Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biophysics and Physical Chemistry Candidate: Kateřina Brokešová Supervisor: Ing. Martin Drastík, Ph. D. Thesis title: The use of core-shell columns for fluconazole determination A novel HPLC method for determination of fluconazole in dissolution test samples was developed and partly validated. A matrix formed by lactic and glycolic acid copolymer branched by different compounds was used as a drug carrier. Fluconazol was incorporated as the model drug. The concentration profile of fluconazole was studied by developed HPLC method during the dissolution test. A modern core-shell column Ascentis Express RP-Amide, 10 cm × 3.0 mm; 2.7 μm was employed. A mixture of acetate buffer pH 5.0:methanol (80:20) served as the mobile phase. The flow rate was 0.70 ml/min and the detection wavelength was 260 nm. The temperature of analysis was 50 řC. The retention time of fluconazole was 3.3 min and the whole analysis took just 4 min. Keywords: fluconazole, core-shell column, HPLC, PLGA

Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Pharmaceutical Technology Candidate: Karolína Valíková Supervisor: doc. RNDr. Milan Dittrich, CSc. Diploma thesis title: Formulation and study of the antimicrobial agent A literary overview of selected characteristics of silver nanoparticles is presented in the diploma thesis. Size measurement methods are described, while considerable attention is paid to photon correlation spectroscopy (PCS), which was used in the experimental part of this diploma thesis. Various methods of silver nanoparticle synthesis are outlined. Later part of the text is focused on the application of silver nanoparticles in areas concerning human health - mainly for the purposes of medicine, disinfection and as components of cosmetic products. Possible toxic effects of silver nanoparticles on human organism are also discussed. The focus of the thesis is in the experimental part. Stability of microparticles in suspensions used for product formulation was studied, as well as the PCS instrument's capability to distinguish the size distribution of particles in aqueous medium in highly polydisperse systems and in highly diluted systems. It was proven that microparticles have a spontaneous ability to form flocks, nonionic surfactants in 0.1%...

Charles Univerzity in Prague Faculty of Pharmacy in Hradec Králové Department of Pharmaceutical Technology Candidate: Veronika Müllnerová Consultant: Doc. RNDr. Milan Dittrich, CSc. Title of thesis: Impact of molecular weight and the grade of branching of aliphatic oligoesters on their hydrolytic degradation Theoretical part of the thesis deals with behavior, properties and applications of biodegradable polyesters, mainly copolymers of lactic and glycolic acid (PLGA). This part concerns degradation, erosion and release mechanism. Furthermore, it describes properties that influence the drug release kinetics from systems based on PLGA. The final section of theoretical part is focused on the in situ forming implants, whose carrier of active substance is biodegradable polyester. The experimental part analyzes the influence of different pH of the medium within physiologically common boundaries and also the influence of ionic force on the degree of swelling and polymer erosion. These degradation parameters have been studied on three potential polyester carriers of active substances - PLGA, M3 (terpolymer of lactic and glycolic acid with mannit) and T3 (terpolymer of lactic and glycolic acid with tripentaerythritol). Polymer bodies were kept in temperature of 37řC inside phosphate-citrate buffers with...

Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biophysics and Physical Chemistry Candidate: Andrea Malovaná Supervisor: Ing. Martin Drastík, Ph. D. Diploma thesis: Simplification of terbinafine HPLC analysis of samples based on biodegradable polyesters A HPLC method for determination of terbinafine in samples consisting of copolymers of lactic and glycolic acid was optimized and validated. The development of the method was based on the finding of suitable chromatographic conditions for separation of terbinafine. The separation was performed on the Ascentis Express ES-CN, 15 cm × 4.6 mm; 2.7 μm core- shell column. The mixture of the citrate phosphate buffer pH 4 and acetonitrile in ratio 40:60 (v/v) was chosen as the mobile phase. The mobile phase flow rate was set to 1.4 ml/min and the temperature to 30 řC. The injection volume of samples containing terbinafine was 5 µl. The UV detection at 226 nm was employed. The retention time of terbinafine was 3.3 min. The whole analysis was completed within 4 min. The method was validated, following parameters were tested: column efficiency, factor of symmetry, LOD, LOQ, linearity, repeatability and robustness. Keywords: terbinafine, HPLC, core-shell column, PLGA

BSc. Martina Hloušková Supervisor: Ing. Martin Drastík, Ph.D. The aim of this diploma thesis was the optimization and validation of a HPLC method for miconazole determination in samples supplied by the Department of Pharmaceutical Technology. A gradual miconazole release dependent on the composition of the copolymere of glycolic and lactic acid was studied. HPLC analysis was performed using a modern core-shell Column Ascentis Express RP- Amide, 10 cm x 3.0 mm; 2.7 μm. Optimized analytical conditions were: mobile phase methanol:water 70:30, flow rate 0.8 ml/min, temperature 45 řC, injection 5 l and UV detection at 220 nm. Miconazole retention time was 5.65 min. The entire analysis was carried out in 7 minutes. When the optimal conditions of analysis were determined, the method could be validated. The following parameters were monitored during validation: linearity, selectivity, efficiency, LOD, LOQ, repeatability and tailing factor. All of the monitored parameters met the requirements of the Czech Pharmacopoeia.

O presente trabalho apresenta resultados do estudo que objetivou desenvolver blendas poliméricas compostas por dois polimeros biodegradáveis, o poli (butileno succinato - co - adipato) (PBSA) copolímero de poliésteres alifáticos e a policaprolactona (PCL). O estudo desenvolveu a tecnologia para a incorporação de nanowhiskers de celulose (NWC) enxertados com PCL (NWC - g - PCL), com objetivo de melhorar a adesão nanowhisker/matrix, e otimizar as propriedades mecânicas destes polímeros. As composições utilizadas foram 30PCL/70PBSA e 50PCL/50PBSA (em % de massa), com 1, 3 e 5% de NWC - g - PCL. As blendas e compósitos foram preparadas na forma de filmes por evaporação de solvente clorofórmio (casting) e via extrusão e moldagem por injeção, e caracterizadas quanto a suas propriedades térmicas, mecânicas e morfológicas através de calorimetria exploratória diferencial (DSC), análise termogravimétrica (TGA) e análise termodinâmica mecânica (DMTA), ensaio mecânico de tração e microscopia eletrônica de varredura (MEV). Os resultados obtidos através das análises de espectroscopia no infravermelho (IV) e difração de raios-X (DRX) sugerem a enxertia de cadeias de PCL nos NWC. Os polímeros puros, na forma de filmes, obtidos por evaporação de solvente, apresentaram estabilidade térmica semelhantes com início de decomposição em 290°C e decomposição máxima em torno de 420°C. Os filmes dos compósitos apresentaram uma diminuição da estabilidade térmica com o aumento dos teores de NWC - g - PCL nas formulações. As curvas DSC apresentaram dois picos de fusão e de cristalização o que indica a imiscibilidade dos polímeros, as análises térmicas dos filmes, mostraram também um aumento no grau de cristalinidade conforme os teores de nanowhiskers enxertados aumentaram, isso para composição 30/70. No caso dos compósitos com composição 50/50 (PCL/PBSA) houve uma diminuição do grau de cristalinidade dos polímeros. A imiscibilidade dos polímeros nos filmes foi confirmada também através das análises DMTA onde dois picos distintos de tanδ foram encontrados, para as composições 30/70, próximos das temperaturas de transição vítrea dos dois polímeros. As mesmas composições processadas via extrusão, e corpos de prova moldados por injeção, apresentaram uma queda na estabilidade térmica detectada pelas análises TGA. Houve um aumento no grau de cristalinidade da PCL conforme maior quantidade de nanowhisker nos compósitos. A maior rigidez do material foi confirmada a partir dos ensaios mecânicos de tração, onde o módulo elástico aumentou aproximadamente duas vezes com o aumento da porcentagem de nanocargas nos compósitos, assim como a diminuição da porcentagem de alongamento e a resistência à tração não sofreu grandes alterações. / In this dissertation results of the study aimed to develop polymer blends composed of two biodegradable polymers, poly (butylene succinate - co - adipate) (PBSA) and aliphatic polyester polycaprolactone (PCL). The study developed the technology for the incorporation of cellulose nanowhiskers (NWC) grafted PCL (NWC - g - PCL), aiming to improve adherence nanowhisker / matrix, and improve mechanical properties of these polymers. The compositions used were 30PCL/70PBSA and 50PCL/50PBSA (in mass%), with 1, 3 and 5% of NWC - g - PCL. The blends and composites were prepared as films by evaporation of solvent chloroform (casting) and via extrusion and injection molding, and characterized their thermal, mechanical and morphological means of differential scanning calorimetry (DSC), thermogravimetric analysis (TGA) mechanical and thermodynamic analysis (DMTA), mechanical tests and scanning electron microscopy (SEM). The results obtained by analysis of infrared spectroscopy (IR) and X -ray diffraction (XRD) suggest the grafting of PCL chains in the NWC. The pure polymers in the form of films obtained by solvent evaporation, showed similar thermal stability with decomposition onset at 290°C and decomposition maximum at around 420°C. The films of the composites showed a decrease in thermal stability with increased levels NWC - g - PCL in the formulations. The DSC curves show two melting peaks and crystallization which indicates immiscibility of the polymers, the thermal analysis of the film showed also an increase in degree of crystallinity as the content of grafted nanowhiskers increased, this composition to 30/70. In the case of composites with composition 50/50 (PCL / PBSA) there was a decrease in the degree of crystallinity of polymers. The immiscibility of the polymers in the film was also confirmed by analysis DMTA where tanδ two distinct peaks were found for compositions 30/70, close to the glass transition temperatures of the two polymers. The same compositions processed via extrusion, and specimens injection molded, showed a decrease in thermal stability detected by TGA analysis. There was an increase in the degree of crystallinity of the PCL as the largest quantity of nanowhisker composites. The rigidity of the material was confirmed from the tensile test, where the elastic modulus increased approximately twice the percentage increase of nanofiller in the composites, as well as the decrease in percentage elongation and tensile strength has not changed much.

Na presente tese foram preparados poliésteres derivados de dois materiais renováveis o ácido 2,5-furanodicarboxílico (AFD) e a isoidida. Foram também preparados monômeros dihidroxílados para uso em poliesterificações e em reações com diisocianatos contendo um aduto de Diels-Alder (DA) formado pela reação de um grupo furano e uma maleimida (reação entre uma bismaleimida e o álcool furfurílico). Esses diois foram utilizados na preparação de poliésteres e poliuretanos termorreversíveis. A termorreversibilidade dos materiais preparados advém do fato de que os adutos de DA são termorreversíveis (rDA). Portanto, ao aquecer os poliésteres preparados com aduto-DA são originados monômeros difuncionais com grupos furano e/ou maleimida. O produto da rDA pode então ser repolimerizado, mas não via reações de poliesterificação ou isocianato/hidroxila, mas pelo acoplamento furano/maleimida (DA). Por fim uma nova rota para a obtenção de copolímeros aleatórios foi investigada. Essa rota consiste em provocar a rDA de uma mistura de dois homopolímeros distintos obtidos por polimerização em etapas contendo no interior de suas unidades repetitivas adutos de Diels-Alder e em seguida provocar a sua repolimerização via DA para dar origem a um copolímero aleatório. Os materiais foram caracterizados por suas estruturas químicas por Espectroscopia na região do infravermelho com transformada de Fourier (FTIR), ressonância magnética nuclear de próton (RMN ¹H). Foram feitos ensaios de cromatografia de permeação de gel (GPC), onde notou-se uma massa média numérica e ponderal para os poliésteres obtidos a partir do AFD e também para o poliéster e poliuretano termorreversíveis de aproximadamente 1600 g.mol¹. Nos ensaios de calorimetria exploratória diferencial (DSC) foi obtido Tgs variados para os poliésteres obtidos a partir do AFD indo de 88 a 159ºC, de 80ºC para o poliuretano termorreversível e de 106ºC para o poliéster termorreversível. Para a análise termogravimétrica (TGA) foi observado temperaturas de degradação para os poliésteres obtidos a partir do AFD em torno de 280ºC. Para a análise térmica dinâmico-mecânicas (DMTA) obteve-se Tg\'s em 117, 123 e 120ºC para o poliéster, o poliuretano e o copolímero termorreversíveis. / In this thesis it was prepared polyesters of two renewable monomers, the 2.5-furandicarboxylic acid and isoidide. Dihydroxy monomers were also prepared for use in polysterification and in reactions with diisocyanates, both containing Diels-Alder adduct in the molecule formed by the reaction of a furan group and a maleimide (reaction between bismaleimide and furfuryl alcohol). These diols were used in the preparation of thermoreversible polyesters and polyurethanes. The thermoreversibility of these materials comes from the fact that the DA adducts are thermoreversible (rDA). Therefore, heating these polymers give rise to difunctional monomers with an adduct-DA with furan and/or maleimide functions. The product of the rDA may then be polymerized not via polysterification reactions or isocyanate/hydroxyl, but by furan/maleimide coupling (DA) restoring the DA adduct. Finaly a new route to obtain random copolymers was investigated. This route consists in the rDA of a mixture of two different homopolymers obtained by polymerization in stages containing adducts producing monomers with furan and maleimide functions that can be polymerization via DA reaction to give a random copolymer. The materials were characterized by their chemical structures by infrared spectroscopy (FTIR), nuclear magnetic resonance of proton (¹H-NMR), gel permeation chromatography (GPC), experiments were made, where it was noticed a number and weight average mass for the polyesters obtained from the AFD and also for the thermoreversible polyester and polyurethane 1600 gmol¹. In differential scanning calorimetry tests (DSC) was obtained Tg\'s for various polyesters obtained from the AFD ranging from 88 to 159°C, 80°C for the thermoreversible polyurethane and 106°C for the thermoreversible polyester. For the thermogravimetric analysis (TGA) was observed degradation temperatures for the polyesters obtained from the AFD around 280°C. For dynamic mechanical thermal analysis (DMTA) Tg was obtained in 117, 123 and 120°C for polyester, polyurethane and copolymer thermoreversible.

Thesis (M.S.)--Massachusetts Institute of Technology, Dept. of Mechanical Engineering, 1982. / MICROFICHE COPY AVAILABLE IN ARCHIVES AND ENGINEERING / Vita. / Includes bibliographical references. / by Ronald James Salig. / M.S.

Os polihidroxialcanoatos constituem um grupo de poliésteres acumulados por inúmeras bactérias na forma de grânulos intracelulares, que podem representar até 80% da massa seca celular. No presente trabalho, foi avaliado o comportamento de diferentes linhagens quanto a capacidade de metabolizarem ácidos graxos livres e óleo de soja para a produção de biopolímeros e, posteriormente, em biorreator, algumas linhagens selvagens e recombinantes foram testadas na presença ou não de um inibidor da b-oxidação. Experimentos em frascos agitados, mostraram valores de teor de PHA em até cerca de 70% na biomassa total com cerca de 5% de monômeros de cadeia média permitindo selecionar as linhagens Cupriavidus necator e Burkholderia cepacia para os ensaios seguintes. Experimentos em biorreator mostraram que, as variáveis manipuláveis quanto ao fluxo de óleo de soja, de co-substratos e o inibidor de b-oxidação não influenciaram, significativamente, no rendimento de P3HB/3HAmcl formado quando utilizado Burkholderia cepacia IPT-048, entretanto, contribuíram na síntese de 3HV. Durante os ensaios, o aumento do número de cópias do gene phaB em Cupriavidus necator, aparentemente, não contribuíram para o aumento do rendimento de 3HHx, porém, outras variáveis manipuláveis deverão ser propostas para a confirmação. / The poly-3-hydroxyalkanoates (PHA) are a group of polyesters accumulated for several bacteria in the intracellular granule form, that can represent up to 80% of the dry mass cellular. The main advantage of the biopolymers on the synthetic materials is its degradation in the environment. Recycable raw materials can be used as carbon sources for the production biodegradable polymer. Some polymers have appeared in literature having mechanical characteristic considered appropriate in such a way for use in packings (flexible and covering films), how base for controlled release of asset to be applied in the pharmacological-medical and foods area. The P3HB-co-3HAmcl are 3-hydroxybutyrate copolymers and 3-hydroxyacyl of 6 or more carbon atoms which has aroused interest for to present intermediate properties between HB and HAmcl, having taken care of the requirements for diverse applications. Some strains are distinguished, however, assays in bottles agitated with the Cupriavidus sp. and Burkholderia sp. strains, using as substratum greasy free acid and soy oil, had led the promising results. In the present work, first, was evaluated the behavior of different strains (Cupriavidus necator DSM545 IPT-026, C. necator H16 IPT-027, Pseudomonas sp. IPT-066, Burkholderia cepacia IPT-048 and B. sacchari IPT-189) how much to metabolizer capacity of free fatty acids and soybean oil for the production of biopolymers and, later, in tank bioreactor some wild strains and recombinant had been tested together or not of an inhibitor of the boxidation (acid acrylic). Experiments in bottles with agitation, had shown values of PHA in about 70% of the total biomass with about 5% monomers chain average allowing to select the Burkholderia cepacia IPT-048 and Cupriavidus necator DSM545 strains for the following assays. Experiments in tank bioreactor had shown that, the interactions between the substratum, co-substratum and the inhibitor of boxidation had influenced in the amount of formed P3HB/3HAmcl when used the Burkholderia cepacia IPT-048 strains, contributing for the synthesis of 3HV and 3HHx. When used only the soybean oil (5g/L) was gotten an amount of HHx with about 6 mol% with Burkholderia cepacia strains. In this same condition was verified that after some determined period also proved monomer HV in an amount of 0.20 mol%. Adding the acid acrylic (0.18 g/L) were gotten 1.50 mol% of HHx, however, differently of the previous condition the amount of HV varied of 0.91 - 2.77 mol%. A conclusion for the condition soybean oil and acrylic acid is that, the strains is using the acrylic fatty acid as carbon source, or either, the formation of acrylil-CoA with consequent formation of chains with 5 carbons. Similar results had been gotten when analyzed extracted polymer, or either, after the stage of extraction with chloroform was verified that the amount varied of 1.60 - 2.14 mol%. When added to a cosubstratum in the phase of accumulation (caproic fatty acid) and absence of the acrylic acid, it did not have the increase of HHx in relation to the previous variable, as much that, the amount of monomer reached about 0.50 mol%. In this condition, the presence of the acrylic fatty acid also did not contribute for the HV synthesis, not having been observed monomers during the assays. In the following phase, in which we work with Cupriavidus necator and a recombinant (Cupriavidus necator::phaB) strains, was verified that the gotten amount of HHx had been similar, about 0.30 mol%. Our hypothesis, we believe that the increase of the number of copies of the gene phaB could contribute for the increase of the carbon flow in the direction to raise the amount of 3HB how much of other monomers. As in the assays in bottles with agitation substrat caproic and caprylic fatty acids they had been distinguished also when availabled 2g/L and the soybean oil of in a concentration of 5g/L, we analyze the strains recombinant with soybean oil and caprylic fatty acid with 5 g/L. The insistence in the use of the caprylic fatty acid was based on the fact of the possibility of strains to oxidate part of fatty acids for the production of cells and energy and, at the moment that had the nitrogen limitation, to begin the accumulation phase dividing two carbons for the production of acetyl-CoA and the remain of the chain was stored in the form of 3HHx. The caprylic fatty acid disponibility it did not have the monomer attainment, therefore, Cupriavidus necator and Cupriavidus necator::phaB had its replyed inhibited capacity by the substratum. In the added soybean oil bottles with and without the acrylic fatty acid the behavior it was the opposite to the observed with caprylic fatty acid. During the incubation period, for both the strains, the amount of PHA, and consequently, the percentage of 3HB in the dry mass cellular was of 22.95% (100 mol%) and 25.90% (100 mol%) in 28ª and 24ª hours, respectively. In this condition, in the recombinant strains, also was observed the presence of HHx between 32ª and 49ª hour with a maxim amount of 1.10 mol%. From the results it can be concluded that the Burkholderia cepacia is considered a promising strains for the attainment of HHx and HV allowing that soybean oil with and without the acrylic fatty acid, respectively. The increase in the number of copies of the gene phaB in Cupriavidus necator apparently did not contribute with the increase of 3HHx efficiency however, other handling variables will be proposed for confirmation.

Ces travaux ont pour objet l’élaboration de nouvelles structures poreuses non tissées antibactériennes. Différentes stratégies ont été développées : l’une a consisté à élaborer des mats poreux par electrospinning en utilisant un polymère biosourcé et biocompatible et l’autre voie consistait à modifier un support fibreux provenant d’un masque de protection respiratoire commercial. La méthode des assemblages par interactions ioniques en superposant de façon alternative les couches de polymères cationiques et les polymères anioniques à la surface du filtre médian en polypropylène (PP) a permis d’élaborer de nouvelles structures ayant de bonnes propriétés antioxydantes et antibactériennes. Le polymère anionique, dérivé du polymère de cyclodextrine présente l’avantage de pouvoir encapsuler un agent antimicrobien biosourcé, le carvacrol. Une autre approche a consisté à modifier des supports en PP avec de l'acide tannique, un polyphénol d'origine naturelle. Dans cette étude, deux stratégies ont été mises en place afin de fonctionnaliser le PP avec de l’acide tannique (AT). La première est l’extrusion réactive du PP avec l’AT en présence (ou non) de peroxyde de dicumyle (DCP) pour greffer directement l’acide tannique sur le PP. La deuxième stratégie consiste à polymériser l’AT au travers d’une couche poreuse de PP extraite d’un masque de protection commercial, afin de permettre l’immobilisation physique de l’AT à la surface du mat fibreux en PP. Le greffage en surface via un procédé “grafting from” a également été étudié. Ces matériaux ont montré de bonnes propriétés antiradicalaires. / The purpose of this work is to develop new antibacterial non-woven porous structures. Different strategies were developed: one was to develop porous structures by electrospinning using a biosourced and biocompatible polymer, the other was to modify a fibrous support from a commercial respiratory protection mask. Assembling materials using ionic interactions by alternatively superposing cationic polymer layers and anionic polymers on the surface of the polypropylene (PP) median filter allowed to develop new structures with good antioxidant and antibacterial properties. The anionic polymer, derived from the cyclodextrin polymer, has the advantage of being able to encapsulate a bio-based antimicrobial agent, carvacrol. Another approach was to modify PP filters with tannic acid, a naturally occurring polyphenol. In this study, two strategies were implemented to functionalize PP with tannic acid (TA). The first is the reactive extrusion of PP with TA in the presence (or not) of dicumyl peroxide (DCP) to directly graft tannic acid onto PP. The second strategy consists in polymerizing the TA through a porous layer of PP extracted from a commercial mask, in order to allow the physical immobilization of the TA on the surface of the PP fibrous mat. Surface grafting using a "grafting from" process was also studied. These materials have shown good anti-free radical properties.