Desenvolvimento, caracterização e funcionalização de nanofilmes a partir de galactomanana e alginato

Orientadora: Profª Drª Maria Rita SierakowskiCo-orientador: Prof. Dr. Gero DecherTese (doutorado) - Universidade Federal do Paraná, Setor de Ciências Biológicas, Programa de Pós-Graduação em Bioquímica. Defesa: Curitiba, 22/11/2011Inclui bibliografiasResumo: As galactomananas (GM) podem modificar a viscosidade dos meios aquosos e o alginato (AL) em presenca de ions Ca2+ geleifica, apresenta varias aplicacoes, entre as quais, a formacao de filmes finos. As propriedades fisico quimicas desses dois polissacarideos em solucao, incluindo a interacao entre eles, foram estudadas, com o ob etivo de desenvolver materiais nanoestruturados na forma de filmes finos e utiliza-los para futuras aplicacoes biologicas. A GM empregada foi extraida de sementes de Leucaena leucocephala (GML) e, apos purificacao, apresentou um rendimento de 19,3% (m/m). O AL foi adquirido comercialmente. Ambos foram caracterizados como sendo homogeneos e com conformacao ao acaso (0,5<ƒ¿<0,8). Apresentaram razao molar Man/Gal 1,7 e M/G 1; e de 7,1x105 e 2,9x105g/mol, respectivamente, para a GML e o AL os quais, em analises de toxicidade, indicaram pouca alteracao na viabilidade celular. O comportamento de solucoes aquosas foi caracterizado por espectroscopia de fluorescencia (sonda de pireno) e forneceu os valores de CAC, 1,0 mg/mL para a GML e 0,57 mg/mL para o AL. Analises por dicroismo circular confirmaram a interacao da mistura GML-AL com albumina (ALB), atraves da alteracao nos espectros do arranjo das cadeias dos polimeros. A mistura GML-AL (15 mg/mL, 50%, m/m) em analises reologicas mostrou a formacao de um gel viscoelastico em presenca de ions Ca2+ quando em pH 7,2 e em 11,2. As analises por microscopia de fluorescencia dos derivados dos dois polissacarideos comprovaram que a mistura GML-AL foi adsorvida sobre a lamina de vidro e nao foi removida durante as lavagens. A formacao de filmes finos em monocamadas com a adsorcao de GML sobre substratos de Si-aminofuncionalizados foi avaliada por isotermas e elipsometria, onde se determinou a concentracao (1mg/mL) o pH (4,0) e a espessura (D, 1,5 nm). Sobre os filmes de GML foram adsorvidas proteinas como a ALB (D, 6,2 nm em pH 5,5) e a Concanavalina A (D, 1,6 nm em pH 4,5) e, tambem, diferentes sorotipos do virus da dengue, sendo que o tipo 2 foi o que formou filmes mais espessos (D 3,7 nm em pH 7,2). A mistura GML-AL que tem caracteristicas de um polianion, comprovada pelo potencial zeta, foi aplicada em filmes multicamadas (LBL) obtidos atraves de diferentes tecnicas de deposicao e com diferentes polications poli(alilamina hidroclorada) (PAH), poli-lisina (PLL) ou quitosana (CHI). Por analises de elipsometria e AFM os filmes contendo PAH foram os mais espessos e menos rugosos (rms 6 nm), e, por analises de QCM, os filmes com PLL foram os menos rigidos, e os com CHI foram o que tiveram maior mobilidade de agua entre as camadas. Os filmes LBL foram avaliados em testes de adesao celular e todos permitiram o crescimento e o espalhamento das celulas (HeLa) em suas superficies, mesmo apos 8 dias de contato. Esses resultados sugerem uma potencial aplicacao dos filmes monocamadas de GML para o desenvolvimento de biomateriais, incremento de biosensores e dispositivos iotecnologicos; e os filmes LBL do polianion GML-AL como um potencial modelo para estudos de crescimento de celulas, e em aplicacoes terapeuticas para engenharia de tecidos.Abstract: Galactomannans (GM) can modify aqueous solution viscosities and sodium alginate (AL) gelatinized in presence of calcium ions, is also widely used in applications such as thin films formation. The physical-chemical properties of these polysaccharides in solutions, including their interaction, were evaluated aimed to develop nanostructured materials, such as thin films and use them into future biological applications. GM was extracted from the seeds of Leucaena leucocephala (GML) and showing a yield of 19.35% (w/w) after purification. AL was obtained from a commercial source. Both of them were characterized as homogeneous with random coil conformation (0,5<á<0,8), Man/Gal 1,7 and M/G 1 molar ratio and of 7,1 x105 and 2,9 x105g/mol, respectively, for GML and AL. The GML and AL cytotoxicity analysis showed almost no change in cell viability. The CAC values of 1,0 mg/mL for GML and 0,57 mg/mL for AL were obtained by fluorescence spectroscopies (pyrene probe). Circular dichroism confirmed the GML-AL mixture interaction with ALB through the spectra changes in the polymers chains conformations. The GML-AL mixture (15 mg/mL, 50%, w/w) with calcium ions at pH 7.2 and 11.2 showed a viscoelastic gel behaviour by rheological analysis. The fluorescence microscopy from the two derivate polysaccharides proved that the GML-AL mixture was adsorbed onto glass and was not removed during the wash steps. The GML isotherms adsorption onto amine-functionalized silicon formed a monolayer thin film, with the concentration of 1 mg/mL, pH of 4.0 and a 1.5 nm thickness set by ellipsometry. Proteins were adsorbed onto GML films, such as ALB (D: 6.2 nm, pH 5.5) and Concanavalin A (D: 1.6 nm, pH 4.5), and also different serotypes of dengue virus, being the virus type 2 the one that obtained the highest adsorption value (D: 3.7 nm, pH 7.2). The GML-AL mixture characterized as a polyanion by zeta potential measurements was applied in multilayers films (LBL). LBL films were obtained by different deposition techniques and using different polycations, poly(allylamine hydrocloride) (PAH), poly-L-lysine (PLL) or chitosan (CHI). By ellipsometry and AFM, films containing PAH were the thickest measured (RMS 6 nm), and from QCM-D analysis films with PLL were the less rigid and the CHI were the one that had the higher mobility of water between the layers. The LBL films were evaluated in cell adhesion experiments in which all of them allowed the cell growth and spreading (Hela cells) in their surface even after 8 days of contact. These results suggest a potential application of GML monolayers films for the evelopment of biomaterials, for a rapid estimation of biosensors; and for biotechnological devices; and the application of LBL films using GML-AL as polyanion as a potential model for studies of cell growth, and therapeutic for tissue engineering

Estudos de interação entre galactomanana e alginato e possíveis aplicações /

Orientadora: Maria Rita SierakowskiDissertação (mestrado) - Universidade Federal do Paraná, Setor de Ciências Biológicas, Programa de Pós-Graduação em Bioquímica. Defesa: Curitiba, 2007Inclui bibliografi

Formation, drug-release kinetics and solution-stability of N-acetyl-N-carboxymethyl chitosan nanoparticles as potential drug carriers

Nano-aggregates of N-acetyl-N-carboxymethyl chitosan (NCac) were studied at 0.5 mg.mL-1 using pyrene fluorimetry analysis in 0.1 mol.L-1 phosphate buffer (pH 7.4). The size and morphology of the aggregates were determined by dynamic light scattering and atomic force microscopy. The stability of the particles for periods up to 20 h in buffer was determined. Camptothecin was entrapped in the particles using various methods and the rate constant for drug release (k) was determined. Lower k values indicate strong interactions between the drug and the hydrophobic core of the polymeric micelles

Desenvolvimento, caracterização e funcionalização de nanofilmes a partir de galactomanana e alginato

Orientadora: Profª Drª Maria Rita SierakowskiCo-orientador: Prof. Dr. Gero DecherTese (doutorado) - Universidade Federal do Paraná, Setor de Ciências Biológicas, Programa de Pós-Graduação em Bioquímica. Defesa: Curitiba, 22/11/2011Inclui bibliografiasResumo: As galactomananas (GM) podem modificar a viscosidade dos meios aquosos e o alginato (AL) em presenca de ions Ca2+ geleifica, apresenta varias aplicacoes, entre as quais, a formacao de filmes finos. As propriedades fisico quimicas desses dois polissacarideos em solucao, incluindo a interacao entre eles, foram estudadas, com o ob etivo de desenvolver materiais nanoestruturados na forma de filmes finos e utiliza-los para futuras aplicacoes biologicas. A GM empregada foi extraida de sementes de Leucaena leucocephala (GML) e, apos purificacao, apresentou um rendimento de 19,3% (m/m). O AL foi adquirido comercialmente. Ambos foram caracterizados como sendo homogeneos e com conformacao ao acaso (0,5<ƒ¿<0,8). Apresentaram razao molar Man/Gal 1,7 e M/G 1; e de 7,1x105 e 2,9x105g/mol, respectivamente, para a GML e o AL os quais, em analises de toxicidade, indicaram pouca alteracao na viabilidade celular. O comportamento de solucoes aquosas foi caracterizado por espectroscopia de fluorescencia (sonda de pireno) e forneceu os valores de CAC, 1,0 mg/mL para a GML e 0,57 mg/mL para o AL. Analises por dicroismo circular confirmaram a interacao da mistura GML-AL com albumina (ALB), atraves da alteracao nos espectros do arranjo das cadeias dos polimeros. A mistura GML-AL (15 mg/mL, 50%, m/m) em analises reologicas mostrou a formacao de um gel viscoelastico em presenca de ions Ca2+ quando em pH 7,2 e em 11,2. As analises por microscopia de fluorescencia dos derivados dos dois polissacarideos comprovaram que a mistura GML-AL foi adsorvida sobre a lamina de vidro e nao foi removida durante as lavagens. A formacao de filmes finos em monocamadas com a adsorcao de GML sobre substratos de Si-aminofuncionalizados foi avaliada por isotermas e elipsometria, onde se determinou a concentracao (1mg/mL) o pH (4,0) e a espessura (D, 1,5 nm). Sobre os filmes de GML foram adsorvidas proteinas como a ALB (D, 6,2 nm em pH 5,5) e a Concanavalina A (D, 1,6 nm em pH 4,5) e, tambem, diferentes sorotipos do virus da dengue, sendo que o tipo 2 foi o que formou filmes mais espessos (D 3,7 nm em pH 7,2). A mistura GML-AL que tem caracteristicas de um polianion, comprovada pelo potencial zeta, foi aplicada em filmes multicamadas (LBL) obtidos atraves de diferentes tecnicas de deposicao e com diferentes polications poli(alilamina hidroclorada) (PAH), poli-lisina (PLL) ou quitosana (CHI). Por analises de elipsometria e AFM os filmes contendo PAH foram os mais espessos e menos rugosos (rms 6 nm), e, por analises de QCM, os filmes com PLL foram os menos rigidos, e os com CHI foram o que tiveram maior mobilidade de agua entre as camadas. Os filmes LBL foram avaliados em testes de adesao celular e todos permitiram o crescimento e o espalhamento das celulas (HeLa) em suas superficies, mesmo apos 8 dias de contato. Esses resultados sugerem uma potencial aplicacao dos filmes monocamadas de GML para o desenvolvimento de biomateriais, incremento de biosensores e dispositivos iotecnologicos; e os filmes LBL do polianion GML-AL como um potencial modelo para estudos de crescimento de celulas, e em aplicacoes terapeuticas para engenharia de tecidos.Abstract: Galactomannans (GM) can modify aqueous solution viscosities and sodium alginate (AL) gelatinized in presence of calcium ions, is also widely used in applications such as thin films formation. The physical-chemical properties of these polysaccharides in solutions, including their interaction, were evaluated aimed to develop nanostructured materials, such as thin films and use them into future biological applications. GM was extracted from the seeds of Leucaena leucocephala (GML) and showing a yield of 19.35% (w/w) after purification. AL was obtained from a commercial source. Both of them were characterized as homogeneous with random coil conformation (0,5<á<0,8), Man/Gal 1,7 and M/G 1 molar ratio and of 7,1 x105 and 2,9 x105g/mol, respectively, for GML and AL. The GML and AL cytotoxicity analysis showed almost no change in cell viability. The CAC values of 1,0 mg/mL for GML and 0,57 mg/mL for AL were obtained by fluorescence spectroscopies (pyrene probe). Circular dichroism confirmed the GML-AL mixture interaction with ALB through the spectra changes in the polymers chains conformations. The GML-AL mixture (15 mg/mL, 50%, w/w) with calcium ions at pH 7.2 and 11.2 showed a viscoelastic gel behaviour by rheological analysis. The fluorescence microscopy from the two derivate polysaccharides proved that the GML-AL mixture was adsorbed onto glass and was not removed during the wash steps. The GML isotherms adsorption onto amine-functionalized silicon formed a monolayer thin film, with the concentration of 1 mg/mL, pH of 4.0 and a 1.5 nm thickness set by ellipsometry. Proteins were adsorbed onto GML films, such as ALB (D: 6.2 nm, pH 5.5) and Concanavalin A (D: 1.6 nm, pH 4.5), and also different serotypes of dengue virus, being the virus type 2 the one that obtained the highest adsorption value (D: 3.7 nm, pH 7.2). The GML-AL mixture characterized as a polyanion by zeta potential measurements was applied in multilayers films (LBL). LBL films were obtained by different deposition techniques and using different polycations, poly(allylamine hydrocloride) (PAH), poly-L-lysine (PLL) or chitosan (CHI). By ellipsometry and AFM, films containing PAH were the thickest measured (RMS 6 nm), and from QCM-D analysis films with PLL were the less rigid and the CHI were the one that had the higher mobility of water between the layers. The LBL films were evaluated in cell adhesion experiments in which all of them allowed the cell growth and spreading (Hela cells) in their surface even after 8 days of contact. These results suggest a potential application of GML monolayers films for the evelopment of biomaterials, for a rapid estimation of biosensors; and for biotechnological devices; and the application of LBL films using GML-AL as polyanion as a potential model for studies of cell growth, and therapeutic for tissue engineering

Estudos de interação entre galactomanana e alginato e possíveis aplicações /

Orientadora: Maria Rita SierakowskiDissertação (mestrado) - Universidade Federal do Paraná, Setor de Ciências Biológicas, Programa de Pós-Graduação em Bioquímica. Defesa: Curitiba, 2007Inclui bibliografi

Galactomannan thin films as supports for the immobilization of Concanavalin A and/or dengue viruses

The immobilization of the glucose/mannose-binding lectin from Concanavalia ensiformis seeds (ConA) onto a monolayer made of a galactomannan extracted from Leucaena leucocephala seeds (GML), which was adsorbed onto - amino-terminated surfaces, was investigated by means of ellipsometry and atomic force microscopy. The mean thickness of GML monolayer, which polysaccharide consists of linear 1 -&gt; 4-linked beta-D-mannopyranosil units partially substituted at C-6 by alpha-D-galactopyranosyl units, amounted to (1.5 +/- 0.2) nm. ConA molecules adsorbed onto GML surfaces forming (2.0 +/- 0.5) nm thick layers. However, in the presence of mannose the adsorption failed, indicating that ConA binding sites were blocked by mannose and were no longer available for mannose units present in the GML backbone. The GML film was also used as support for the adsorption of three serotypes of dengue virus particles (DENV-1, DENV-2 and DENV-3), where DENV-2 formed the thickest film (4 +/- 2) nm. The adsorbed layer of DENV-2 onto ConA-covered GML surfaces presented mean thickness values similar to that determined for DENV-2 onto bare GML surfaces. The addition of free mannose units prevented DENV-2 adsorption onto ConA-covered GML films by similar to 50%, suggesting competition between virus and mannose for ConA binding sites. This finding suggests that if ConA is also adsorbed to GML surface and its binding site is blocked by free mannose, virus particles are able to recognized GML mannose unities substituted by galactose. interactions between polysaccharides thin films, proteins, and viruses are of great relevance since they can provide basis for the development of biotechnological devices. These results indicate that GML is a potential polysaccharide for biomaterials development, as those could involve interactions between ConA in immune system and viruses. (C) 2011 Elsevier B.V. All rights reserved.Conselho Nacional de Pesquisa (CNPq)CNPq (Conselho Nacional de Pesquisa)CAPES (Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior)Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES)Fundacao de Amparo Pesquisa do Estado de Sao Paulo (FAPESP)FAPESP (Fundacao de Amparo Pesquisa do Estado de Sao Paulo