Magister Scientiae - MSc / Glucose oxidase (GOx) and horseradish peroxidase (HRP) are important enzymes for the development of amperometric enzyme linked immunosensors. The selectivity of each enzyme towards its analyte deepens its importance in determining the sensitivity of the resultant immunosensor. In designing immunosensors that have customized transducer surfaces, the incorporation with FAD and iron based enzymes ensures that electron kinetics remains optimal for electrochemical measurement. Various different immobilization strategies are used to produce response signals directly proportional to the concentration of analyte with minimal interferences. The combination of self-assembled monolayers and supramolecular chemistry affords stability and simplicity in immunosensor design. In this work, two electrochemical strategies for the detection of human chorionic gonadotropin (hCG) is presented. This involves the modification of a gold surface with a thiolated β- cyclodextrin epichlorohydrin polymer (βCDPSH) to form a supramolecular inclusion complex with ferrocene (Fc)-functionalised carboxymethyl cellulose polymer (CMC). Cyclic voltammetry indicated that ferrocene is in close proximity to the electrode surface due to the supramolecular complex formed with βCDPSH. Furthermore, strategy (a) for the detection of hCG used α-antihCG labelled (HRP) as reporter conjugate. Strategy (b) maintained the CMC bifunctionalised with Fc and recognition antibody for hCG hormone. However, the system was functionalised with a HRP enzyme and detection is done by using GOx reporter conjugates for in situ production of hydrogen peroxide. The reduction of H2O2 was used for the amperometric detection of hCG by applying a potential of 200 mV. The sensitivity and limit of detection of both strategies were calculated from calibration plots. For strategy (a) the LOD was found to be 3.7283 ng/mL corresponding to 33.56 mIU/mL and a sensitivity of 0.0914 nA ng-1 mL-1. The corresponding values for strategy (b) are 700 pg/mL (6.3 mIU/mL) and 0.94 nA ng-1 mL-1.

Objectives Glyburide, an important drug for type 2 diabetes, has extremely poor aqueous solubility and resulting low bioavailability. This study describes the ability of hydroxybutenyl-β-cyclodextrin (HBenBCD) to form complexes with glyburide, with enhanced solubility and dissolution rate in vitro. Method Glyburide and glyburide-HBenBCD were evaluated in various test media known to simulate human gastrointestinal conditions in the fasted and fed states, respectively. Key findings At ~14 wt% drug load, in the presence of HBenBCD, an almost 400-fold increase in glyburide aqueous solubility was observed. In the presence of HBenBCD, glyburide solubility was also significantly improved in all physiologically relevant test media. Subsequent dissolution experiments confirmed the solubility study results; the dissolution rate and total amount of drug released were significantly increased. Conclusions Complexation with HBenBCD may be an effective way to increase the bioavailability of glyburide.

The design of versatile biohybrid nanosized materials has revealed itself as a promising avenue towards biomedical applications in today´s life sciences. In this regard the combination of components of synthetic and natural origin facilitates an applicability which is supposed to be far beyond the sum of their single components. These biohybrid structures (BHS) can be built by a huge variety of building blocks including solid or soft nanoparticles, peptides/proteins, polynucleotides or low molecular weight drugs. Along with the latter the attachment of biologically active entities or imaging moieties, e. g. enzymes, fluorescence markers or targeting motifs display thereby a key step towards the development of carrier systems for drug delivery purposes. Among the soft nanoparticles especially dendritic polymers such as perfectly branched dendrimers or hyperbranched polymers are considered as ideal building blocks, since they allow an easy tailoring of crucial properties such as solubility, biocompatibility or bioactivity by means of surface functionalization. Especially in the field of targeted drug delivery the crucial role of sizes and size distributions of carriers has been highlighted recently, since it critically influences important factors such as circulation time or biodistribution within the body. The ability of avidin to form high molecular weight associates with biotinylated macromolecules as well as its inherent properties makes it a suitable candidate for passive and active targeting in combination with biotinylated (bio-)polymers. Furthermore, along with the covalent attachment of bioactive moieties, non-covalent attachment is a frequently used approach, because it is assumed to only require stoichiometric mixing. In context of the latter molecular recognition processes such as the avidin-biotin, β-cyclodextrin-adamantane or Ni(II)-NTA-histidine-tag interactions have shown to be fruitful strategies for the attachment of bioactive entities. The overall aim of this work was to fabricate BHS based on dendritic glycopolymers with varied sizes in the nano- and micrometer range as models for biomedical applications e. g. carriers for drug delivery. Therefore the molecular recognition of avidin with biotin derivatives and β-cyclodextrin with adamantane derivatives was utilized in order to tailor final sizes, functionality or catalytic activity of those BHS.

The design of versatile biohybrid nanosized materials has revealed itself as a promising avenue towards biomedical applications in today´s life sciences. In this regard the combination of components of synthetic and natural origin facilitates an applicability which is supposed to be far beyond the sum of their single components. These biohybrid structures (BHS) can be built by a huge variety of building blocks including solid or soft nanoparticles, peptides/proteins, polynucleotides or low molecular weight drugs. Along with the latter the attachment of biologically active entities or imaging moieties, e. g. enzymes, fluorescence markers or targeting motifs display thereby a key step towards the development of carrier systems for drug delivery purposes. Among the soft nanoparticles especially dendritic polymers such as perfectly branched dendrimers or hyperbranched polymers are considered as ideal building blocks, since they allow an easy tailoring of crucial properties such as solubility, biocompatibility or bioactivity by means of surface functionalization. Especially in the field of targeted drug delivery the crucial role of sizes and size distributions of carriers has been highlighted recently, since it critically influences important factors such as circulation time or biodistribution within the body. The ability of avidin to form high molecular weight associates with biotinylated macromolecules as well as its inherent properties makes it a suitable candidate for passive and active targeting in combination with biotinylated (bio-)polymers. Furthermore, along with the covalent attachment of bioactive moieties, non-covalent attachment is a frequently used approach, because it is assumed to only require stoichiometric mixing. In context of the latter molecular recognition processes such as the avidin-biotin, β-cyclodextrin-adamantane or Ni(II)-NTA-histidine-tag interactions have shown to be fruitful strategies for the attachment of bioactive entities. The overall aim of this work was to fabricate BHS based on dendritic glycopolymers with varied sizes in the nano- and micrometer range as models for biomedical applications e. g. carriers for drug delivery. Therefore the molecular recognition of avidin with biotin derivatives and β-cyclodextrin with adamantane derivatives was utilized in order to tailor final sizes, functionality or catalytic activity of those BHS.

Naphthenic acids (NAs) are a group of carboxylic acids that are found in hydrocarbon deposits such as the oil sands bitumen. These compounds are a well-known corrosive agent and a toxic component in the oil sands process water (OSPW). Due to Alberta’s zero discharge policy, OSPW cannot be released and must be stored until toxic components like NAs are remediated. One technique that has shown potential is to physically adsorb NAs onto a copolymer generated from economical biomaterials. Therefore, the project can be divided into three sections: 1) Synthesis of β-cyclodextrin (β-CD) copolymer for the sorption of p-nitrophenol (PNP); 2) Synthesis of chitosan-based copolymers (Chi-Glu) for the sorption of PNP; 3) Sorption of carboxylates and NAs using Chi-Glu copolymers. PNP sorption was used as a probe to understand the physicochemical properties of the copolymers. In the first section, β-CD was reacted with sebacoyl chloride (SCl) and terephthaloyl chloride (TCl) at various mole ratios. Characterization was done using Fourier Transform Infrared Spectroscopy (FT-IR), thermogravimetric analysis (TGA), 1H NMR spectroscopy (1H NMR), elemental analysis (CHN), and nitrogen porosimetry. Copolymers synthesized at mole ratios of β-CD to SCl from 1:1 to 1:3 were hydrolyzed at acidic and basic conditions. Therefore, sorption studies were not done at these ratios. The same occurred for 1:1 to 1:3 TCl copolymers. Sorption studies with PNP at pH 4.6 demonstrated enhanced sorption capacity when comparing with a standard: granular activated carbon (GAC). The sorption capacity, Qm (mmol/g), ordered from largest to smallest is 1:9 SCl>1:9 TCl>1:6 SCl> GAC> 1:6 TCl. Chi-Glu copolymers were synthesized by cross-linking glutaraldehyde with pristine chitosan. A systematic study on the effects reaction conditions have on the sorption capacity of the materials was done. Three conditions were changed: pH, temperature, and mole ratios. Chi-Glu copolymers were synthesized at various chitosan to glutaraldehyde mole ratios (1:400, 1:700, 1:1000). Sufficient time was allowed for the aging process. Characterization was done using TGA, FT-IR, CHN, and nitrogen porosimetry. Sorption study with PNP were done at pH = 7.0 and 9.0. At pH = 7.0 sorption capacity appears to correlate to the quantity of homo-polymerized glutaraldehyde: 1:700>1:1000>1:400. While at pH = 9.0, the sorption capacity is inversely proportional to the degree of crosslinking: 1:400>1:700>1:1000. By increasing the pH at the shrinkage phase, PNP was weakly bound onto the Chi-Glu copolymer. Varying temperature before gelation caused a decrease in the sorption capacity with PNP. Sorption studies involving carboxylates and NAs were done at pH = 9.0 at ambient temperature using Chi-Glu copolymers (1:400, 1:700, and 1:1000) and chitosan. Three carboxylates were chosen to reflect the diverse components in NAs. Varying degrees of cyclization (Z = 0, -2, -4) and lipophilic surface area were the main criteria for carboxylates. The sorption capacity depended mainly on the lipophilic surface area (LSA) with sorption capacity highest for 2-hexyldecanoic acid (S1) which has the largest LSA and lowest for, trans-4-pentylcyclohexanecarboxylic acid (S2) and dicyclohexylacetic acid (S3). Unfortunately, cross-linking with glutaraldehyde does not enhance sorption as pristine chitosan retained a higher sorption capacity compared to Chi-Glu copolymers. Acros and Fluka NAs were chosen for sorption and no significant sorption was recorded for any copolymers. Problems involving the micellization process can explain the lack of sorption.

A genisteína é uma isoflavona da soja que vem sendo investigada pelo seu potencial antienvelhecimento, baseado nas suas atividades antioxidante, estrogênica e inibidora de proteínas tirosina-quinase. A associação da genisteína com β- ciclodextrina com a formação de um complexo elevou a hidrossolubilidade da isoflavona. A permeabilidade intrínseca da genisteína foi avaliada, bem como sua permeabilidade quando aplicada a partir de gel de hidróxi propilmetilcelulose (HPMC) a 3 %, pelo método de célula de Franz. As associações com β-ciclodextrina, em géis de HPMC 3 %, também foram avaliadas e foi observado um incremento da penetração em favor do complexo produzido em meio aquoso. Devido ao seu alto coeficiente de partição (log P) - 4,36 - a genisteína demonstrou a capacidade de formar reservatórios nas estruturas internas da pele favorecendo sua ação antienvelhecimento na pele. / Genistein is a soy isoflavone that has been investigated for its antiaging potencial, based on antioxidant, estrogenic and proteins tirosin-kinase inhibitor activities. The association of genistein with β-cyclodextrin resulted in a complex formation enhanced the isoflavone hidrosolubity. Genistein instrinsic permeability was evaluated, as well permebility from hydroxypropyl methylcelullose (HPMC) 3 % gel, wtih Franz cell method. The associations with β-cyclodextrin, in HPMC 3 % gels were also evaluated with the observation of na enhancing effect of cyclodextrin in the complex produced in aqueous media. Because its high partition coefficient (log P) - 4,36 - genistein was able to form reservoir in the internal skin layers, favorable to its antiagin action.

O coumestrol, isoflavonóide presente na alfafa (Medicago sativa L., Fabaceae), é um potente agente antioxidante e estrogênico, podendo ser empregado para fins de prevenção do envelhecimento cutâneo, bem como na reposição hormonal para mulheres pós-menopausa. A sua baixa hidrossolubilidade motivou a investigação de sua associação, em mistura física ou complexado, à b-ciclodextrina (1:1). A presente pesquisa desenvolveu e validou dois métodos analíticos (UV e CLAE) para a quantificação do coumestrol e de suas associações. A caracterização da interação entre o coumestrol e a b-ciclodextrina foi realizada por MEV, DSC, IV, RMN e modelagem molecular. Visando a sua aplicação tópica, foram realizados ensaios de permeação cutânea em pele de orelha de suínos, em células de difusão tipo Franz, de maneira intrínseca e em matriz de gel HPMC F4M. As análises de caracterização da interação indicaram a existência de complexo de inclusão tanto na mistura física como no complexo, mostrando a possibilidade de inclusão do anel A ou B do coumestrol na cavidade da b-ciclodextrina. A inclusão do anel B do coumestrol na extremidade das hidroxilas secundárias da b-ciclodextrina (borda mais larga) mostrou-se favorecida, uma vez que apresentou a menor energia. Na permeação intrínseca, a mistura física apresentou maior quantidade permeada em 8 horas (22,6 μg/cm2) e maior fluxo (3,3 μg/cm2/h) do que o coumestrol ou o complexo coumestrol:b-ciclodextrina. Já a partir do gel, o complexo coumestrol:b-ciclodextrina apresentou a maior quantia permeada (2,7 μg/cm2), bem como o maior tempo de latência que os demais. O coumestrol complexado mostrou maior solubilidade e apresentou razoável permeabilidade cutânea na forma de gel, mostrando-se promissor para aplicação tópica em pele. / Coumestrol, isoflavonoid present in alfalfa (Medicago sativa L, Fabaceae), is a potent antioxidant and estrogenic agent, which could be employed for the cutaneous aging prevention, as well as, in hormonal replacement for pos-menopausal women. Its low solubility in water gave rise to its association investigation, in physical mixture or complexed with b- cyclodextrin (1:1). The present research developed and validated two analytical methods (UV and HPLC) for the quantification of coumestrol and its associations. The characterization of the interaction between coumestrol and b-cyclodextrin was accomplished by SEM, DSC, IR, NMR and molecular modelling. Seeking the topical application, cutaneous permeation assays were accomplished in swine ear skin, in Franz type diffusion cells, in an intrinsic way and in a gel matrix of HPMC F4M. The characterization analyses indicated the existence of an inclusion compound, as much in the physical mixture as in the complex, showing the inclusion possibility of the A or B rings of coumestrol enter in the cavity of b-cyclodextrin. The inclusion of the B ring of coumestrol in the wider rim of b-cyclodextrin showed to be favorable, due to its lower energy. In the intrinsic permeation, the physical mixture presented larger amount permeated in 8 hours of study (22,6 μg/cm2) and larger flow (3,3 μg/cm2/h) than that of coumestrol or its coumestrol:b-cyclodextrin complex. With respect to the gel permeation, the complex coumestrol:b-cyclodextrin presented the higher permeation amount (2,7 μg/cm2), as well as the largest lag-time than the others. Coumestrol showed higher solubility when complexated with b-cyclodextrin and it presented reasonable cutaneous permeability in the gel form, being promising to topical application in skin.

Made available in DSpace on 2016-08-29T15:34:27Z (GMT). No. of bitstreams: 1 tese_4971_Dissertação_Nahun Thiagor.pdf: 1844062 bytes, checksum: 8e3e54f1750887fb5387ffffb0103629 (MD5) Previous issue date: 2011-08-16 / No presente estudo é estabelecida uma correlação entre alguns tensoativos, um derivado de ciclodextrina, oligossacarídeo cíclicos originado da ação da ciclodextrina glicosiltransferase, a β-ciclodextrina metilada e as linhagens de câncer de ovário A 2780 e OVCAR 3, tentando abrir a possibilidade de utilização da ciclodextrina associada a medicamentos quimioterápicos padronizados à terapia contra o câncer de ovário, que é apontado como uma das principais causas de óbito entre as malignidades ginecológicas. O câncer de ovário apresenta alta taxa de mortalida, além de possuir vários subtipos histo-clínicos, a cada ano aumenta cada vez mais o número de pacientes diagnosticadas, assim, é indispensável à continuidade das pesquisas científicas relacionadas a este tipo de câncer, visto que os conhecimentos até então elaborados são de extrema significância, porém insuficientes para que seja estabelecida uma cura. Através da viabilidade celular pelo método do MTT, onde se utiliza o sal de tetrazol para expressar de forma quantitativa a proliferação e sobrevivência das células e o método de BRADFORD, para normalização de dados, tenta-se estabelecer a influência de alguns tensoativos e do derivado de ciclodextrina sobre e entre as linhagens de câncer de ovário. A metil-β-ciclodextrina e os tensoativos SDS, TX100 E TW20 induziram uma redução da atividade mitocondrial dose-dependente em cultura de células de câncer de ovário da linhagem A 2780 e OVCAR 3. O derivado de ciclodextrina demonstrou indução gradativa da redução da atividade mitocondrial para duas das oito concentrações testadas (0,2% e 0,4%) com viabilidade celular próxima a 60% na linhagem A 2780. Na linhagem OVCAR 3 a metil-β-ciclodextrina apresenta potencial citotóxico capaz de inviabilizar 25% do crescimento celular em concentrações superiores a 0,003125%, a viabilidade celular perante as mesmas condições de tratamento é menor nesta linhagem quando comparada a linhagem A 2780. Devido às propriedades da ciclodextrinas os resultados das análises comparativas de viabilidade celular, nas linhagens de câncer de ovário, estes resultados apontam para possibilidades maiores em futuros estudos, podendo a pesquisa aqui apresentada ser tomada como referência. / This study sets up a correlation between some surfactants, a derivate of cyclodextrin, cyclic oligosaccharide originated from the action of the glycosyltransferase cyclodextrin, a methyllated β-cyclodextrin and the lines of ovarian cancer A 2780 and OVCAR 3, trying to offering the possibility of using cyclodextrin associated with standard chemotherapy drugs in therapy against ovarian cancer, which is pointed as one of the main causes of death among gynecological ills. Ovarian cancer is connected with high mortality rate and has several histo-clinical subtypes, raising the number of patient diagnosed each year, therefore there are the needy of continuing the scientific research which this kind of cancer is related, once the knowledge developed so far are extremely significant, yet it still is insufficient to generate a cure. Through the cellular viability by MTT method, which uses salt of tetrazol to express quantitatively the proliferation and survival of cells, and Bradford method for standardization data, it attempts to establish the influence of surfactants and some of the derivate of cyclodextrin on and between the lines of ovarian cancer. The methyl-β-cyclodextrin and the surfactant SDS, TX100 and TW20 induced a reduction in dose-dependent mitochondrial activity in cell cultures of ovarian cancer line A 2780 and OVCAR 3. The cyclodextrin derivate has demonstrated induction of gradual reduction of the mitochondrial activity of two from eight concentrations tested (0,2% and 0,4%) with cell viability of approximately 60% in strain A 2780. In the line OVCAR 3 the methyl-β-cyclodextrin shows potential cytotoxic able to tamper 25% of cell growth in conditions of treatment is reduced in this strain when compared with strain A 2780. Due to the properties of cyclodextrins, results of comparative analyzes of cell viability, in the strains of ovarian cancer, indicates to greater possibilities in future studies, enabling the research presented here as a reference.

O coumestrol, isoflavonóide presente na alfafa (Medicago sativa L., Fabaceae), é um potente agente antioxidante e estrogênico, podendo ser empregado para fins de prevenção do envelhecimento cutâneo, bem como na reposição hormonal para mulheres pós-menopausa. A sua baixa hidrossolubilidade motivou a investigação de sua associação, em mistura física ou complexado, à b-ciclodextrina (1:1). A presente pesquisa desenvolveu e validou dois métodos analíticos (UV e CLAE) para a quantificação do coumestrol e de suas associações. A caracterização da interação entre o coumestrol e a b-ciclodextrina foi realizada por MEV, DSC, IV, RMN e modelagem molecular. Visando a sua aplicação tópica, foram realizados ensaios de permeação cutânea em pele de orelha de suínos, em células de difusão tipo Franz, de maneira intrínseca e em matriz de gel HPMC F4M. As análises de caracterização da interação indicaram a existência de complexo de inclusão tanto na mistura física como no complexo, mostrando a possibilidade de inclusão do anel A ou B do coumestrol na cavidade da b-ciclodextrina. A inclusão do anel B do coumestrol na extremidade das hidroxilas secundárias da b-ciclodextrina (borda mais larga) mostrou-se favorecida, uma vez que apresentou a menor energia. Na permeação intrínseca, a mistura física apresentou maior quantidade permeada em 8 horas (22,6 μg/cm2) e maior fluxo (3,3 μg/cm2/h) do que o coumestrol ou o complexo coumestrol:b-ciclodextrina. Já a partir do gel, o complexo coumestrol:b-ciclodextrina apresentou a maior quantia permeada (2,7 μg/cm2), bem como o maior tempo de latência que os demais. O coumestrol complexado mostrou maior solubilidade e apresentou razoável permeabilidade cutânea na forma de gel, mostrando-se promissor para aplicação tópica em pele. / Coumestrol, isoflavonoid present in alfalfa (Medicago sativa L, Fabaceae), is a potent antioxidant and estrogenic agent, which could be employed for the cutaneous aging prevention, as well as, in hormonal replacement for pos-menopausal women. Its low solubility in water gave rise to its association investigation, in physical mixture or complexed with b- cyclodextrin (1:1). The present research developed and validated two analytical methods (UV and HPLC) for the quantification of coumestrol and its associations. The characterization of the interaction between coumestrol and b-cyclodextrin was accomplished by SEM, DSC, IR, NMR and molecular modelling. Seeking the topical application, cutaneous permeation assays were accomplished in swine ear skin, in Franz type diffusion cells, in an intrinsic way and in a gel matrix of HPMC F4M. The characterization analyses indicated the existence of an inclusion compound, as much in the physical mixture as in the complex, showing the inclusion possibility of the A or B rings of coumestrol enter in the cavity of b-cyclodextrin. The inclusion of the B ring of coumestrol in the wider rim of b-cyclodextrin showed to be favorable, due to its lower energy. In the intrinsic permeation, the physical mixture presented larger amount permeated in 8 hours of study (22,6 μg/cm2) and larger flow (3,3 μg/cm2/h) than that of coumestrol or its coumestrol:b-cyclodextrin complex. With respect to the gel permeation, the complex coumestrol:b-cyclodextrin presented the higher permeation amount (2,7 μg/cm2), as well as the largest lag-time than the others. Coumestrol showed higher solubility when complexated with b-cyclodextrin and it presented reasonable cutaneous permeability in the gel form, being promising to topical application in skin.

A genisteína é uma isoflavona da soja que vem sendo investigada pelo seu potencial antienvelhecimento, baseado nas suas atividades antioxidante, estrogênica e inibidora de proteínas tirosina-quinase. A associação da genisteína com β- ciclodextrina com a formação de um complexo elevou a hidrossolubilidade da isoflavona. A permeabilidade intrínseca da genisteína foi avaliada, bem como sua permeabilidade quando aplicada a partir de gel de hidróxi propilmetilcelulose (HPMC) a 3 %, pelo método de célula de Franz. As associações com β-ciclodextrina, em géis de HPMC 3 %, também foram avaliadas e foi observado um incremento da penetração em favor do complexo produzido em meio aquoso. Devido ao seu alto coeficiente de partição (log P) - 4,36 - a genisteína demonstrou a capacidade de formar reservatórios nas estruturas internas da pele favorecendo sua ação antienvelhecimento na pele. / Genistein is a soy isoflavone that has been investigated for its antiaging potencial, based on antioxidant, estrogenic and proteins tirosin-kinase inhibitor activities. The association of genistein with β-cyclodextrin resulted in a complex formation enhanced the isoflavone hidrosolubity. Genistein instrinsic permeability was evaluated, as well permebility from hydroxypropyl methylcelullose (HPMC) 3 % gel, wtih Franz cell method. The associations with β-cyclodextrin, in HPMC 3 % gels were also evaluated with the observation of na enhancing effect of cyclodextrin in the complex produced in aqueous media. Because its high partition coefficient (log P) - 4,36 - genistein was able to form reservoir in the internal skin layers, favorable to its antiagin action.