A amilose, na presença de agentes complexantes adequados, tende a formar complexos de inclusão. Os mesmos são carregadores promissores, já que os ligantes aprisionados podem ser libertados posteriormente, o que conduz a muitas aplicações. Porém, a utilização da amilose nativa (AM) na formação dos complexos é limitada devido a sua baixa solubilidade em água. Sendo assim, estudos envolvendo a modificação desta molécula tornam-se promissores. O objetivo da presente tese foi preparar complexos de inclusão a partir da amilose nativa (AM) e modificada (AMA) usando como ligantes Rifampicina (RIF), Rodamina B (RB) e o Azul de Bromotimol (AB). Primeiramente, dentre os métodos existentes para modificação, escolheu-se a acetilação. O grau de acetilação foi investigado e a estrutura da amilose foi caracterizada por meio de FTIR, MEV, TGA e DSC. Quando comparada à AM, AMA apresentou maior solubilidade em água. A presença das bandas de absorção no FTIR à 1727, 1240 e 1122 cm-1, confirmaram a acetilação. Os resultados de MEV sugeriram que a superfície lisa da AM foi transformada em uma superfície mais áspera em AMA e as análises de TGA e DSC mostraram uma estrutura instável para a mesma. Após esta etapa os complexos foram produzidos e a influência da temperatura e dos ligantes foi avaliada através da caracterização físico-química. UV-vis, DLS, PZ e MEV foram as técnicas usadas neste processo. As análises de UV confirmaram a formação dos complexos e aqueles desenvolvidos na temperatura de 65°C foram mais eficientes. Dentre estes destacam-se aqueles complexos preparados com RIF. O diâmetro hidrodinâmico médio (dh) dos complexos medidos por DLS variou entre 70 e 100 nm, indicando que os mesmos podem ser utilizados em sistema de liberação controlada. Comparando o dh da AM e AMA, observou-se que os tamanhos são maiores após a complexação, o que pode indicar que para AMA ou a interação forma complexos mais compactos, ou os ligantes não interagiram com a AMA. Análise de PZ mostrou que os complexos AM-RB e AM-AB apresentam alta estabilidade (PZ < -30 mV) e que os demais complexos apresentam valores de PZ próximos da neutralidade, o que pode melhorar a adsorção dos mesmos em sistemas biológicos. Os complexos AMA-RB e AMA-AB não formaram complexos no estado sólido e os demais formaram uma estrutura amorfa após precipitação. Em conclusão, este estudo levou ao desenvolvimento de um método eficaz para a preparação de complexos de inclusão de amilose. / In the presence of suitable complexing agents, amylose tends to form inclusion complexes. This polymer is considered a promisor carrier since the ligands confined in its chains can be released later, leading to various applications. However, the use of native amylose (AM) in complexes formation is restricted due to its low water solubility. Therefore, studies regarding amylose modification become promising. The aim of this thesis was preparing inclusion complexes made of native (AM) and modified (AMA) amylose using rifampicin (RIF), rhodamine B (RB), and bromothymol blue (AB) as ligands. At first, acetylation was the chosen modification among the modified methods described in the literature. The acetylation degree was investigated and the modified macromolecule was characterized using FTIR, SEM, TGA, and DSC analysis. Compared with AM, AMA presented increased water solubility. The presence of absorption bands at 1727, 1240, and 1121 cm-1 confirmed the acetylation. SEM images suggested that the smooth surface of AM was turned into a rougher surface in AMA, while TGA and DSC results showed a less stable structure for AMA. After this step, the complexes were prepared and the influence of the temperature and ligand type was evaluated through physicochemical characterization. UV-Vis, DLS, PZ, and SEM were the techniques used in this process. UV-Vis analysis confirmed complexes formation, revealing that the ones prepared at 65°C were more efficient. Among those, complexes prepared with RIF stand out. The average hydrodynamic diameter (dh) of the complexes measured by DLS ranged from 70 to 100 nm, indicating that these complexes can be used in controlled release systems. Comparing the dh of AM and AMA, it was observed that the sizes were larger after complexation, which may indicate more compact complexes or no interaction between AMA and ligands. ZP results showed that AM-RB and AM-AB complexes presented high stability (PZ < -30 mV), while the others presented PZ values near neutrality, which can increase their adsorption in biological systems. AMA-RB and AMA-AB did not form complexes in solid state, while the others formed an amorphous structure after precipitation. In conclusion, this study leaded to an effective method development for the amylose inclusion complexes preparation.

Orientador: Valdecir Farias Ximenes / Banca: Rosângela Gonçalves Peccinini Machado / Banca: Regina Lúcia Moraes Moreau / Resumo: Este trabalho teve como objetivo estudar os efeitos da rifampicina (RMP) sobre os parâmetros farmacocinéticos da isoniazida (INH), sobre a produção de seus metabólitos e sobre a sua hepatotoxicidade. Foram utilizados 140 ratos (Wistar, machos, peso médio 250g) que receberam, por gavagem, durante 21 dias: Grupo I - água estéril (n = 20); Grupo II - INH (100mg/Kg) (n = 50); Grupo III - RMP (100mg/Kg) (n = 20) e Grupo IV- INH (100mg/Kg) + RMP (100mg/Kg) ( n= 50). Anteriormente ao início do experimento, o sangue de todos os animais foi coletado pela cauda para determinação da atividade sérica de AST e ALT, cujos valores foram considerados como basais. No 21o do experimento os animais foram sacrificados por decapitação e o material biológico obtido foi utilizado para a determinação da atividade de AST e ALT e para a análise dos parâmetros farmacocinéticos da INH nos grupos II e IV. A cinética da isoniazida e de seus metabólitos foi investigada com base na relação concentração plasmática x tempo a partir de amostras seriadas de sangue em 10 tempos diferentes (0; 15þ; 30þ; 45þ; 60þ; 1,5h; 3h; 6h; 12h; e 24h); para cada tempo de coleta foram empregados 5 ratos (5 replicatas). As amostras de soro foram desproteinizadas com ácido tricloroacético 10%, derivatizeda com cinamaldeído 1% e analisada por HPLC...(Resumo completo, clicar acesso eletrônico abaixo). / Abstract: The aim of the present study was to evaluate the hepatotoxicity, pharmacokinetic parameters and biotransformation of isoniazid when rats were treated with isoniazid (INH); rifampicin (RMP); and INH + RMP. Daily doses of the tuberculostatic drugs were administrated intragastrically to the animals (Wistar rats) for one period of 21 days as follow: sterile water (group I, control); INH (100mg/Kg) (group II), RMP (100mg/Kg) (group III); INH (100mg/Kg) + RMP (100mg/Kg) (group IV). The serum levels of the biomarkers aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were determined before the administration of the drugs (basal) and after the 21 days treatments. On day 21, blood samples were obtained before and 15þ; 30þ; 45þ; 60þ; 1,5; 3h; 6h; 12h and 24 hours after the dose. (five animals for each point). The blood samples were deproteinized with 10% trichloroacetic acid, derivatized by 1% cinnamaldehyde and analyzed by liquid chromatograph. For the determination of the acetylated metabolites acetylisoniazid (AcINH) and acetylhydrazine (AcHz) a previous hydrolysis with 6 M hydrochloride acid was performed. The results are presented as mean and SEM. The pharmacokinetic parameter of the INH and its metabolites AcINH and hydrazine (Hz) were compared between the groups (p < 0.05, Student t-test)...(Complete abstract, click electronic address below). / Mestre

A amilose, na presença de agentes complexantes adequados, tende a formar complexos de inclusão. Os mesmos são carregadores promissores, já que os ligantes aprisionados podem ser libertados posteriormente, o que conduz a muitas aplicações. Porém, a utilização da amilose nativa (AM) na formação dos complexos é limitada devido a sua baixa solubilidade em água. Sendo assim, estudos envolvendo a modificação desta molécula tornam-se promissores. O objetivo da presente tese foi preparar complexos de inclusão a partir da amilose nativa (AM) e modificada (AMA) usando como ligantes Rifampicina (RIF), Rodamina B (RB) e o Azul de Bromotimol (AB). Primeiramente, dentre os métodos existentes para modificação, escolheu-se a acetilação. O grau de acetilação foi investigado e a estrutura da amilose foi caracterizada por meio de FTIR, MEV, TGA e DSC. Quando comparada à AM, AMA apresentou maior solubilidade em água. A presença das bandas de absorção no FTIR à 1727, 1240 e 1122 cm-1, confirmaram a acetilação. Os resultados de MEV sugeriram que a superfície lisa da AM foi transformada em uma superfície mais áspera em AMA e as análises de TGA e DSC mostraram uma estrutura instável para a mesma. Após esta etapa os complexos foram produzidos e a influência da temperatura e dos ligantes foi avaliada através da caracterização físico-química. UV-vis, DLS, PZ e MEV foram as técnicas usadas neste processo. As análises de UV confirmaram a formação dos complexos e aqueles desenvolvidos na temperatura de 65°C foram mais eficientes. Dentre estes destacam-se aqueles complexos preparados com RIF. O diâmetro hidrodinâmico médio (dh) dos complexos medidos por DLS variou entre 70 e 100 nm, indicando que os mesmos podem ser utilizados em sistema de liberação controlada. Comparando o dh da AM e AMA, observou-se que os tamanhos são maiores após a complexação, o que pode indicar que para AMA ou a interação forma complexos mais compactos, ou os ligantes não interagiram com a AMA. Análise de PZ mostrou que os complexos AM-RB e AM-AB apresentam alta estabilidade (PZ < -30 mV) e que os demais complexos apresentam valores de PZ próximos da neutralidade, o que pode melhorar a adsorção dos mesmos em sistemas biológicos. Os complexos AMA-RB e AMA-AB não formaram complexos no estado sólido e os demais formaram uma estrutura amorfa após precipitação. Em conclusão, este estudo levou ao desenvolvimento de um método eficaz para a preparação de complexos de inclusão de amilose. / In the presence of suitable complexing agents, amylose tends to form inclusion complexes. This polymer is considered a promisor carrier since the ligands confined in its chains can be released later, leading to various applications. However, the use of native amylose (AM) in complexes formation is restricted due to its low water solubility. Therefore, studies regarding amylose modification become promising. The aim of this thesis was preparing inclusion complexes made of native (AM) and modified (AMA) amylose using rifampicin (RIF), rhodamine B (RB), and bromothymol blue (AB) as ligands. At first, acetylation was the chosen modification among the modified methods described in the literature. The acetylation degree was investigated and the modified macromolecule was characterized using FTIR, SEM, TGA, and DSC analysis. Compared with AM, AMA presented increased water solubility. The presence of absorption bands at 1727, 1240, and 1121 cm-1 confirmed the acetylation. SEM images suggested that the smooth surface of AM was turned into a rougher surface in AMA, while TGA and DSC results showed a less stable structure for AMA. After this step, the complexes were prepared and the influence of the temperature and ligand type was evaluated through physicochemical characterization. UV-Vis, DLS, PZ, and SEM were the techniques used in this process. UV-Vis analysis confirmed complexes formation, revealing that the ones prepared at 65°C were more efficient. Among those, complexes prepared with RIF stand out. The average hydrodynamic diameter (dh) of the complexes measured by DLS ranged from 70 to 100 nm, indicating that these complexes can be used in controlled release systems. Comparing the dh of AM and AMA, it was observed that the sizes were larger after complexation, which may indicate more compact complexes or no interaction between AMA and ligands. ZP results showed that AM-RB and AM-AB complexes presented high stability (PZ < -30 mV), while the others presented PZ values near neutrality, which can increase their adsorption in biological systems. AMA-RB and AMA-AB did not form complexes in solid state, while the others formed an amorphous structure after precipitation. In conclusion, this study leaded to an effective method development for the amylose inclusion complexes preparation.

The drugs are considered pollutant emergent, a time that its presence in aquatic environments is each bigger time. This research has as objective to develop electrochemical methodologies that allow to monitor and to degrade the rifampicin in water of public supplying. In the stage of monitor a voltammetric method was developed using a biosensor of chitosan for rifampicin analysis. Parallel to this study was development and optimized a electrodegradation method using electrode of the type Dimensionally Stable Anodes DSA® (Ti/RuO2 - TiO2). The voltammetric behavior of the rifampicin in the biosensor of chitosan using itself it technique of differential pulse voltammetry on indicates the presence of a peak of irreversible oxidation in 0,492 V (versus Ag/AgCl) corresponding the electronic transference of two electrons for molecule. The study of the effect of the speed of sweepings and pH of the way they indicate to be about a electrodic process with diffusional and dependent control of pH. The voltammetric method of determination of the rifampicin presents a recovery of 110% for 1,0 10-5 mol.L-1. From the experiments of electrodegradation of the rifampicin under electrodes DSA® it was evaluated influence of the chain density, getting as excellent value 120 mA cm-2. For the different assays in pHs, it was possible to observe that the half reacionais with pHs 8 and 3 favor the electrolysis of the rifampicin for this type of reactor in NaCl as electrolyte has supported with electrodegradation of 100% of organic molecules of the rifampicina in only 72s and 84s in these half reationais. Being that in only 48s the molecule of the rifampicin total is degraded, in turn, its by-products have a time of 24 electrodegradation of and 36s. The application in water of supplying with accompaniment of the loss of color was made through spectrophotometry in the region of UV-Visible, in =370 nm. It approximately had a complete removal of the color and total reduction of the absorbance in the electrodegradation of the rifampicin in 96 and 84s of electrolysis in the two studied ways, pH 3 and 8 respectively. / Os fármacos são considerados poluentes emergentes, uma vez que sua presença em ambientes aquáticos é cada vez maior. Essa pesquisa tem como objetivo desenvolver metodologias eletroquímicas que permitam monitorar e degradar a rifampicina em água de abastecimento público. Na etapa de monitoramento foi desenvolvido um método voltamétrico empregando um biossensor de quitosana para análise de rifampicina. Paralelo a este estudo foi desenvolvimento e otimizado um método de eletrodegradação empregando eletrodo do tipo Ânodo Dimensionalmente Estável ADE® (Ti/RuO2 TiO2). O comportamento voltamétrico da rifampicina no biossensor de quitosana empregando-se a técnica de voltametria de pulso diferencial evidencia a presença de um pico de oxidação irreversível em 0,492 V (vs Ag/AgCl) correspondente a transferência eletrônica de dois elétrons por molécula. O estudo do efeito da velocidade de varredura e do pH do meio indicaram tratar-se de um processo eletródico com controle difusional e dependente do pH. O método voltamétrico de determinação da rifampicina apresentou uma recuperação de 110% para 1,0 10-5 mol.L-1. Nos experimentos de eletrodegradação da rifampicina sob eletrodos ADE® Foi avaliada a influência da densidade de corrente, obtendo como valor ótimo 120 mA cm-2. Para os ensaios em diferentes valores de pH, foi possível observar que os meios reacionais com pH 8 e 3 favoreceram a eletrólise da rifampicina para este tipo de reator em NaCl como eletrólito suporte com eletrodegradação de 100% das moléculas orgânicas da rifampicina em apenas 72s e 84s nestes meios racionais. Sendo que em apenas 48s a molécula da rifampicina foi totalmente degradada, por sua vez, seus subprodutos tiveram um tempo de eletrodegradação de 24 e 36s. A aplicação em água de abastecimento com acompanhamento da perda de cor foi feita por espectrofotometria na região do UV-Vis, em =370 nm. Houve uma remoção completa da cor e redução total da absorbância na eletrodegradação da rifampicina em aproximadamente 96 e 84s de eletrólise nos dois meios estudados, pH 3 e 8 respectivamente.

Este trabalho descreve o desenvolvimento de novos métodos cromatográficos em conjunto com técnicas de microextração para determinação de parabenos em produtos cosméticos e leite humano e rifampicina em amostras de plasma. Destaca-se também o desenvolvimento da fase extratora molecularmente impressa para extração em fase sólida (SPE) de parabenos em amostras de leite humano. O capítulo I descreve a avaliação da extração sortiva em barra de agitação (SBSE) em conjunto com a cromatografia líquida (LC) com detecção espectrofotométrica (LC-UV) para análises de parabenos em produtos cosméticos para fins de controle de qualidade. As variáveis do processo de extração SBSE, tais como tempo e temperatura de extração, pH e força iônica da amostra e condições de dessorção foram otimizadas para aumentar a sensibilidade analítica do método e diminuir o tempo de análise. O método SBSE/LC-UV desenvolvido apresentou faixa linear correspondente ao limite de quantificação (LQ) a 2,50 µg. mg-1 e coeficientes de determinação maiores que 0,993. Os valores de coeficiente de variação resultantes das análises de precisão interensaios foram 5,0% para todos os parabenos analisados e a exatidão variou de 90 a 100%. O método desenvolvido foi aplicado para análises de amostras de produtos cosméticos comerciais (cremes hidratantes, antitranspirantes em creme e filtros solares). O método SBSE/LC-UV padronizado e validado resultou nas seguintes vantagens em relação aos métodos convencionais: simplicidade no preparo de amostra, redução do volume de solvente orgânico e da quantidade de amostra utilizada. O capítulo II descreve o desenvolvimento da fase extratora molecularmente impressa para SPE de parabenos em amostras de leite humano e análises por cromatografia em fase líquida com detecção espectrofotométrica (SPE/LC-UV). Os polímeros de impressão molecular, materiais sintéticos baseados em sistemas biomiméticos, foram sintetizados através do processo sol-gel tendo como molde, o benzilparabeno. Após a remoção do molde, cavidades seletivas (sítios de reconhecimento molecular) ao benzilparabeno e análogos estruturais foram reveladas. O método SPE/LC desenvolvido apresentou faixa linear correspondente ao limite de quantificação a 150 ng. mL-1 e coeficientes de determinação 0,992. Os valores de coeficiente de variação resultantes das análises de precisão interensaios foram 13% e a exatidão variou de 86 a 117%. A aplicabilidade do método SPE/LC foi demonstrada através das análises de amostras de leite humano de lactantes. Segundo os parâmetros de validação analítica avaliados, o método padronizado SPE/LC-UV é adequado para análises de parabenos em amostras de leite humano. O capítulo III descreve a avaliação da microextraçao em fase sólida no capilar (in-tube SPME) acoplada à cromatografia líquida (LC) com detecção espectrofotométrica (LC-UV) para análises de rifampicina em amostras de plasma para fins de monitorização terapêutica. As variáveis in-tube SPME, tais como fase estacionária da coluna capilar, volume de amostra, ciclos aspirar/dispensar e vazão foram otimizadas visando obter maior eficiência do processo. O método in-tube SPME/LC desenvolvido apresentou faixa linear correspondente ao limite de quantificação (0,1 µg. mL-1) a 100 µg. mL-1 e coeficientes de determinação igual a 0,998. Os valores de coeficiente de variação resultantes das análises de precisão interensaios foram 2% e a exatidão variou de 83 a 92%. A aplicabilidade do método in-tube SPME/LC, padronizado e validado foi comprovada através das análises de amostras de plasma de pacientes em tratamento com rifampicina. O método in-tube SPME/LC resultou na automação do processo de análise, maior precisão analítica, menor tempo de análise, menor volume de solvente orgânico e de fluido biológico. / This work describes the development of chromatographic methods in combination with microextraction techniques for the determination of parabens in cosmetic and human milk samples and of rifampicin in plasma samples. The design of a molecularly imprinted extraction phase for the solid-phase extraction (SPE) analysis of parabens in human milk samples is also reported. Chapter I describes the evaluation of the stir bar sorptive extraction (SBSE) in combination with liquid chromatography (LC) with UV detection (LC-UV) for the analysis of parabens in cosmetics aiming at quality control. The SBSE variables such as extraction time, temperature, pH of the matrix, ionic strength, and desorption conditions have been optimized, in order to establish the parabens partition equilibrium in a short analysis time. The linear range of the SBSE/LC method lay from LOQ to 2.5 µg. mg-1, with a coefficient of determination higher than 0.993. The interday precision of the SBSE/LC method presented a coefficient of variation lower than 5%, and the accuracy ranged from 90 to 99%. The effectiveness of the proposed method for the analysis of commercial cosmetic products such as body creams, antiperspirant creams, and sunscreens has been proven. Therefore, the combination of SBSE with liquid desorption, followed by LC analysis, provides a simple, and selective tool for the analysis of parabens in cosmetic products using minimized volume of organic solvent as well as very small amount of the target sample. Chapter II presents the development of a molecularly imprinted extraction phase for the SPE analysis of parabens in human milk samples. The molecularly imprinted polymers, which are synthetic materials based on biomimetic systems, were synthesized by the sol-gel technology in the presence of the template (benzilparaben). After template removal, selective cavities (benzilparaben) were revealed for parabens selective extraction. The linear range of the SPE/LC method lay from LOQ to 150 ng. mL-1, with a coefficient of determination higher than 0.992. The interday precision of the SPE/LC method presented a coefficient of variation lower than 13%, and the accuracy ranged from 86 to 117%. The effectiveness of the proposed method for the analysis of human milk samples has been demonstrated. According to the evaluated analytical validation parameters, the SPE/LC-UV method is suitable for the analysis of parabens in human milk samples. Chapter III depicts the evaluation of solid-phase microextraction in the capillary (in-tube SPME) in combination with liquid chromatography (LC) with UV detection (LC-UV) for the analysis of rifampicin in plasma samples for therapeutic drug monitoring. The in-tube SPME/LC method was linear over the LOQ (0.1) to 100 g. mL-1 range, with a linear coefficient value (r2) of 0.998. The inter-assay precision presented coefficient of variation 2%, and the accuracy ranged from 83 to 92%. The effectiveness and practicability of the proposed method have been by analysis of plasma samples from ageing patients undergoing therapy with rifampicin. The in-tube SPME/LC method allowed for automated continuous sample preparation (extraction, concentration, desorption, and injection of analytes) and minimized the analysis time as well as the volumes of organic solvent and biological fluid.

Philosophiae Doctor - PhD / Tuberculosis (TB) remains a global epidemic despite the fact that treatment has been available since the 1950’s. This disease is highly contagious and spreads via transmission of the Mycobacterium Tuberculosis (MTB) tubercle via coughing, sneezing and spitting. The disease has various side effects including weight loss, fatigue and even death. To date no cure has been found for TB and thus optimisation of treatment is a constant focus in health related research. TB is highly prevalent in South Africa due to the increased level of patients who are co-infected with HIV. Treatment for TB consists of first line drugs including isoniazid (INH), ethambutol (ETH), pyrazinamide (PYR) and rifampicin (RIF). These drugs are highly effective but also produce many adverse drug reactions (ADR’s) over the 6-month course of treatment. These reactions lead to patients not completing the course, losing quality of life and ultimately adding to the development of drug resistant strains of TB. A method of minimising these ADR’s is the development of a phenotype sensor, which is able to determine the metabolic profile of patients. Metabolic profiles play a huge role in the efficacy of treatment by tailoring treatment in order for patients to stay within the therapeutic range of treatment. This would in turn minimise both toxicity and ineffective treatment. Various methods for the quantification of drugs have been developed such as high performance liquid chromatography (HPLC), mass spectrometry (MS) and ultra-violet visible spectroscopy (UV-vis). / 2023-12-01

Philosophiae Doctor - PhD / Introduction: Tuberculosis (TB) is a serious bacterial infections caused by the Mycobacterium Tuberculosis (MTB) organism affecting mainly the lungs. Occasionally, MTB bacilli may be transported out of the pulmonary region and infect peripheral organs causing extra-pulmonary tuberculosis. Many therapeutic agents were developed over the years to combat TB, however the rapid emergence of resistant strains hampered their use. Furthermore, most of the current anti-TB drugs experience many challenges, which can be summarized in treatment regimen factors, drug-drug interactions, and physicochemical characteristics factors (such as hydrophobicity and low permeability into alveolar macrophages). These challenges have a significant role in treatment failure and the emergence of resistant TB. Due to the lack of newly discovered anti-TB drugs, and the absence of effective vaccines, many scientists have suggested the use of novel modalities for the current anti-TB drugs to enhance their efficacy and overcome some of the drawbacks. One of these modalities is nanotechnology-based drug delivery systems. Most of the anti-TB drugs experience low drug distribution to the lung and particularly alveolar macrophages within which the MTB resides, leading to treatment failure. Employing nanoparticles as drug delivery systems can have a significant impact on improving the pharmacokinetic profile of anti-TB drugs, the feasibility of different routes of administration, enhancing drug permeability, controlled/sustained drug release, and targeting specific disease sites. Collectively, these impacts will aid in enhancing drug concentration at the site of infection and reduce dosing and regimen duration. Dendrimers, such as polyamidoamine (PAMAM) dendrimers, are synthetic polymeric nanoparticles that have unique features that afford a dendrimer-conjugate complex the possibility to overcome the most common hurdles associated with drug delivery and treatment of diseases. Obstacles associated with solubility, permeability, inadequate biodistribution associated side effects may be enhanced. Manipulating the outermost surface functional groups with various ligands and polymers, will enhance the dendrimer properties and targeting potential. Aim: This study aims to develop a novel pulmonary delivery system for the anti-TB drug rifampicin using surface-modified G4 PAMAM dendrimer nanoparticles (polyethylene glycol (PEG) or mannose moieties), to improve drug solubility, prolong-release, enhance permeability into the macrophages, and decrease the toxicity of the drug-dendrimer conjugates. Methods: PAMAM dendrimers having increasing concentrations of poly(ethylene glycol) (PEG) 2 kDa or mannose residues were synthesized. The 4-nitrophenyl chloroformate was used as an activator in the case of PEG functionalization, while for the mannose conjugation the 4-isothiocyanatophenyl alpha-D-mannopyranoside (4-ICPMP) directly interacted with the primary amines of the dendrimer. The conjugated PEG polymers and mannose moieties on the dendrimer periphery were confirmed using FTIR and 1H NMR analytical techniques. Thereafter, rifampicin was loaded into the native and surface-modified dendrimers via a simple dissolution solvent evaporation method. Rifampicin-loaded dendrimers were then characterized using several analytical techniques namely; FTIR, DSC, NMR, SEM, and DLS. The polymer encapsulation efficiency (EE%) and percentage of drug loading (DL%) were determined directly using a validated HPLC method. In vitro drug release was studied at pH 7.4 and pH 4.5. The MTT technique was used to assess the cytotoxicity of the dendrimer formulations against raw 264.7 cell lines. Finally, the uptake of dendrimer nanoparticles by raw macrophages was studied using a flow cytometer and fluorescence microscopy techniques. Results: The percentage coverage of 4.0 G PAMAM dendrimer peripheral with PEG was achieved in a range of 38% - 100%, while for mannose moieties was from 44% - 100%. The EE% of unmodified dendrimer was 7.5% (w/w). The EE% of PEGylated dendrimers ranged from 65.0% - 78.75% (w/w), whereas for mannosylated dendrimers was from 43.43% - 57.91% (w/w). The size of the unloaded dendrimer nanoparticles was less than 25 nm, a gradual increase in the size after drug conjugation followed. The zeta potential of dendrimers was positive with values greater than 12 mV, the nanoparticle's zeta potential decreased upon increasing the density of PEG/mannose and after drug loading. FTIR and NMR data showed that rifampicin molecules were conjugated to the dendrimer at three sites; at the surface amines via electrostatic linkages, within the PEG/mannose, and into the dendrimer interior. SEM images of dendrimer nanoparticles confirmed the spherical shape of particles, and DSC data verified drug entrapment. Drug release was found to be affected by the pH of the medium and the extent of dendrimer functionalization. At the physiologic pH, surface-modified dendrimers showed a slower release rate compared to the unmodified dendrimer and free drug. Among surface-modified dendrimers, the release rate was inversely associated with the density of PEG/mannose molecules. At pH 4.5, a relatively higher drug release from all formulations was observed which suggests a burst release inside the alveolar macrophages. Toxicity studies showed that the unmodified dendrimer experienced time-dependent and concentration-dependent cytotoxicity against raw 264.7 cells. The toxicity gradually decreased upon increasing the density of PEG/mannose, and negligible toxicity was detected for formulations with 100% functionalization. Dendrimer nanoparticles were successfully internalized into raw cells after 24 hrs of incubation. The order of nanoparticles permeability was PEG 100% < PEG 85% < PEG 70% < PEG 49% < PEG 38% < unmodified dendrimer < mannose 44% < mannose 69% < mannose 93% < mannose 100%. The significant increase in the uptake of mannosylated dendrimers was due to the interaction with lectin receptors at the surface of raw macrophages, whereas the lower internalization of PEGylated dendrimers was due to the shielding of the surface positive charges. Conclusion: The in-vitro and ex-vivo data studies suggested that the developed novel surface-modified G4 PAMAM dendrimers are suitable drug carriers in terms of biocompatibility, release behaviour, and site-specific delivery of the anti-TB drug rifampicin.

V průběhu projektu byly vyvinuty unikátní kolagenní pěny z kolagenu získaného z kůže sladkovodní ryby (kapr obecný, Cyprinus carpio). Pomocí síťování karbodiimidem byl překonán problém s nestabilitou kolagenní matrix z kolagenu získávaného z chladnokrevných živočichů při tělesné teplotě savců. Následně byly pěny impregnovány antibiotiky (gentamicin a vankomycin) a opětovně lyofilizovány, což je postup, který zajišťuje požadovanou koncentraci antibiotika bez rizika následného vymytí při dalších technologických krocích. Uvedený produkt je, na rozdíl od přípravků z nesíťovanéhokolagenu, stabilní i při sterilizaci gamma zářením. Finální sterilizovaný produkt byl testován in vivo na potkaním modelu infikované rány. Byla prokázána efektivita v léčbě potenciálně letální infekce Pseudomonas aeruginosa a kmene Stafylococcus aureus rezistentní k meticilinu (MRSA). Vzhledem k vysoké potřebě profylaxe a terapie infekcí pooperačních a jiných ran právě výše uvedenými polyrezistentními původci se jedná o slibný prostředek k budoucímu klinickému využití. Zkušenosti, které jsme získali v průběhu uvolnování ATB z kolagenních pěn budou v dalším vyvoji použity pro impregnaci zevní kolagenní vrstvy cévní protézy, čímž bychom mohli eliminovat jednu z největších nevýhod a rizik spojených s použitím umělých materiálu a tím je...

Bacteremia initiated by Staphylococcus aureus infections can be a serious medical problem. Although a number of different antibiotics are used to combat staphylococcal infections, resistance has continued to develop. Combination therapy for certain infections has been used to reduce the emergence of resistance when a single agent has become ineffective. We hypothesize that the use of rifampicin and ciprofloxacin in combination with azithromycin, known for its inhibitory effects on the bacterial ribosome, can create potential synergistic effects resulting from indirect effects on ribosomal subunit synthesis. To determine this we measured the effects of single and multiple antibiotics on cell growth rates, cell viability, and synthesis rates for DNA, RNA, and protein. We then measured synthesis rates of ribosomal subunits and the amounts of gyrase and RNAP. Effects of the antibiotic combinations on 70S ribosomes was assayed and the amounts of RNA and degradation was measured. We lastly studied the effects of these antibiotic combinations on mutation frequency in Staphylococcus aureus. Our data have shown support not only for the use of antibiotic combination therapy but have provided strong evidence of an increase in the inhibition of bacterial ribosome assembly in Staphylococcus aureus. The reduction of 50S ribosomal subunit synthesis and 23S ribosomal RNA in cells grown in the presence of azithromycin, already known for it’s inhibitory effects on the 50S subunit synthesis, in combination with rifampicin or in combination with rifampicin and ciprofloxacin was observed. This also resulted in a reduction or elimination in the frequency of resistant cells when grown in the presence of these combinations. These studies have shed light on the mechanism of action involved and synergistic effects occurring in combination antibiotic treatments and how ribosomal subunit assembly is affected. The insights gained through this research provide necessary information needed for the design of more potent antibiotic combinations. This will create a better understanding and new methods for eliminating the spread of harmful pathogens such as Staphylococcus aureus.

Thesis (PhD)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Rifampicin resistance in clinical isolates of Mycobacterium tuberculosis develops through selection of bacterial variants harbouring mutations in the rpoB gene. These mutations infer a fitness-cost in the absence of antibiotic pressure, however, fitness-levels of rifampicin-resistant strains can be restored by compensatory mutations in rpoA and rpoC. This study was the first to investigate the epidemiological relevance of these compensatory mutations in clinical M. tuberculosis isolates collected in South Africa. Through targeted DNA sequencing, we demonstrated a strong association between rpoC mutations and transmission, and the rpoB S531L mutation. Our study emphasises the epidemiological relevance of compensatory evolution in response to the emergence of rifampicin resistance, and illustrates how compensatory mutations may be selected as a function of epistatic interactions. Recently a hypothesis has been developed which suggests that the activation of efflux systems through exposure to rifampicin may explain the observed spectrum of rifampicin resistance phenotypes. To elucidate whether rifampicin dependent activation of efflux systems also increases energy production, the RNA expression profiles of candidate energy metabolism genes were investigated. This study demonstrated that rifampicin exposure induced an overall increase in the expression of energy metabolism genes. Our findings suggest that the response to rifampicin is not universal and may depend on other genomic mutations. From these results we conclude that the stress response induced by exposure to rifampicin increases the energy production which fuels efflux activity thereby enabling the cell to extrude rifampicin in an energy dependent manner. This also provides a platform to explain the mechanism by which the newly developed drug, TMC207, increases the rate of culture conversion when used in combination with second-line anti-TB drugs. We propose that inhibition of ATP synthesis by TMC207 will deprive the efflux pumps and transporter genes of energy, which will result in the accumulation of second-line anti-TB drugs within the bacilli, leading to more efficient binding of the second-line drugs to their targets and ultimately to cell death. To identify the genetic basis governing the level of rifampicin resistance, we sequenced the genomes of MDR clinical isolates and in vitro generated rifampicin resistant mutants. Only minor genetic changes in addition to the rpoB mutation were identified in the genomes of in vitro rifampicin resistant mutants which displayed varying levels of resistance. This suggests that these mutants may either use alternative regulatory mechanisms or have acquired SNPs outside the genetic regions investigated in this study to modulate rifampicin resistance levels. In contrast, the genomes of clinical MDR isolates from the Low Copy Clade showed considerable variability in genes involved in cell wall, cellular processes and lipid metabolism, while the genomes from the Beijing Clade displayed variability in genes known to confer drug resistance and compensatory mechanisms. These results suggest that the structure and processes of the cell wall, as well as lipid metabolism plays a critical role in determining the intra-cellular concentration of rifampicin. Finally, this study illustrated the complexity in the physiology of M. tuberculosis resistant to rifampicin, whereby multiple mechanisms are employed by the bacteria to modulate its resistance levels. / AFRIKAANSE OPSOMMING: Rifampisien weerstandigheid in kliniese isolate van Mycobacterium tuberculosis ontwikkel deur die seleksie van bakteriële variante wat mutasies in die rpoB geen het. Alhoewel hierdie mutasies lei tot „n afname in fiksheid van die bakterieë in die teenwoordigheid van antibiotika, kan die fiksheids vlakke van rifampisien weerstandige stamme herstel word deur vergoedende mutasies in rpoA en rpoC. Hierdie is die eerste studie wat die epidemiologiese relevansie van hierdie vergoedende mutasies in kliniese M. tuberculosis isolate wat in Suid-Afrika versamel is, ondersoek. Deur middel van doelgerigte DNA volgordebepaling het ons „n sterk assosiasie tussen rpoC mutasies en transmissie, en die rpoB S31L mutasie getoon. Hierdie studie beklemtoon die epidemiologiese relevansie van regstellende evolusie na aanleiding van die ontwikkeling van rifampisien weerstandigheid en illustreer hoe regstellende mutasies geselekteer mag word as „n funksie van epistatiese interaksies. „n Hipotese is onlangs ontwikkel wat voorstel dat blootstelling aan rifampisien uitvloei sisteme in die bakterium aktiveer, wat moontlik die waargenome spektrum van rifampisien weerstandige fenotipes kan verklaar. Ons het die RNA uitdrukkingsprofiele van kandidaat-energiemetabolisme gene ondersoek om te bepaal of rifampisien afhanklike aktivering van uitvloei sisteme ook energieproduksie verhoog. Hierdie studie demonstreer dat rifampisien-blootstelling „n algehele verhoging in die uitdrukking van energiemetabolisme gene induseer. Ons bevindinge stel voor dat die reaksie van die sel op rifampisien blootstelling nie universeel is nie, en moontlik ook afhanklik is van ander genomiese mutasies. Uit hierdie resultate kan ons aflei dat die stres respons wat geïnduseer word deur rifampisien-blootstelling energieproduksie verhoog, wat weer die uitvloei aktiwiteit aanvuur, en gevolglik die sel in staat stel om rifampisien op „n energie-afhanklike wyse uit te dryf. Dit bied ook „n basis om die meganisme te verklaar waardeur die nuwe middel, TMC207, die tempo van kultuuromskakeling verhoog wanneer dit saam met tweede-linie anti-TB middels gebruik word. Ons stel voor dat die inhibisie van ATP sintese deur TMC207 die uitvloeipompe en transporteerder gene van energie ontneem. Gevolglik veroorsaak dit „n ophoping van tweedelinie anti-TB middels binne-in die bakterium, wat geleentheid bied vir meer effektiewe binding tussen die middels en hulle teikens en uiteindelik seldood veroorsaak. Ons het DNA volgordes bepaal van die genome van MDR kliniese isolate en in vitro selekteerde rifampisienweerstandige mutante om sodoende die genetiese grondslag waarop die vlak van rifampisienweerstandigheid beheer word, te identifiseer. Slegs klein verskille, bo en behalwe die rpoB mutasie, is geïdentifiseer in die genome van in vitro rifampisien weerstandige mutante wat verskillende vlakke van weerstandigheid getoon het. Dit dui aan dat hierdie mutante of ander regulatoriese meganismes gebruik, of hulle het enkelnukleotied polimorfismes buite die genetiese area wat in hierdie studie ondersoek is, waarmee rifampisien weerstandigheid gemoduleer word. In teenstelling hiermee het die genome van kliniese MDR isolate van die “Low Copy Clade” aansienlike variasie getoon in gene wat betrokke is by die selwand, sellulêre prosesse en lipiedmetabolisme. Verder het die genome van die Beijing genotipe variasie in gene getoon wat betrokke is by middelweerstandigheid en regstellende meganismes. Hierdie resultate dui aan dat die struktuur en prosesse van die selwand, asook lipiedmetabolisme, „n kritiese rol speel in die bepaling van die intrasellulêre konsentrasie van rifampisien. Opsommend, hierdie studie toon verskeie meganismes aan wat deur die bakterieë gebruik word om weerstandigheidsvlakke te moduleer en die kompleksiteit van die fisiologie van M. tuberculosis wat weerstandig is teen rifampisien. / The National Research Foundation (NRF) / South African Medical Research Council (MRC) / Harry Crossley Foundation