Made available in DSpace on 2014-11-10T11:09:59Z (GMT). No. of bitstreams: 0 Previous issue date: 2014-03-18Bitstream added on 2014-11-10T11:58:20Z : No. of bitstreams: 1 000773399_20150318.pdf: 1093846 bytes, checksum: bd937cc46ca78add01ded3bc8b95b99f (MD5) Bitstreams deleted on 2015-02-13T12:47:48Z: 000773399_20150318.pdf,Bitstream added on 2015-02-13T12:48:19Z : No. of bitstreams: 1 000773399_20160601.pdf: 1093828 bytes, checksum: d50b90fb3506b31c4c02f17278a97ae5 (MD5) Bitstreams deleted on 2015-04-13T18:26:31Z: 000773399_20160601.pdf,Bitstream added on 2015-04-13T18:27:08Z : No. of bitstreams: 1 000773399_20160601.pdf: 1093828 bytes, checksum: d50b90fb3506b31c4c02f17278a97ae5 (MD5) / A modificação da superfície de nanopartículas de óxido de ferro tem atraído interesse da comunidade científica devido às várias aplicações deste material em diferentes áreas do conhecimento. A biomedicina é uma área em crescente avanço no uso das nanopartículas, pois a sinergia entre as propriedades magnéticas e a biocompatibilidade da superfície modificada permite o uso destes materiais em diagnóstico e tratamento de doenças, como na liberação controlada de drogas. A aplicação de um campo magnético para guiar as nanopartículas magnéticas conjugadas com fármaco é uma técnica promissora, capaz de reduzir problemas associados a tradicional forma de administração de drogas, aumentando o bem estar de pacientes submetidos a este tratamento. O presente trabalho consiste na funcionalização de nanopartículas magnéticas a base de óxido de ferro pela dextrana derivatizada, utilizando o método da polimerização por miniemulsão inversa, para estudos de liberação de doxorrubicina. As nanopartículas magnéticas de maghemita foram obtidas a partir da oxidação da magnetita sintetizada por coprecipitação, enquanto a dextrana foi derivatizada pelo glicidil metacrilato com intuito de ancorar grupos vinílicos sobre sua estrutura. Estes materiais foram caracterizados por técnicas espectroscópicas, tais como infravermelho, Raman, ressonância magnética de H1 e C13. O óxido de ferro foi encapsulado na dextrana derivatizada através da polimerização por miniemulsão inversa. Nesta etapa, alguns parâmetros de síntese, tais como o tipo e concentração de surfactante, fração de monômeros e teor de óxido de ferro utilizado, foram variados para obter um sistema estável, permitindo seu uso em aplicações biomédicas. O sistema caroço@casca obtido apresentou eficiência de incorporação superior a 80% de doxorrubicina. Entretanto, a porcentagem de liberação da droga por difusão foi baixa durante o período... / The modification of the iron oxide nanoparticles surface has attracted interest of the scientific community due to the large number of applications of this material in different areas of knowledge. The biomedicine is one area in growing improvement in the use of nanoparticles, because the synergy between the magnetic properties and the biocompatibility of the modified surface allows the use these materials in diagnostic and treatment of diseases, as well as the controlled drug delivery. The application of magnetic field to guide the magnetic nanoparticles conjugated with drugs is one promising technique, capable of reduce the problems associated with the traditional way of drugs administration, increasing the welfare of the patients on these treatments. The present work consists in the functionalization of magnetic nanoparticles based on iron oxide by derivatized dextran, using the miniemulsion polymerization method, for doxorubicin release studies. The magnetic nanoparticles of maghemite were obtained from the magnetite oxidation synthesized by coprecipitation, while the dextran was derivatized by the glycidyl methacrylate with the intention of anchor vinyl groups on the surface. These materials were characterized by spectroscopic techniques, as infrared, Raman, magnetic resonance of H1 e C13. The iron oxide was encapsulated in the derivatized dextran through inverse polymerization by miniemulsion. In this phase, some parameters of synthesis, as the type and the surfactant concentration, monomers fraction and percentage of iron oxide, were varied to obtain a stable system, allowing its use in biomedical application. The core@shell system obtained showed incorporation efficiency over 80% of doxorubicin, although the drug release percentage from diffusion was low during the analyzed period.

Made available in DSpace on 2016-06-07T17:11:59Z (GMT). No. of bitstreams: 0 Previous issue date: 2014-03-18. Added 1 bitstream(s) on 2016-06-07T17:16:30Z : No. of bitstreams: 1 000773399.pdf: 2412044 bytes, checksum: bbf98ec27e8e36a7e1d6408d06d16292 (MD5) / A modificação da superfície de nanopartículas de óxido de ferro tem atraído interesse da comunidade científica devido às várias aplicações deste material em diferentes áreas do conhecimento. A biomedicina é uma área em crescente avanço no uso das nanopartículas, pois a sinergia entre as propriedades magnéticas e a biocompatibilidade da superfície modificada permite o uso destes materiais em diagnóstico e tratamento de doenças, como na liberação controlada de drogas. A aplicação de um campo magnético para guiar as nanopartículas magnéticas conjugadas com fármaco é uma técnica promissora, capaz de reduzir problemas associados a tradicional forma de administração de drogas, aumentando o bem estar de pacientes submetidos a este tratamento. O presente trabalho consiste na funcionalização de nanopartículas magnéticas a base de óxido de ferro pela dextrana derivatizada, utilizando o método da polimerização por miniemulsão inversa, para estudos de liberação de doxorrubicina. As nanopartículas magnéticas de maghemita foram obtidas a partir da oxidação da magnetita sintetizada por coprecipitação, enquanto a dextrana foi derivatizada pelo glicidil metacrilato com intuito de ancorar grupos vinílicos sobre sua estrutura. Estes materiais foram caracterizados por técnicas espectroscópicas, tais como infravermelho, Raman, ressonância magnética de H1 e C13. O óxido de ferro foi encapsulado na dextrana derivatizada através da polimerização por miniemulsão inversa. Nesta etapa, alguns parâmetros de síntese, tais como o tipo e concentração de surfactante, fração de monômeros e teor de óxido de ferro utilizado, foram variados para obter um sistema estável, permitindo seu uso em aplicações biomédicas. O sistema caroço@casca obtido apresentou eficiência de incorporação superior a 80% de doxorrubicina. Entretanto, a porcentagem de liberação da droga por difusão foi baixa durante o período... / The modification of the iron oxide nanoparticles surface has attracted interest of the scientific community due to the large number of applications of this material in different areas of knowledge. The biomedicine is one area in growing improvement in the use of nanoparticles, because the synergy between the magnetic properties and the biocompatibility of the modified surface allows the use these materials in diagnostic and treatment of diseases, as well as the controlled drug delivery. The application of magnetic field to guide the magnetic nanoparticles conjugated with drugs is one promising technique, capable of reduce the problems associated with the traditional way of drugs administration, increasing the welfare of the patients on these treatments. The present work consists in the functionalization of magnetic nanoparticles based on iron oxide by derivatized dextran, using the miniemulsion polymerization method, for doxorubicin release studies. The magnetic nanoparticles of maghemite were obtained from the magnetite oxidation synthesized by coprecipitation, while the dextran was derivatized by the glycidyl methacrylate with the intention of anchor vinyl groups on the surface. These materials were characterized by spectroscopic techniques, as infrared, Raman, magnetic resonance of H1 e C13. The iron oxide was encapsulated in the derivatized dextran through inverse polymerization by miniemulsion. In this phase, some parameters of synthesis, as the type and the surfactant concentration, monomers fraction and percentage of iron oxide, were varied to obtain a stable system, allowing its use in biomedical application. The core@shell system obtained showed incorporation efficiency over 80% of doxorubicin, although the drug release percentage from diffusion was low during the analyzed period.

Orientador: Miguel Jafelicci Junior / Banca: Marcio José Tiera / Banca: Carolina Vautier Teixeira Giongo / Resumo: A modificação da superfície de nanopartículas de óxido de ferro tem atraído interesse da comunidade científica devido às várias aplicações deste material em diferentes áreas do conhecimento. A biomedicina é uma área em crescente avanço no uso das nanopartículas, pois a sinergia entre as propriedades magnéticas e a biocompatibilidade da superfície modificada permite o uso destes materiais em diagnóstico e tratamento de doenças, como na liberação controlada de drogas. A aplicação de um campo magnético para guiar as nanopartículas magnéticas conjugadas com fármaco é uma técnica promissora, capaz de reduzir problemas associados a tradicional forma de administração de drogas, aumentando o bem estar de pacientes submetidos a este tratamento. O presente trabalho consiste na funcionalização de nanopartículas magnéticas a base de óxido de ferro pela dextrana derivatizada, utilizando o método da polimerização por miniemulsão inversa, para estudos de liberação de doxorrubicina. As nanopartículas magnéticas de maghemita foram obtidas a partir da oxidação da magnetita sintetizada por coprecipitação, enquanto a dextrana foi derivatizada pelo glicidil metacrilato com intuito de ancorar grupos vinílicos sobre sua estrutura. Estes materiais foram caracterizados por técnicas espectroscópicas, tais como infravermelho, Raman, ressonância magnética de H1 e C13. O óxido de ferro foi encapsulado na dextrana derivatizada através da polimerização por miniemulsão inversa. Nesta etapa, alguns parâmetros de síntese, tais como o tipo e concentração de surfactante, fração de monômeros e teor de óxido de ferro utilizado, foram variados para obter um sistema estável, permitindo seu uso em aplicações biomédicas. O sistema caroço@casca obtido apresentou eficiência de incorporação superior a 80% de doxorrubicina. Entretanto, a porcentagem de liberação da droga por difusão foi baixa durante o período... / Abstract: The modification of the iron oxide nanoparticles surface has attracted interest of the scientific community due to the large number of applications of this material in different areas of knowledge. The biomedicine is one area in growing improvement in the use of nanoparticles, because the synergy between the magnetic properties and the biocompatibility of the modified surface allows the use these materials in diagnostic and treatment of diseases, as well as the controlled drug delivery. The application of magnetic field to guide the magnetic nanoparticles conjugated with drugs is one promising technique, capable of reduce the problems associated with the traditional way of drugs administration, increasing the welfare of the patients on these treatments. The present work consists in the functionalization of magnetic nanoparticles based on iron oxide by derivatized dextran, using the miniemulsion polymerization method, for doxorubicin release studies. The magnetic nanoparticles of maghemite were obtained from the magnetite oxidation synthesized by coprecipitation, while the dextran was derivatized by the glycidyl methacrylate with the intention of anchor vinyl groups on the surface. These materials were characterized by spectroscopic techniques, as infrared, Raman, magnetic resonance of H1 e C13. The iron oxide was encapsulated in the derivatized dextran through inverse polymerization by miniemulsion. In this phase, some parameters of synthesis, as the type and the surfactant concentration, monomers fraction and percentage of iron oxide, were varied to obtain a stable system, allowing its use in biomedical application. The core@shell system obtained showed incorporation efficiency over 80% of doxorubicin, although the drug release percentage from diffusion was low during the analyzed period. / Mestre

Indiana University-Purdue University Indianapolis (IUPUI) / Nanoparticles (NPs) are an emerging technology in biomedical engineering with opportunities in diagnostics, imaging, and drug delivery. NPs can be prepared from a wide range of organic and/or inorganic materials. They can be fabricated to exhibit different characteristics for biomedical applications. The goal of this thesis was to develop NPs with tunable surface properties for selective cell targeting. Specifically, polyelectrolyte complexes composed of heparin (Hep, a growth factor binding glycosaminoglycan) and poly-L-lysine (PLL, a homopolymeric lysine) were prepared via a pulse sonication method. The Hep/PLL core NPs were further layered with additional Hep, tetrazine (Tz) modified Hep, or dextran sulfate (DS). The addition of Tz handle on Hep backbone permitted easy modification of NP surface with norbornene (NB) modified motifs/ligands, including inert poly(ethylene glycol) (PEG), cell adhesive peptides (e.g., RGD), and/or fluorescent marker. Both Hep and DS coated NPs could be readily internalized by J774A.1 monocytes/macrophages, whereas PEGylated NPs effectively reduced cellular uptake/recognition. The versatility of this NP system was further demonstrated by laying DS on the Hep/PLL NP surface. DS-coated NPs were recognized by J774A.1 cells more effectively. Furthermore, DS-layered NPs seemed to reduce IL-10 production on a per cell basis, suggesting that these NPs could be used to alter polarization of macrophages.

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The extracellular enzyme dextransucrase (α 1,6-glucan: D-fructose 2-glucosyltransferase EC 2.4.1.5) was concentrated and purified from the fermentor culture of Leuconostoc mesenteroides (ATCC 10830) by a three-step procedure: centrifugation, membrane ultrafiltration and dialysis, and gel permeation chromatography. Two enzyme preparations which contained dextransucrase activity of 0.95 U/ml and 1.59 U/ml were used for this study. The contaminating enzyme levansucrase was determined to be less than 2%. Shear inactivation study was performed on a batch reactor after subjecting the enzyme solution to a shear rate of 1046 second⁻¹ in the couette viscometer for several different periods of shear exposure time. The kinetic data showed dextransucrase lost part of its catalytic power due to shear inactivation. A correlation curve of the remaining dextransucrase activity versus a dimensionless group-shear strain was generated in the absence of substrate. The data revealed there was not inactivation until shear strain reached 10⁵. Two sets of the correlation curves were generated on the same system but with the presence of substrate at two different levels of enzyme loading. Both results indicated less shear inactivation effect in the presence of substrate and this protective effect on substrate binding strongly endorses Ebert and Schenk's hypothesis that there are substrate induced conformation changes of the enzyme molecule. In order to find any long range effect on the catalytic specificity of the enzyme molecule associated with shear inactivation, the dextransucrase solution with different shear histories was incubated with sucrose. The synthesized dextrans were then precipitated out by a 83% ethanol-water mixture. When the molecular structure of the dextran precipitates was analyzed on ¹³C NMR spectroscopy at 90°C, it was found that there was no change in the polymer structure. But one of the spectra which corresponded to the dextransucrase preparation with the longest shear history contained resonance peaks of levan. All these findings lead to the conclusion that shear induced conformational changes of the enzyme molecule can not alter their catalytic specificity, and that the sensitivity toward shear inactivation is much greater by dextransucrase than by the small quantity of levansucrase present. / Ph. D.

O diagnóstico precoce de tumores e metástase constitui atualmente o elemento de maior impacto dentro das políticas de saúde públicas contra o câncer. No câncer de mama e melanoma, a técnica de biópsia de linfonodo sentinela, para o diagnostico de metástase, tem sido muito utilizada evitando a dissecção total dos nodos da região anatômica afetada, e permitindo definir com precisão o procedimento terapêutico a utilizar. O objetivo principal deste trabalho centrou-se no desenvolvimento de conjugados radiomarcados de dextran-manose para diagnóstico, utilizando o núcleo de tecnécio altamente estável, [99mTc(CO)3]+. A cisteína, ligante tridentado, foi incorporada na estrutura dos conjugados, como agente quelante do Tecnécio-99m. As condições de marcação definidas para os produtos avaliados garantiram altos valores de pureza radioquímica (>90%) e atividade específica (>59,9 MBq/nmol) assim como uma alta estabilidade in vitro. Os conjugados de dextran-cisteína-manose demonstraram uma captação superior (4 vezes maior) nos nodos linfáticos em relação aos homólogos que não possuíam manose na estrutura. O conjugado de dextran-cisteína-manose de 30 kDa radiomarcado (99mTc-DCM2) foi o traçador com melhor desempenho biológico entre os avaliados à diferentes atividades injetadas. Demonstrou-se que concentrações superiores a 1 M favorecem a retenção do produto nos nodos linfáticos. As comparações com radiofármacos já utilizados no Brasil (Dextran-500 e Fitato) para detecção de linfonodo sentinela evidenciaram a superioridade do 99mTc-DCM2. / Early diagnosis of tumors and metastasis is the current cornerstone in public health policies directed towards the fights against cancer. In breast cancer and melanoma, the sentinel lymph node biopsy has been widely used for diagnoses of metastasis. The minor impact in patient of this technique compared with total nodes dissection and the accurate definition of therapeutic strategies have powered its spreading. The aim of this work was the development of radiolabeled dextran-mannose conjugates for diagnosis using the stable technetium core [99mTc(CO)3]+. Cysteine, a trident ligand, was attached to the conjugates backbone, as a chelate for 99mTc labeling. Radiolabeling conditions established for all products considered in this study showed high radiochemical purities (> 90%) and specific activities (>59,9 MBq/nmol) as well and high stability obtained through in vitro tests. The lymphatic node uptake increased significantly (4-folds) when mannose units were added to the conjugates compared with those without this monosaccharide. The radiolabeled cysteine-mannose-dextran conjugate with 30 kDa (99mTc-DCM2) showed the best performance at different injected activities among the studied tracers. Concentrations of this radiocomplex higher than 1 M demonstrated an improvement of lymph node uptakes. Comparisons of 99mTc-DCM2 performance with commercial radiopharmaceuticals in Brazil market for lymph node detection showed its upper profile.

O diagnóstico precoce de tumores e metástase constitui atualmente o elemento de maior impacto dentro das políticas de saúde públicas contra o câncer. No câncer de mama e melanoma, a técnica de biópsia de linfonodo sentinela, para o diagnostico de metástase, tem sido muito utilizada evitando a dissecção total dos nodos da região anatômica afetada, e permitindo definir com precisão o procedimento terapêutico a utilizar. O objetivo principal deste trabalho centrou-se no desenvolvimento de conjugados radiomarcados de dextran-manose para diagnóstico, utilizando o núcleo de tecnécio altamente estável, [99mTc(CO)3]+. A cisteína, ligante tridentado, foi incorporada na estrutura dos conjugados, como agente quelante do Tecnécio-99m. As condições de marcação definidas para os produtos avaliados garantiram altos valores de pureza radioquímica (>90%) e atividade específica (>59,9 MBq/nmol) assim como uma alta estabilidade in vitro. Os conjugados de dextran-cisteína-manose demonstraram uma captação superior (4 vezes maior) nos nodos linfáticos em relação aos homólogos que não possuíam manose na estrutura. O conjugado de dextran-cisteína-manose de 30 kDa radiomarcado (99mTc-DCM2) foi o traçador com melhor desempenho biológico entre os avaliados à diferentes atividades injetadas. Demonstrou-se que concentrações superiores a 1 M favorecem a retenção do produto nos nodos linfáticos. As comparações com radiofármacos já utilizados no Brasil (Dextran-500 e Fitato) para detecção de linfonodo sentinela evidenciaram a superioridade do 99mTc-DCM2. / Early diagnosis of tumors and metastasis is the current cornerstone in public health policies directed towards the fights against cancer. In breast cancer and melanoma, the sentinel lymph node biopsy has been widely used for diagnoses of metastasis. The minor impact in patient of this technique compared with total nodes dissection and the accurate definition of therapeutic strategies have powered its spreading. The aim of this work was the development of radiolabeled dextran-mannose conjugates for diagnosis using the stable technetium core [99mTc(CO)3]+. Cysteine, a trident ligand, was attached to the conjugates backbone, as a chelate for 99mTc labeling. Radiolabeling conditions established for all products considered in this study showed high radiochemical purities (> 90%) and specific activities (>59,9 MBq/nmol) as well and high stability obtained through in vitro tests. The lymphatic node uptake increased significantly (4-folds) when mannose units were added to the conjugates compared with those without this monosaccharide. The radiolabeled cysteine-mannose-dextran conjugate with 30 kDa (99mTc-DCM2) showed the best performance at different injected activities among the studied tracers. Concentrations of this radiocomplex higher than 1 M demonstrated an improvement of lymph node uptakes. Comparisons of 99mTc-DCM2 performance with commercial radiopharmaceuticals in Brazil market for lymph node detection showed its upper profile.