Vaccination plays a very important part in daily life. It is essential to get vaccinated at an early age. The conventional parented method used is not always effective and not cost efficient. It requires qualified personnel and sterile conditions for administration of the vaccines. The aim of this study was to investigate the effect of chitosan, N-trimethyl chitosan chloride (TMC) and Emzaloid™ particles on the local and systemic immune response of mice after oral vaccination with Diphtheria toxoid (DT). The different formulations used were chitosan microparticles (± 10 µm), chitosan nanoparticles (± 400 nm), TMC microparticles (± 5 µm), Emzaloid microparticles (± 4 µm) and Emzaloid nanoparticles (± 500 nm). All of these formulations proved to be very good delivery systems and can entrap large amounts of the antigen. Balb/c mice were used to determine the local and systemic immune response of these formulations. The mice were vaccinated orally on three consecutive days in week 1 and 3 with 40 Lf DT per week with a total volume of 300 µl. Blood samples were taken from the mice and analysed for a systemic immune response (IgG). The same mice were used to determine the local immune response (IgA). Faeces were collected from each mouse on day 1, 3, 4, 6, 14 and 20 for analysis. An enzyme-linked immunosorbent assay (ELISA) was used to determine IgG and IgA titers. It can be concluded that chitosan nanoparticles was the only formulation with a higher response than that of the currently used vaccine. Emzaloid nanoparticles showed no significant difference in response when compared to the currently used vaccine. All the other formulations showed a much smaller response than that of the conventional method of vaccination. / Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2005.

Vaccination plays a very important part in daily life. It is essential to get vaccinated at an early age. The conventional parented method used is not always effective and not cost efficient. It requires qualified personnel and sterile conditions for administration of the vaccines. The aim of this study was to investigate the effect of chitosan, N-trimethyl chitosan chloride (TMC) and Emzaloid™ particles on the local and systemic immune response of mice after oral vaccination with Diphtheria toxoid (DT). The different formulations used were chitosan microparticles (± 10 µm), chitosan nanoparticles (± 400 nm), TMC microparticles (± 5 µm), Emzaloid microparticles (± 4 µm) and Emzaloid nanoparticles (± 500 nm). All of these formulations proved to be very good delivery systems and can entrap large amounts of the antigen. Balb/c mice were used to determine the local and systemic immune response of these formulations. The mice were vaccinated orally on three consecutive days in week 1 and 3 with 40 Lf DT per week with a total volume of 300 µl. Blood samples were taken from the mice and analysed for a systemic immune response (IgG). The same mice were used to determine the local immune response (IgA). Faeces were collected from each mouse on day 1, 3, 4, 6, 14 and 20 for analysis. An enzyme-linked immunosorbent assay (ELISA) was used to determine IgG and IgA titers. It can be concluded that chitosan nanoparticles was the only formulation with a higher response than that of the currently used vaccine. Emzaloid nanoparticles showed no significant difference in response when compared to the currently used vaccine. All the other formulations showed a much smaller response than that of the conventional method of vaccination. / Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2005.

Sickle cell disease is a hereditary blood disorder caused by a point mutation in the gene encoding hemoglobin. This mutation causes hemoglobin molecules to polymerize during de-oxygenation of erythrocytes producing rod-shaped polymers that bend and distort the red blood cell membrane, making it more rigid and “sickled”. This sickling causes red blood cells to lose their flexibility and ability to navigate small capillaries and also enhances the production of pro-inflammatory membrane-derived microparticles, leading to chronic inflammation and many complications such as peripheral artery disease, stroke, myocardial infarction, vasculitis and even death. Sphingolipids are a class of lipids containing a backbone of sphingoid bases and are integral components of erythrocyte and microparticle membranes. Many of these lipids are known to mediate biological processes, but their expression, distribution and orientation in erythrocytes during sickle cell disease has never been explored. Sphingomyelin, the most abundant sphingolipid in the red blood cell membrane is hydrolyzed by sphingomyelinase to produce ceramide, which has been shown to alter membrane dynamics and enhance microvessel formation. Additionally, ceramide can be further metabolized to form sphingosine and sphingosine 1-phosphate, which is a bioactive ligand for 5 known G-protein coupled receptors present on most blood and vascular cells that modulates cell motility, proliferation, migration and phenotype. Prior to this work, it was not understood how sphingolipid metabolism contributes to vascular inflammation in sickle cell disease. Together, this body of work has elucidated key enzymatic and lipidomic alterations in sphingolipid metabolism (i.e. the activation of acid sphingomyelinase on red blood cells) that result in the production of sphingolipid-rich erythrocyte-derived microparticles, which enhance inflammatory cell activation. Our work has elucidated novel pharmacological targets to reduce microparticle generation and subsequent vascular inflammation in sickle cell disease.

During embryo development, extracellular matrix (ECM) remodeling by matrix metalloproteinases (MMPs) and promotes downstream cell specifications. Pluripotent stem cell (PSC) aggregates can recapitulate various aspects of embryogenesis in vitro, and incorporation of biomaterial microparticles also provides an ideal platform to study cell-biomaterial interactions. Stem cell interactions with ECM-based biomaterials can impact tissue remodeling and differentiation propensity via modulation of MMP activity. This work investigated the MMP activity and subsequent mesenchymal differentiation of embryonic stem cell (ESC) aggregates with incorporated gelatin methacrylate (GMA) MPs with either low (20%) or high (90%) cross-linking densities, corresponding to faster or slower degradation rate, respectively. GMA MP incorporation increased total MMP and MMP-2 levels within 3D ESC aggregates in a substrate-dependent manner. GMA MP-incorporated aggregates also expressed higher levels of epithelial-to-mesenchymal transition markers and displayed enhanced mesenchymal morphogenesis than aggregates without MPs, and the MP-mediated effects were completely abrogated with MMP inhibitor treatment. This work predicts that control of proteolytic responses via introducing ECM-based MPs may offer a novel avenue to engineer the ECM microenvironment to modulate stem cell differentiation.

This work deals with a preparation of superparamagnetic nano- and microparticles with hydrophilic surfaces for bioapplications. The wok is divided into three parts in consonance with experimentally solved problem. First part describes a choice and an optimalization of synthesis of iron oxides nanoparticles with appropriate toxicological, morphological and physico-chemical properties, which surface can be post synthetically modified. Maghemite - γ-Fe2O3 particles were prepared by consequent oxidation of mangnetite - Fe3O4 as an initial substance for a preparation of materials for diagnostics and separations. A conventional alkaline coprecipitation method of magnetite preparation was modified to produce nanoparticles with narrowed size distribution without use of surfactants during their synthesis. Prepared maghemite nanoparticles were cca. 6 nm in diameter and their saturation magnetization was Ms ~ 70 A·m2 ·kg-1 . Such observed value is far higher in comparison with the state of the art and argues thus a proposition the Ms depends not on the preparation method, but only on the nanoparticles size. Maghemite nanoparticles morphology was evaluated by picture analysis of SEM and TEM micrographs, hydrodynamic size and zetapotential was measured with DLS. The structure of the maghemite was confirmed with...

Micropartículas de quitosana contendo DMAE foram preparadas utilizando o método de coacervação simples. A morfologia das partículas foi observada utilizando um Microscópio Eletrônico de Varredura. O tamanho das partículas foi medido por técnica de espalhamento de luz. As partículas obtidas possuem forma esférica e superfície irregular. Apresentaram uma distribuição de tamanho entre 419 e 528 nm. O pequeno índice de polidispersividade sugere que a distribuição do tamanho de partícula é homogêneo. O rendimento do processo e eficiência de encapsulação foram de 90% e 63%, respectivamente. O desenvolvimento de um novo sistema de liberação requer uma metodologia analítica para a identificação e quantificação do fármaco. Neste trabalho um método simples por cromatografia de fase reversa desenvolvido para a análise do DMAE é apresentado. O DMAE foi analisado utilizando- se uma coluna Merck RP 18 column 5 m (125 x 4 mm D.I.). A fase móvel foi tampão fosfato pH 7,4; acetonitrila (99,5:0,5 v/v) a um fluxo de 0,5 mL.min-1. O comprimento de onda de detecção foi de 208nm à temperatura ambiente (25oC). Linearidade foi obtida para uma faixa de 1,5x10-4 a 6,0x10-4 mol.L-1. Para os ensaios intra e inter dia o coeficiente de variação foi menor que 10%. Este novo método desenvolvido para a quantificação do DMAE apresentou sensibilidade e seletividade, demonstrando ser um método vantajoso e confiável para a realização dos estudos propostos. O método de encapsulação mostrou-se adequado para a preparação de micropartículas de quitosana contendo DMAE, porque apresentou alto rendimento e excelente eficiência de encapsulação. / Chitosan microparticles containing DMAE were prepared by using the simple coacervation method. A scanning electron microscopy (SEM) was used to observe microparticles morphology. Particle size was measured by laser light scattering. Particles presented spherical shape, irregular surface and size distribution between 419 and 528 nm. The small polydispersity index suggested that size distribution is homogeneous. Process yield and encapsulation efficiency were 90% and 63%, respectively. The development of a new drug delivery system requires analytical methods for identification and quantification of this drug. In the present study a simple reversed-phase high performance liquid chromatography developed for DMAE assay is presented. The DMAE was analyzed using/by means of a 5 ?m Merck RP 18 column (125 x 4 mm I.D.). The mobile phase was phosphate buffer pH 7,4; acetonitrile (99,5:0,5 v/v) at a flow rate of 0,5 mL.min-1. Detection was carried out at 208nm at room temperature (25oC). Linearity was obtained from 1,5x10-4 to 6,0x10-4 mol.L-1. Intra and inter- assay coefficient of variation was less than 10 %. This new method developed to assay DMAE presented sensibility and selectivity, providing a useful and reliable means to perform the proposed studies. The encapsulation method was proven suitable to the preparation of chitosan microparticles containing DMAE because it presented high yields and excellent encapsulation efficiency.

Novos sistemas de liberação de fármacos têm sido desenvolvidos com o intuito de melhorar a eficácia terapêutica de muitos fármacos no tratamento de diferentes patologias. Os sistemas microparticulados têm despertado grande interesse devido a suas propriedades, suas vantagens sobre os sistemas de distribuição convencional e, conseqüentemente, melhoria na adesão ao tratamento. As micropartículas de quitosana são exploradas como sistemas de liberação sítio-específico, devido suas propriedades biodegradáveis, biocompatíveis e mucoadesivas. Assim, visando o tratamento da tuberculose, que atualmente é causa prevalente de morte considerando as doenças infecciosas no mundo, neste trabalho micropartículas constituídas de quitosana foram desenvolvidas para veicular isoniazida. A isoniazida, uma hidrazida do ácido isonicotínico, é uma dos fármacos mais poderosos entre os fármacos de primeira linha utilizados no tratamento da tuberculose devido à sua alta eficiência, baixa dose e de baixo custo, tornando-o um bom candidato para o desenvolvimento de uma formulação de liberação sítio-específica. Por spray-drying, as micropartículas inertes e com isoniazida obtidas apresentaram-se esféricas e com ampla distribuição de tamanho de partículas que variou entre 5 e 12 m, potencial zeta positivo e elevada eficiência de encapsulação. O perfil liberação in vitro em tampão fosfato salino (PBS) pH 7,4 apresentou formação de produto de degradação, sendo confirmado por espectrometria de massa como o ácido isonicotínico. Entretanto, a isoniazida mostrou ter uma liberação rápida (1 hora) a partir das micropartículas de quitosana em meio aquoso. Dessa forma, as micropartículas de quitosana desenvolvidas constituem um sistema promissor para veiculação da isoniazida e administração pulmonar e, finalmente, melhoria da terapia da tuberculose. Ademais, adequações do método deverão ser testadas considerando a via de administração pretendida, bem como será necessária a realização de estudos in vivo para avaliar o comportamento dos sistemas desenvolvidos em condições fisiológicas reais e a biodisponibilidade e biodistribuição do fármaco no organismo. / New drug delivery systems have been developed in order to improve the therapeutic efficacy of many drugs in the treatment of different pathologies. Microparticles have attracted great interest due to its properties, its advantages over conventional delivery systems and, consequently, better treatment adherence. The chitosan-based microparticles are exploited as delivery systems for site-specific, because their biodegradable, biocompatible and mucoadhesive properties. Thus, chitosan-based microspheres containing isoniazid were developed to target the treatment of tuberculosis, which is currently prevalent cause of death considering infectious diseases in the world. Isoniazid, an isonicotinic acid hydrazide, is one of the most powerful drugs among first-line drugs used to treat tuberculosis due to its high efficiency, low dose and low cost, making it a good candidate for the development of site-specific delivery system. By spray-drying, the isoniazid-loaded and free microparticles obtained presented wide size particle distribution with particle size ranging between 5 and 12 m, spherical morphology, positive zeta potential and high encapsulation efficiency. The in vitro release profile in phosphate buffered saline (PBS) pH 7.4 showed the formation of degradation product, which was confirmed by mass spectrometry as isonicotinic acid. However, isoniazid release from the microparticles was fast (1 hour) in water. Thus, the developed chitosan microparticles are a promising system as a vehicle to isoniazid and pulmonary administration, and ultimately to improve the therapy of tuberculosis. Moreover, adaptations of the method should be tested considering the intended route of administration and it will be necessary to perform in vivo studies to evaluate the behavior of the systems developed under physiological conditions and actual bioavailability and biodistribution of the drug in the body.

The bottom-up assembly of functional devices requires novel building blocks to facilitate the incorporation of functional and structural hierarchy. Anisotropic building blocks can substantially broaden the creation of self-assembled devices with unique properties because of their morphological and/or chemical asymmetry. In this regard, we have created microspheres with one hemispherical face exposing silica and the other exposing gold. These microspheres were formed by the shadow deposition of gold onto silica microspheres. The two chemical surfaces allowed use of different surface reactions—silane chemistry for the silica side and thiol chemistry for the gold side—for immobilizing different oligonucleotide sequences on each of the two faces. These dual-functionalized microspheres were used in the selective orthogonal assembly of fluorophore-tagged target oligonucleotides. The DNA-directed assembly was confirmed by confocal microscopy of the microspheres. In essence, employing DNA as the linker molecule, these “Janus” particles can be assembled into various novel 1-D, 2-D, and 3-D structures, which are difficult to realize using symmetric building blocks. / Singapore-MIT Alliance (SMA)

The effect of decompression stress on circulating microparticles (MPs) from leukocytes (LMP), platelets (PMP), and endothelial cells (EMP) was investigated in fifteen male naval clearance divers. Venous blood samples were obtained 30 min before and 75 min after exposure to 81msw for 20 min. MPs were isolated by differential centrifugation and immunophenotyped using multiparameter flow cytometry. Venous gas emboli (VGE) were assessed using Doppler ultrasound every 40 min post-dive and subsequently graded using the Kisman Integrated Severity Score (mean KISS=21.92, indicating moderate level of VGE). Following the dive there was increased expression of CD41a, CD106, CD62P and CD31 on MPs, while CD45 and CD141 expression decreased. A positive correlation was found between KISS and CD41a expression post-dive. These results indicate that decompression stress activated platelets, producing PMPs and resulting in potential vascular disruption or injury. The inclusion of MP measures in future DCS-related research may help identify biomarkers of DCS.

The effect of decompression stress on circulating microparticles (MPs) from leukocytes (LMP), platelets (PMP), and endothelial cells (EMP) was investigated in fifteen male naval clearance divers. Venous blood samples were obtained 30 min before and 75 min after exposure to 81msw for 20 min. MPs were isolated by differential centrifugation and immunophenotyped using multiparameter flow cytometry. Venous gas emboli (VGE) were assessed using Doppler ultrasound every 40 min post-dive and subsequently graded using the Kisman Integrated Severity Score (mean KISS=21.92, indicating moderate level of VGE). Following the dive there was increased expression of CD41a, CD106, CD62P and CD31 on MPs, while CD45 and CD141 expression decreased. A positive correlation was found between KISS and CD41a expression post-dive. These results indicate that decompression stress activated platelets, producing PMPs and resulting in potential vascular disruption or injury. The inclusion of MP measures in future DCS-related research may help identify biomarkers of DCS.