| Name: | Description: | Size: | Format: | |
|---|---|---|---|---|
| 5.05 MB | Adobe PDF |
Authors
Abstract(s)
Um dos grandes desafios a nível da saúde altamente é a obesidade, afetando cerca de 500 milhões de pessoas em todo o mundo. Com a obesidade, aumenta o aparecimento de doenças como os diabetes tipo 2, doenças cardíacas, acidentes vasculares e cancros. Uma das abordagens destinadas a revolucionar a ingestão de alimentos e a mudança no tratamento da obesidade é a utilização de péptidos de soja, com a capacidade de suprimir a ingestão de alimentos e inibir o esvaziamento gástrico, conferindo assim, o efeito de saciedade. No entanto, até o momento, os inibidores de protease mostraram efeitos limitados nos estudos de intervenção humana. O encapsulamento desses compostos saciantes tem sido explorado de forma limitada, apesar da sua natureza proteica os tornar suscetíveis à digestão por proteases. Uma estratégia inteligente para o desenvolvimento de nanotransportadores, permitindo a liberação sob demanda de inibidores de tripsina, é o seu encapsulamento em biopolímeros mucoadesivos, que podem ajudar a melhorar a biodisponibilidade no trato gastrointestinal. Neste contexto, pretende-se estudar os biopolímeros mucoadesivos mais adequados para a distribuição de péptidos no trato gastrointestinal. Este estudo iniciou-se com a síntese das nanopartículas a partir de biopolímeros não tóxicos como o quitosano (CH), o ácido hialurónico (HA) e a condroitina de sódio (CS), através do método da coacervação. As nanopartículas foram de seguida caracterizadas físico-químicamente e quanto à mucoadesividade, foram selecionadas para este trabalho as de CHβ/TPP/HA uma vez que, apresentavam as características pretendidas. Para comprovar que as nanopartículas eram mucoadesivas, foi realizado um estudo de internalização das partículas, onde estas foram incubadas durante 4h com células Caco2/HT29-MTX diferenciadas e observadas através de microscospia confocal. Este estudo veio mais uma vez provar que as partículas são mucoadesivas, ficando aderidas na superfície das células após lavagem com PBS. Seguidamente foi otimizada a eficiência da encapsulação do péptido saciante de soja (PSS), nas nanopartículas usando várias concentrações deste. Percebeu-se assim que, a percentagem ótima de encapsulamento do PSS foi de 51% que corresponde a uma concentração de 100 mg/mL de PSS. Realizou-se também um estudo da estabilidade das partículas ao longo do tempo e concluise que a altas concentrações de nanopartículas, existe agregação das mesmas, enquanto que diluídas são mais estáveis ao longo do tempo. As nanopartículas mais estáveis são as que não tem PSS encapsulado. No entanto, era necessário diluir o PSS nanoencapsulado e estudar o seu comportamento ao longo do tempo para poder comparar. Para determinar o efeito do encapsulamento na permeabilidade intestinal do composto ativo, realizou-se um estudo da permeabilidade do PSS (livre e encapsulado) usando linhas celulares Caco-2/HT29-MTX. A cultura das linhas celulares foi feita na proporção 9:1 (Caco-2/HT29- MTX) durante 21 dias para mimetizar o epitélio intestinal. Como esperado grande parte do PSS não passa através da monocamada de células, mas quando está nanoencapsulado nas partículas desenvolvidas a permeabilidade aumenta 5 vezes. Procurou-se também estudar a citoxicidade das nanopartículas em células Caco-2, através do estudo de viabilidade celular em MTT e resazurina após 4 e 24h de incubação. Conclui-se que as nanopartículas não eram tóxicas para as células, indicando que não são tóxicas para o trato intestinal. Por fim foi realizado o estudo do encapsulamento do PSS em condições gastrointestinais in vitro, em que o PSS livre e o PSS nanoencapsulado foram expostos às condições gastrointestinais. Os resultados mostram que o PSS se hidrolisa na fase gástrica não chegando à fase intestinal. No caso do PSS nanoencapsulado chega até à fase intestinal, no entanto parte das partículas se desintegram nesta fase. Após o trabalho realizado conclui-se que, as nanopartículas apesar de serem resistentes à fase gástrica, para que o sistema se torne mais eficiente, seria necessário encapsular as nanopartículas em micropartículas para aumentar a estabilidade.
One of the major health challenges at present is the obesity, which affects approximately 500 million people worldwide. With obesity, the diseases like diabetes type 2, heart diseases, cardiac diseases and cancers increased. One approach to revolutionizing food intake is the release of soybean peptides, with the capacity to suppress the food intake and inhibit the gastric emptying, giving the effect of satiety. However, the protease inhibitors have shown limited effects in human intervention studies, at present. The encapsulation of these satiating compounds has been exploited to a limited extent, despite their protein nature rendering them susceptible to protease digestion. One intelligent strategy, for the development of nanocarries, allowing on-demand release of trypsin inhibitors, is their encapsulation in mucoadhesive biopolymers, which may help improve bioavailability in the gastrointestinal tract. In this context, we pretend to study the mucoadhesive biopolymers more appropriate for the distribution of peptides in the gastrointestinal tract. This study begins with the synthesis of nanoparticles from non-toxic biopolymers such as chitosan (CH), the hyaluronic acid (HA) and the sodium chondroitin (CS), through the coacervation method. After, the nanoparticles were characterize physical-chemical and the mucoadhesive was selected for this study the CHβ/TPP/HA because, these nanoparticles had the pretended characteristics. To confirm the theory that the nanoparticles were mucoadhesive, it was made a study of internalization of the particles, where these were incubated during 4 h with differentiated Caco2/HT29-MTX cells and observed by a confocal microscopy. This study proved again that the particles are mucoadhesive, becoming sticking to the surface of the cells after washing with PBS. After, was optimized the efficiency for encapsulation of the soybean satiating peptide (PSS), was encapsulated in the nanoparticles CHβ/TPP/HA, at various concentrations. It was thus realized that the optimal percentage of PSS encapsulation was 51% corresponding to a concentration of 100 mg/mL PSS. A study of the stability of the particles was also carried out over time and it was concluded that at high concentrations of nanoparticles, there is aggregation of the these, while diluted are more stable over time. The most stable nanoparticles are those that do not have encapsulated PSS. However, it was necessary to dilute the nanoencapsulated PSS and study its behavior over time to be able to compare. To determine the effect of encapsulation on the intestinal permeability of the active compound, a permeability study of PSS (free and encapsulated) was performed using Caco- 2/HT29-MTX cell lines. The culture of the cell lines was done in the ratio 9:1 (Caco-2/HT29- MTX) for 21 days to minimize the intestinal epithelium. As expected the major of the PSS doesn’t pass through the monolayer cell, but when it is nanoencapsulated in the developed particles the permeability increases 5 times. We also study the citoxicity of nanoparticles in Caco-2 cells by the study of viability cells in MTT and resazurin after at 4 and 24h of incubation. It is concluded that the applied nanoparticles were not toxics to the cells, and for this reason they are not toxic to the intestinal tract. In the end, was made an in vitro digestion study, where the free PSS and the nanoencapsulated PSS were exposed to gastrointestinal conditions. The results show that the PSS hydrolyzes in the gastric phase not reaching the intestinal phase. In the case of nanoencapsulated PSS it reaches the intestinal stage, however it is understood that some parts of the particles were disintegrated at this point. After study, it was conclude that, although the nanoparticles are resistant to the gastric phase, in order for the system to become more efficient, it would be necessary to encapsulate the nanoparticles in microparticles to increase stability.
One of the major health challenges at present is the obesity, which affects approximately 500 million people worldwide. With obesity, the diseases like diabetes type 2, heart diseases, cardiac diseases and cancers increased. One approach to revolutionizing food intake is the release of soybean peptides, with the capacity to suppress the food intake and inhibit the gastric emptying, giving the effect of satiety. However, the protease inhibitors have shown limited effects in human intervention studies, at present. The encapsulation of these satiating compounds has been exploited to a limited extent, despite their protein nature rendering them susceptible to protease digestion. One intelligent strategy, for the development of nanocarries, allowing on-demand release of trypsin inhibitors, is their encapsulation in mucoadhesive biopolymers, which may help improve bioavailability in the gastrointestinal tract. In this context, we pretend to study the mucoadhesive biopolymers more appropriate for the distribution of peptides in the gastrointestinal tract. This study begins with the synthesis of nanoparticles from non-toxic biopolymers such as chitosan (CH), the hyaluronic acid (HA) and the sodium chondroitin (CS), through the coacervation method. After, the nanoparticles were characterize physical-chemical and the mucoadhesive was selected for this study the CHβ/TPP/HA because, these nanoparticles had the pretended characteristics. To confirm the theory that the nanoparticles were mucoadhesive, it was made a study of internalization of the particles, where these were incubated during 4 h with differentiated Caco2/HT29-MTX cells and observed by a confocal microscopy. This study proved again that the particles are mucoadhesive, becoming sticking to the surface of the cells after washing with PBS. After, was optimized the efficiency for encapsulation of the soybean satiating peptide (PSS), was encapsulated in the nanoparticles CHβ/TPP/HA, at various concentrations. It was thus realized that the optimal percentage of PSS encapsulation was 51% corresponding to a concentration of 100 mg/mL PSS. A study of the stability of the particles was also carried out over time and it was concluded that at high concentrations of nanoparticles, there is aggregation of the these, while diluted are more stable over time. The most stable nanoparticles are those that do not have encapsulated PSS. However, it was necessary to dilute the nanoencapsulated PSS and study its behavior over time to be able to compare. To determine the effect of encapsulation on the intestinal permeability of the active compound, a permeability study of PSS (free and encapsulated) was performed using Caco- 2/HT29-MTX cell lines. The culture of the cell lines was done in the ratio 9:1 (Caco-2/HT29- MTX) for 21 days to minimize the intestinal epithelium. As expected the major of the PSS doesn’t pass through the monolayer cell, but when it is nanoencapsulated in the developed particles the permeability increases 5 times. We also study the citoxicity of nanoparticles in Caco-2 cells by the study of viability cells in MTT and resazurin after at 4 and 24h of incubation. It is concluded that the applied nanoparticles were not toxics to the cells, and for this reason they are not toxic to the intestinal tract. In the end, was made an in vitro digestion study, where the free PSS and the nanoencapsulated PSS were exposed to gastrointestinal conditions. The results show that the PSS hydrolyzes in the gastric phase not reaching the intestinal phase. In the case of nanoencapsulated PSS it reaches the intestinal stage, however it is understood that some parts of the particles were disintegrated at this point. After study, it was conclude that, although the nanoparticles are resistant to the gastric phase, in order for the system to become more efficient, it would be necessary to encapsulate the nanoparticles in microparticles to increase stability.
Description
Keywords
Nanopartículas Quitosano Ácido hialurónico Mucoadesividade Encapsulação Saciedade Péptido de soja Nanoparticles Chitosan Hyaluronic acid Mucoadhesiveness Encapsulation Satiety Soybean peptide