que le poly(acide lactique) ou le poly(butylène téréphtalate). Les mélanges doivent présenter une bonne compatibilité et être mis en œuvre à des températures convenables pour limiter la dégradation du PBS et conserver la ductilité du matériau final, comme c’est le cas des mélanges PBS/PLA. Des combinaisons ont également été envisagées entre les différentes solutions efficaces pour améliorer à la fois les propriétés mécaniques et la stabilité face à l’hydrolyse chimique. / Within the frame of sustainable development, biobased and biodegradable polymers are going to play an important role according to economic and environmental perspectives. The polyolefins currently used in packaging and automotive industries will be replaced by biomaterials. The poly(butylene succinate), an “old” aliphatic polyester, has recently regained interest thanks to its biobased and biodegradable potential and mechanical properties similar to polyolefins. However, this polyester is very sensitive to degradation even at mild ambient conditions and, even though its flexibility is comparable to polyethylene or polypropylene, its modulus is too low. Some modifications of the chemical structure were considered to improve the long-term use of PBS. The synthesis of random copolymers using long-chain fatty acid Pripol 1009 or terephthalic acid allows to reduce significantly the hydrolysis rate and properties are maintained during a longer time. The incorporation of fillers like calcium carbonate and talc also enhance the PBS stability. The addition of calcium carbonate neutralizes carboxyl terminal group, which play an autocatalytic role in the hydrolytic degradation. High aspect ratio of talc increases the gas and liquid diffusion path, reducing permeability and providing better barrier properties to the material. In order to improve Young’s modulus, formulating blends with mineral fillers like calcium carbonate and talc, or with more rigid polyesters like polylactic acid or poly(butylene terephthalate) are efficient. The most interesting results are obtained by using calcium carbonate and polylactic acid, which allow the preservation of PBS’s flexibility. Processing parameters should be maximized to limit the degradation of PBS. Combinations of the most interesting solutions were investigated and lead to materials which fulfill the required specifications.

α-Tocopherol has a well known antioxidant action but is also considered likely to exert significant non-antioxidant effects in cell membranes. Due to its lipophilic nature α-tocopherol inserts into biological membranes where it influences the organisation of the component lipids and may therefore influence biophysical parameters including the membrane dipole potential. The dipole potential has been demonstrated to modulate the function of several membrane associated proteins and perturbation of this physical parameter by α-tocopherol may prove to be a significant non-antioxidant mechanism underlying several of its cellular effects. This study investigates the influence of α-tocopherol, and the non-antioxidant structural analogue α-tocopherol succinate, on the membrane dipole potential employing fluorescence spectroscopy techniques with the dipole potential sensitive probe Di-8-ANEPPS. Similar techniques are utilised with the surface potential sensitive probe FPE to investigate the interaction of the charged α-tocopherol succinate molecule with membranes. α-Tocopherol and α-tocopherol succinate are shown to decrease the dipole potential of egg-phosphatidylcholine vesicles and Jurkat T-lymphocyte cell membranes. This effect is placed in the context of the significant influence of membrane cholesterol oxidation on the dipole potential. 7-ketocholesterol, an oxidised form of cholesterol, significantly influences several cellular processes and is thought to mediate these effects, in part, through its physical effects on the cell membrane. These include altering the composition, and therefore biophysical properties, of rafts; structures which are considered to support the function of a host of membrane proteins. This study attempts to correlate the effect of 7-ketocholesterol on the dipole potential of microdomains with the influence of the oxysterol on the function of two microdomains associated receptors: P-glycoprotein and the insulin receptor, assessed by determining the extent of ligand binding using flow fluorocytometry. α-Tocopherol has been suggested to inhibit the raft-mediated effects of 7-ketocholesterol and the influence of this molecule on the effect of 7-ketocholesterol on the dipole potential are investigated as a potential mechanism for this inhibition. It is hypothesized that α-tocopherols may protect against the deleterious effects of cholesterol oxidation in cell membranes by excluding 7-ketocholesterol from specific microdomains, of which rafts are a subset, acting to preserve their dipole potential and maintain the function of the proteins they support. However, where significant cholesterol oxidation has previously occured the concurrent changes in the microdomain landscape of the membrane is suggested to prevent α-tocopherol succinate from eliciting this protective effect.

Adaptation to chronic hypoxia induces endogenous cardioprotection and increases the heart resistance to ischemia/reperfusion injury. The heart mitochondria, which produce reactive oxygen species (ROS) in addition to ATP, play an important role in these processes. During ischemia/reperfusion, ROS are produced in excessive amounts and damage the cells. However, in lower concentrations, ROS are involved in the signalling pathway of cardioprotection induced by adaptation to chronic hypoxia. In the heart, two mitochondrial subpopulations have been observed, subsarcolemmal mitochondria (SSM) and intermyofibrillar mitochondria (IMFM), which differ in cell localization as well as in morphological and biochemical properties. The aim of this work was to introduce the method of SSM and IMFM isolation in our laboratory and to analyse their antioxidative capacity after adaptation to chronic hypoxia. Adult male Wistar rats were kept either under normoxic conditions or exposed to intermittent high-altitude hypoxia (IHA; 7000 m, 5 days a week/8 hours a day, totally 25 exposures). Mitochondrial subpopulations were isolated from heart left ventricle and their functionality was verified by measuring oxygen consumption and enzyme activities. The IMFM had higher oxygen consumption in comparison with SSM and activities...

The aim of this thesis is to investigate molecular mechanism of reactive oxygen species production by flavin dehydrogenases mitochondrial glycerol-3-phosphate dehydrogenase (mGPDH) and succinate dehydrogenase (SDH). Together, they represent important source of reactive oxygen species in mammalian mitochondria, but the mechanism of electron leak is still poorly understood. Because mechanisms of reactive oxygen species production by other complexes of respiratory chain are better characterized, they can serve as case studies to get insight into mechanisms of reactive oxygen species by flavin dehydrogenases. Relevant knowledge is therefore summarized in the first part of the thesis. To study the production of reactive oxygen species by the isolated flavin dehydrogenases, we used brown adipose tissue mitochondria solubilized by digitonin as a model. Enzyme activity measurements, hydrogen peroxide production studies by Amplex UltraRed fluorescence and luminol luminescence revealed flavin as the most likely source of electron leak in SDH under in vivo conditions, while we propose coenzyme Q binding site as the site of reactive oxygen species production in the case of mGPDH. Distinct mechanism of this production by the two dehydrogenases is also apparent from induction of reactive oxygen species...

A precipitação e cristalização de proteínas são operações unitárias amplamente utilizadas na indústria biotecnológica. A formação de precipitado nestas operações é muitas vezes atingida por meio da adição de um sal que diminui a solubilidade da proteína, ou seja, o chamado fenômeno de saltingout. A principal informação para o estudo da precipitação e da cristalização é o diagrama de fases, em que a solubilidade da proteína é apresentada em função das variáveis do sistema (temperatura, pH, concentração de sal). Outra medida importante é o segundo coeficiente virial, que é uma medida indireta da interação intermolecular. A combinação de ambas as informações é fundamental para a identificação das condições em que fases cristalina ou amorfa são obtidas. O equilíbrio sólido-líquido da lisozima de clara de ovo de galinha em soluções aquosas contendo sais biodegradáveis (succinato de sódio, citrato de sódio e tartarato de sódio) foi estudado por meio da determinação da solubilidade da proteína e do segundo coeficiente virial em função da força iônica. Os dados experimentais do segundo coeficiente virial em função da força iônica foram medidos por meio de cromatografia de autointeração. Estes dados foram correlacionados por modelos apropriados. As informações de solubilidade e do segundo coeficiente virial da proteína foram combinadas para gerar condições experimentais adequadas na identificação da janela de cristalização, i.e., as condições em que uma fase cristalina é obtida. Uma matriz do tipo placa de cultivo foi usada para os experimentos de cristalização. Os resultados experimentais mostraram que as regiões das fases cristalina e amorfa no diagrama de fases são funções simultâneas do segundo coeficiente virial, como parâmetro termodinâmico, e da supersaturação, como parâmetro cinético. Os três sais foram capazes de induzir o efeito salting-out, sendo o succinato de sódio o sal mais eficaz na diminuição da solubilidade da lisozima, seguido pelo tartarato de sódio e pelo citrato de sódio. No caso da lisozima, cristais foram obtidos em soluções de succinato de sódio e tartarato de sódio, e as condições nas quais a fase cristalina é formada foram identificadas; no entanto, para o citrato de sódio somente fase amorfa foi observada. Em conjunto, estes resultados mostram que estes sais biodegradáveis são agentes precipitantes promissores. / The precipitation and crystallization of proteins are unit operations widely used in the biotechnology industry. The formation of a solid phase in these operations is often achieved by adding a salt that lowers the protein solubility, the so-called salting-out phenomenon. The key information to study precipitation or crystallization is the phase diagram, in which the solubility of a protein is presented as a function of system variables (temperature, pH, salt concentration). Another important parameter is the second virial coefficient, which is an indirect measure of intermolecular interactions. The combination of both kinds of information is fundamental to identify the conditions in which crystal and amorphous phases can be formed. The solid-liquid equilibrium of hen egg-white lysozyme in aqueous solutions containing biodegradable salts, viz., sodium citrate, sodium tartrate and sodium succinate, was studied by determining both the protein solubility and the second virial coefficient as a function of ionic strength. Experimental data for second viral coefficient as a function of ionic strength were obtained through self-iteration chromatography. These data were correlated by appropriate models. The protein solubility and the second virial coefficient information were combined to generate adequate experimental conditions to identify the so-called crystallization slot, i.e., the conditions in which a crystalline solid phase is obtained. A plate matrix disposition was used for the crystallization experiments. The experimental results show that the crystal and amorphous regions in the phase diagram depend simultaneously on the second virial coefficient, as a thermodynamic parameter, and on the supersaturation ratio, as a kinetic parameter. All these salts were able to induce salting-out, being sodium succinate the most effective one in decreasing lysozyme solubility, followed by sodium tartrate and sodium citrate. For lysozyme in sodium succinate and sodium tartrate solutions, the conditions for which a crystal phase is formed were identified; however, for sodium citrate only amorphous solid phases were observed.! Overall, these results show that these biodegradable salts are promising precipitating agents.

Applications of Hot Melt Extrusion (HME) in pharmaceuticals have become increasingly popular over the years but nonetheless a few obstacles still remain before wide scale implementation. In many instances these improvements are related to both processing and product performance. It is observed that HME process optimisation is majorly focused on the active pharmaceutical ingredient's (API) properties. Characterising polymeric properties for their suitability in HME should be equally studied since the impact of excipients on both product and process performance is just as vital. In this work, two well-established cellulose ester derivatives: Hydroxy Propyl Methyl Cellulose Acetate Succinate (HPMCAS) and Hydroxy Propyl Methyl Cellulose Phthalate (HPMCP) are studied for their HME suitability. Their thermal, thermodynamic, rheological, thermo-chemical and degradation kinetic properties were evaluated with model plasticisers and APIs. It was found the thermal properties of HPMCP are severely compromised whereas HPMCAS is more stable in the processing zone of 150 to 200 °C. Thermodynamic properties revealed that both polymers share an important solubility parameter range (20-30 MPa P1/2P) where the majority of plasticisers and BCS class II APIs lie. Thus, greater miscibility/solubility can be expected. Further, the processability of these two polymers investigated by rheometric measurements showed HPMCAS possesses better flow properties than HPMCP because HPMCP forms a weak network of chain interactions at a molecular level. However, adding plasticisers such as PEG and TEC the flow properties of HPMCP can be tailored. The study also showed that plasticisers have a major influence on thermo-chemical and kinetic properties of polymers. For instance, PEG reduced polymer degradation with reversal in kinetic parameters whereas blends of CA produced detrimental effects and increased polymer degradation with reduction in onset degradation temperatures. Further, both polymers are observed to be chemically reactive with the APIs containing free -OH, -SOR2RN- and -NH2 groups. Finally, these properties prove that suitability of HPMCP is highly debated for HME and demands great care in use while that of HPMCAS is relatively better than HPMCP in many instances.

Additives, incorporated in film coating formulations, and their process parameters are generally selected using a trial-and-error approach. However, coating problems and defects, especially those associated with aqueous coating systems, indicate the necessity of embracing a quality-by-design approach to identify the optimum coating parameters. In this study, the feasibility of using thermal and rheological measurements to help evaluate and design novel coating formulations has been investigated. Hydroxypropyl methylcellulose acetate succinate (HPMCAS), an enteric coating polymer, was used as the film forming polymer. Differential Scanning Calorimetry (DSC), Dynamic Mechanical Analysis (DMA), and Parallel Plate Shear Rheometery (PPSR) were used to evaluate the effect of different plasticisers on the performance of HPMCAS. The results illustrate that, for identical formulations, the DSC and DMA methods yielded up to 40% differences in glass transition temperature (Tg) values. Moreover, Tg measured using loss modulus signals were always 20-30 oC less than those measured using tan delta results in DMA testing. Absolute and relative Tg values can significantly vary depending on the geometry of the samples, clamp size, temperature ramping rate and the frequency of the oscillations. Complex viscosity data for different formulations demonstrated a variable shear thinning behaviour and a Tg independent ranking. It is, therefore, insufficient to rely purely on Tg values to determine the relative performance of additives. In addition, complex viscosity results, obtained using both the DMA and PPSR techniques at similar temperatures, are shown to be comparable. The results from both techniques were therefore used to produce continuous master curves for the HPMCAS formulations. Additionally, step strain tests showed that HPMCAS chains do not fully III disentangle after 105 seconds as predicted by the Maxwell model. Finally, in situ aqueous-based coating experiments proved that mixtures of triethyl acetyl citrate and acetylated monoglyceride (TEAC/AMG), even without cooling of the suspension, do not cause blocking of the spray nozzle whereas triethyl citrate (TEC) based formulae did. TEAC (alone or in a combination with AMG) exhibits superior wettability to HPMCAS than TEC/AMG formulations and can be used to enhance the efficiency and film quality of the dry coating process.

No description available.

A precipitação e cristalização de proteínas são operações unitárias amplamente utilizadas na indústria biotecnológica. A formação de precipitado nestas operações é muitas vezes atingida por meio da adição de um sal que diminui a solubilidade da proteína, ou seja, o chamado fenômeno de saltingout. A principal informação para o estudo da precipitação e da cristalização é o diagrama de fases, em que a solubilidade da proteína é apresentada em função das variáveis do sistema (temperatura, pH, concentração de sal). Outra medida importante é o segundo coeficiente virial, que é uma medida indireta da interação intermolecular. A combinação de ambas as informações é fundamental para a identificação das condições em que fases cristalina ou amorfa são obtidas. O equilíbrio sólido-líquido da lisozima de clara de ovo de galinha em soluções aquosas contendo sais biodegradáveis (succinato de sódio, citrato de sódio e tartarato de sódio) foi estudado por meio da determinação da solubilidade da proteína e do segundo coeficiente virial em função da força iônica. Os dados experimentais do segundo coeficiente virial em função da força iônica foram medidos por meio de cromatografia de autointeração. Estes dados foram correlacionados por modelos apropriados. As informações de solubilidade e do segundo coeficiente virial da proteína foram combinadas para gerar condições experimentais adequadas na identificação da janela de cristalização, i.e., as condições em que uma fase cristalina é obtida. Uma matriz do tipo placa de cultivo foi usada para os experimentos de cristalização. Os resultados experimentais mostraram que as regiões das fases cristalina e amorfa no diagrama de fases são funções simultâneas do segundo coeficiente virial, como parâmetro termodinâmico, e da supersaturação, como parâmetro cinético. Os três sais foram capazes de induzir o efeito salting-out, sendo o succinato de sódio o sal mais eficaz na diminuição da solubilidade da lisozima, seguido pelo tartarato de sódio e pelo citrato de sódio. No caso da lisozima, cristais foram obtidos em soluções de succinato de sódio e tartarato de sódio, e as condições nas quais a fase cristalina é formada foram identificadas; no entanto, para o citrato de sódio somente fase amorfa foi observada. Em conjunto, estes resultados mostram que estes sais biodegradáveis são agentes precipitantes promissores. / The precipitation and crystallization of proteins are unit operations widely used in the biotechnology industry. The formation of a solid phase in these operations is often achieved by adding a salt that lowers the protein solubility, the so-called salting-out phenomenon. The key information to study precipitation or crystallization is the phase diagram, in which the solubility of a protein is presented as a function of system variables (temperature, pH, salt concentration). Another important parameter is the second virial coefficient, which is an indirect measure of intermolecular interactions. The combination of both kinds of information is fundamental to identify the conditions in which crystal and amorphous phases can be formed. The solid-liquid equilibrium of hen egg-white lysozyme in aqueous solutions containing biodegradable salts, viz., sodium citrate, sodium tartrate and sodium succinate, was studied by determining both the protein solubility and the second virial coefficient as a function of ionic strength. Experimental data for second viral coefficient as a function of ionic strength were obtained through self-iteration chromatography. These data were correlated by appropriate models. The protein solubility and the second virial coefficient information were combined to generate adequate experimental conditions to identify the so-called crystallization slot, i.e., the conditions in which a crystalline solid phase is obtained. A plate matrix disposition was used for the crystallization experiments. The experimental results show that the crystal and amorphous regions in the phase diagram depend simultaneously on the second virial coefficient, as a thermodynamic parameter, and on the supersaturation ratio, as a kinetic parameter. All these salts were able to induce salting-out, being sodium succinate the most effective one in decreasing lysozyme solubility, followed by sodium tartrate and sodium citrate. For lysozyme in sodium succinate and sodium tartrate solutions, the conditions for which a crystal phase is formed were identified; however, for sodium citrate only amorphous solid phases were observed.! Overall, these results show that these biodegradable salts are promising precipitating agents.