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Abstract(s)
Visando o aumento da biodisponibilidade dos compostos bioactivos administrados por via
oral (com especial ênfase para a absorção bucal), o plano de trabalhos deste programa doutoral
visou a optimização de formulações de filmes orais e de micro- e nanopartículas que, conjugados
constituam sistemas inovadores com atividade sinérgica. As moléculas bioactivas seleccionadas
para estudar o comportamento e eficácia das formulações optimizadas foram a cafeína e dois
péptidos presentes na proteína do soro do leite com actividades antihipertensora (sequência:
KGYGGVSLPEW) e relaxante (sequência: YLGYLEQLLR). O processo de optimização das
formulações foi iniciado pela seriação e comparação preliminar dos excipientes para a produção
de filmes e micro/nanopartículas. No primeiro estudo realizado, foram optimizadas e comparadas
duas formulações de filmes (usando-se os polímeros carboximetilcelulose sódica e gelatina tipo
A) como veículos para libertação oral de cafeína. Verificou-se, através da análise por
espectroscopia no infravermelho por transformada de Fourier com reflectância total atenuada
(FTIR-ATR), que a estrutura química da cafeína não fora alterada durante o processo de produção
dos filmes. Concluiu-se também, através do ensaio de dissolução estabelecido pela Farmacopeia
Americana (USP), que os filmes produzidos com gelatina tipo A permitiram uma libertação mais
lenta da cafeína ao passo que os filmes de carboximetilcelulose apresentaram um perfil de
libertação imediata. Em concordância, o valor de permeabilidade aparente da cafeína,
determinada através do ensaio de permeabilidade ex vivo, através de excisões de intestino delgado
de origem porcina, verificou-se superior quando esta foi veiculada pelos filmes de
carboximetilcelulose, comparativamente com os filmes de gelatina tipo A. O tempo de
desintegração de ambas as formulações mostrou-se, contudo, demasiado alto para formulações
orodispersíveis, não ocorrendo desintegração completa após 30 segundos. Ainda na sequência da
escolha do polímero com melhores características para integrar a composição de filmes orais,
uma nova formulação contendo goma-guar foi optimizada por desenho factorial. Os filmes de
goma-guar apresentaram características mecânicas e físico-químicas superiores às verificadas
para os filmes de carboximetilcelulose e gelatina, tomando-se como factores de decisão a
capacidade de absorção de água, a erosão em saliva artificial e o tempo de desintegração
apresentados pelos filmes de goma-guar. Procedeu-se também à optimização (por desenho
fatorial) de uma formulação de micropartículas de alginato que garantissem, em conjunto com os
filmes de goma-guar, uma libertação controlada de cafeína, assim como uma maior
biodisponibilidade da mesma. A associação de micropartículas de alginato aos filmes de gomaguar
– GfB - não induziu alterações das características químicas da cafeína, de acordo com o
verificado por FTIR-ATR, nem toxicidade para as linhas celulares usadas para mimetizar as
mucosas bucal (TR146) e intestinal (Caco-2/HT29-MTX), de acordo com os resultados obtidos
pelo ensaio de viabilidade celular MTT (Brometo de 3-(4,5-Dimethylthiazol-2-yl)-2,5-
Diphenyltetrazólio). Adicionalmente, os perfis de libertação e permeabilidade in vitro (através
das linhas celulares TR146 e Caco-2/HT29-MTX cultivadas em camada) e ex vivo (através de
epitélio intestinal de origem porcina) mostraram-se mais lentos que os observados para as
micropartículas de alginato, filmes de goma-guar ou com a solução controlo de cafeína. A
formulação GfB promoveu o aumento do contacto efectivo entre a cafeína e o epitélio bucal,
oferecendo uma permeação mais completa ao longo do tempo.
De forma a incrementar também a biodisponibilidade do péptido KGYGGVSLPEW com
actividade anti-hipertensora, as micropartículas de alginato foram substituídas por nanopartículas
de ácido poli(láctico-co-glicólico) – PLGA - por estas oferecerem uma eficácia de associação superior, assim como um maior potencial de permeação das membranas biológicas, dado o
tamanho de partícula ser significativamente inferior. A formulação de nanopartículas de PLGA
foi optimizada por desenho factorial. O sistema compreendido pelas nanopartículas de PLGA
associadas aos filmes de goma-guar (GfNp) não comprometeu a viabilidade das linhas celulares
TR146 e Caco-2/HT29-MTX às concentrações testadas. O sistema desenvolvido promoveu a
libertação e permeabilidade controladas do péptido, através das células TR146 e Caco-2/HT29-
MTX cultivadas em camada, comparativamente com os filmes e nanopartículas isoladamente,
assim como com a solução de péptido livre (controlo). Contudo, a permeabilidade aparente
verificou-se superior para a formulação GfNp, comparativamente com as restantes formulações.
Estes resultados deveram-se ao contacto íntimo entre o péptido e o epitélio absorptivo, promovido
pela formulação GfNp. Verificou-se ainda, através da realização do ensaio in vitro da capacidade
de inibição da enzima conversora da angiotensina I, que o péptido transportado por GfNp
apresentava a maior actividade anti-hipertensora após ser sujeito à simulação do tracto
gastrointestinal, comparativamente com o péptido transportado pelas nanopartículas ou filme,
isoladamente, ou com a solução de péptido livre.
O sistema previamente optimizado para a libertação do péptido antihipertensor foi também
usado de forma a incrementar a biodisponibilidade do péptido relaxante alfa-casozepina
(sequência: YLGYLEQLLR). Através do ensaio MTT, foi possível concluir que nenhuma das
formulações comprometeu a viabilidade da linha celular TR146 e da co-cultura Caco-2 /HT29-
MTX. Por isso, a permeabilidade do péptido, sujeito às condições do tracto gastrointestinal
simulado, através dos modelos in vitro bucal e intestinal foi estudada. Verificou-se que a
associação de nanopartículas de PLGA com filmes de goma-guar promoveu um aumento da
permeabilidade face às nanopartículas e filmes não conjugados, assim como com o péptido em
solução (controlo). Estes resultados estão correlacionados com o incremento da mucoadesão
conferida pela associação das nanopartículas de PLGA com os filmes de goma-guar, verificada
através da análise da adesividade e trabalho de adesão à língua de vaca.
Validada a efectividade das formulações para a libertação e permeabilidade de cafeína e
péptidos bioactivos, foram realizados estudos preliminares de modo a verificar a estabilidade da
formulação GfB e a compreender a opinião de potenciais futuros consumidores face aos produtos
desenvolvidos. As formulações foram sujeitas a condições de degradação acelerada (i.e. 40 ºC e
75% de humidade relativa) de acordo com a International Conference of Harmonization (ICH),
não se verificando alterações químicas da cafeína em nenhum dos tempos de amostragem
(imediatamente após a preparação da formulação e após 3, 6 e 9 meses) através da análise do
espectro obtido por ATR-FTIR, assim como dos tempos de retenção em HPLC-UV. Verificou-se
ainda um aumento significativo do conteúdo em água de GfB ao longo dos tempos de
amostragem. Por fim, um estudo por focus group e um estudo de análise sensorial com um painel
naive permitiram compreender a adequabilidade dos sabores propostos, assim como a tolerância
à acidez e amargor por parte do consumidor. Observou-se uma ligeira tendência para a aceitação
do sabor a menta e alguma tolerância ao amargor e acidez quando a menta foi usada na
formulação.
Os sistemas para libertação oral de compostos bioactivos, desenvolvidos e optimizados no
âmbito desta tese, induziram melhorias significativas no comportamento farmacocinético in vitro
dos compostos veiculados. De facto, a associação de filmes orais com micro- ou nanopartículas
pode representar um novo sistema de libertação que ofereça maior efectividade e adesão por parte
do consumidor/utente.
Aiming for the protection and absorption enhancement of bioactive compounds administered by oral route (with special focus on buccal absorption), this thesis had as goals, the optimization of oral films and micro/nanoparticles which, through conjugation of both, worked as innovative oral delivery systems with synergic activity. The bioactive molecules selected to study the behaviour and efficacy of the optimized formulations were caffeine and two whey-derived peptides with antihypertensive and relaxing activities (sequences: KGYGGVSLPEW and YLGYLEQLLR, respectively). The optimization process began with the selection and preliminary comparison of the characteristics of the excipients to be used to prepare films. Indeed, the first study included the optimization and comparison of two film formulations as carriers for the oral release of caffeine, prepared using sodium carobymethylcellullose and type A gelatine as polymers. It was observed by the analysis of the spectra obtained by Fourier-transformed infrared spectroscopy with attenuated total reflectance (FTIR-ATR) that caffeine chemical structure was not altered during the film production process. It was also observed, through the dissolution assay established by the United States Pharmacopoeia (USP), that type A gelatine films offered a slow caffeine release, whereas caroboxymethylcellulose films offered a burst release profile. Accordingly, the apparent permeability of caffeine observed from the ex vivo permeability assay, across small intestine tissues from porcine origin, was higher for carboxymethylcellullose films than for type A gelatine films. Nonetheless, disintegration time of both formulations was too high to meet the criteria of orodispersible formulations, taking longer than 30 s to achieve total disintegration. Still in the process of choosing the best polymer to integrate the composition of oral films, a new formulation containing guar gum as polymer was optimized by factorial design. Guar gum films presented superior mechanical and physico-chemical characteristics than carobxymethylcellullose or type A gelatine films, mainly regarding water-uptake capacity, erosion in artificial saliva and disintegration time. Furthermore, a formulation of alginate microparticles was also optimized by factorial design to associate with guar gum films and guarantee the controlled release of caffeine, as well as an increased bioavailability. The association of alginate beads to guar gum films – GfB – did not induce alterations in the chemical characteristics of caffeine, as outlined in the data obtained by FTIR-ATR, nor cytotoxicity to the cell lines used to mimic the buccal (TR146) or intestinal (Caco-2/HT29-MTX) mucosa, as determined by MTT ((3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide). Additionally, the release and in vitro (through TR146 and Caco-2/HT29-MTX cell lines) and ex vivo (through porcine intestinal mucosa) permeability profiles of caffeine from GfB was slower when compared with alginate microparticles, guar gum films or caffeine control solution. GfB also increased the effective contact between caffeine and buccal epithelia, offering a more complete permeation along time. Further, aiming to increment the bioavailability of the peptide KGYGGVSLPEW with antihypertensive activity, alginate beads were replaced with poly(lactic-co-glycolic) acid (PLGA) nanoparticles, since the last offer a higher association efficiency and a higher permeability of biologic membranes, due to the significantly lower particle size. The formulation of PLGA nanoparticles was optimized by factorial design. The delivery system comprising PLGA nanoparticles into guar gum films (GfNp) did not compromise the viability of the cell lines TR146 and Caco-2/HT29-MTX at tested concentrations. Moreover, GfNp promoted a slower peptide release and in vitro permeability across TR146 and Caco-2/HT29-MTX cell layers when compared with the films and nanoparticles alone or with a free peptide solution (control). However, apparent permeability was higher for GfNp when compared with remaining formulations. Results may be due to the intimate contact between the peptide and the epithelia, promoted by GfNp. It was also possible to observe that the peptide carried by GfNp presented a higher in vitro capacity to inhibit the angiotensin-converting enzyme after being subjected to the simulation of gastrointestinal tract, therefore presenting higher antihypertensive potential, when compared with the peptide carried by the nanoparticles or films alone or with the control solution (free peptide). The previously optimized system as carrier and delivery system for the antihypertensive peptide was also used to enhance the bioavailability of the relaxing peptide alpha-casozepine (sequence: YLGYLEQLLR). It was possible to conclude, through MTT assay, that none of the formulations compromised cell viability of TR146 cell line or Caco-2/HT29-MTX co-culture. Moreover, peptide permeability across in vitro buccal and intestinal epithelial models, while being subjected to simulated gastrointestinal tract, was higher and faster (higher apparent permeability) for the association of guar gum films with PLGA nanoparticles, when compared with PLGA nanoparticles or guar gum films alone or with the free peptide solution (control). Obtained results are related with the increased mucoadhesion conferred by the association of PLGA nanoparticles with guar gum films, verified through the analysis of adhesivity and work of adhesion to cow tongue. After validation of the effectivity of the formulations regarding release and permeability of caffeine and bioactive peptides, preliminary studies were performed to understand the stability of GfB and the opinion of potential future consumers of the developed products. Formulations were subjected to accelerated degradation conditions according to the International Conference of Harmonization (ICH) and it was verified, through analysis of spectra (by FTIR-ATR spectroscopy) and retention times (by HPLC-UV), that no chemical alterations of caffeine molecule carried by GfB were observed in any of the set time points (i.e. immediately after preparation of GfB and 3, 6 and 9 months after preparation, under accelerated degradation conditions – 40 ºC and 75% of relative humidity). Moreover, an increased water content was observed along the three time points. Further, a focus group and a sensory analysis study with a naïve panel allowed to understand the suitability of the flavours but also the tolerability to acidity and bitterness by the consumer. A slight tendency to the acceptance of mint flavour and some tolerance to bitterness and acidity was verified when mint was used in the formulation. Developed and optimized oral delivery systems in the scope of the present thesis induced significant improvements on the in vitro pharmacokinetic behaviour of carried bioactive molecules. Indeed, the association of oral films with micro- and nanoparticles may represent conceptually new delivery systems that offer higher effectivity and consumer/patient compliance.
Aiming for the protection and absorption enhancement of bioactive compounds administered by oral route (with special focus on buccal absorption), this thesis had as goals, the optimization of oral films and micro/nanoparticles which, through conjugation of both, worked as innovative oral delivery systems with synergic activity. The bioactive molecules selected to study the behaviour and efficacy of the optimized formulations were caffeine and two whey-derived peptides with antihypertensive and relaxing activities (sequences: KGYGGVSLPEW and YLGYLEQLLR, respectively). The optimization process began with the selection and preliminary comparison of the characteristics of the excipients to be used to prepare films. Indeed, the first study included the optimization and comparison of two film formulations as carriers for the oral release of caffeine, prepared using sodium carobymethylcellullose and type A gelatine as polymers. It was observed by the analysis of the spectra obtained by Fourier-transformed infrared spectroscopy with attenuated total reflectance (FTIR-ATR) that caffeine chemical structure was not altered during the film production process. It was also observed, through the dissolution assay established by the United States Pharmacopoeia (USP), that type A gelatine films offered a slow caffeine release, whereas caroboxymethylcellulose films offered a burst release profile. Accordingly, the apparent permeability of caffeine observed from the ex vivo permeability assay, across small intestine tissues from porcine origin, was higher for carboxymethylcellullose films than for type A gelatine films. Nonetheless, disintegration time of both formulations was too high to meet the criteria of orodispersible formulations, taking longer than 30 s to achieve total disintegration. Still in the process of choosing the best polymer to integrate the composition of oral films, a new formulation containing guar gum as polymer was optimized by factorial design. Guar gum films presented superior mechanical and physico-chemical characteristics than carobxymethylcellullose or type A gelatine films, mainly regarding water-uptake capacity, erosion in artificial saliva and disintegration time. Furthermore, a formulation of alginate microparticles was also optimized by factorial design to associate with guar gum films and guarantee the controlled release of caffeine, as well as an increased bioavailability. The association of alginate beads to guar gum films – GfB – did not induce alterations in the chemical characteristics of caffeine, as outlined in the data obtained by FTIR-ATR, nor cytotoxicity to the cell lines used to mimic the buccal (TR146) or intestinal (Caco-2/HT29-MTX) mucosa, as determined by MTT ((3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide). Additionally, the release and in vitro (through TR146 and Caco-2/HT29-MTX cell lines) and ex vivo (through porcine intestinal mucosa) permeability profiles of caffeine from GfB was slower when compared with alginate microparticles, guar gum films or caffeine control solution. GfB also increased the effective contact between caffeine and buccal epithelia, offering a more complete permeation along time. Further, aiming to increment the bioavailability of the peptide KGYGGVSLPEW with antihypertensive activity, alginate beads were replaced with poly(lactic-co-glycolic) acid (PLGA) nanoparticles, since the last offer a higher association efficiency and a higher permeability of biologic membranes, due to the significantly lower particle size. The formulation of PLGA nanoparticles was optimized by factorial design. The delivery system comprising PLGA nanoparticles into guar gum films (GfNp) did not compromise the viability of the cell lines TR146 and Caco-2/HT29-MTX at tested concentrations. Moreover, GfNp promoted a slower peptide release and in vitro permeability across TR146 and Caco-2/HT29-MTX cell layers when compared with the films and nanoparticles alone or with a free peptide solution (control). However, apparent permeability was higher for GfNp when compared with remaining formulations. Results may be due to the intimate contact between the peptide and the epithelia, promoted by GfNp. It was also possible to observe that the peptide carried by GfNp presented a higher in vitro capacity to inhibit the angiotensin-converting enzyme after being subjected to the simulation of gastrointestinal tract, therefore presenting higher antihypertensive potential, when compared with the peptide carried by the nanoparticles or films alone or with the control solution (free peptide). The previously optimized system as carrier and delivery system for the antihypertensive peptide was also used to enhance the bioavailability of the relaxing peptide alpha-casozepine (sequence: YLGYLEQLLR). It was possible to conclude, through MTT assay, that none of the formulations compromised cell viability of TR146 cell line or Caco-2/HT29-MTX co-culture. Moreover, peptide permeability across in vitro buccal and intestinal epithelial models, while being subjected to simulated gastrointestinal tract, was higher and faster (higher apparent permeability) for the association of guar gum films with PLGA nanoparticles, when compared with PLGA nanoparticles or guar gum films alone or with the free peptide solution (control). Obtained results are related with the increased mucoadhesion conferred by the association of PLGA nanoparticles with guar gum films, verified through the analysis of adhesivity and work of adhesion to cow tongue. After validation of the effectivity of the formulations regarding release and permeability of caffeine and bioactive peptides, preliminary studies were performed to understand the stability of GfB and the opinion of potential future consumers of the developed products. Formulations were subjected to accelerated degradation conditions according to the International Conference of Harmonization (ICH) and it was verified, through analysis of spectra (by FTIR-ATR spectroscopy) and retention times (by HPLC-UV), that no chemical alterations of caffeine molecule carried by GfB were observed in any of the set time points (i.e. immediately after preparation of GfB and 3, 6 and 9 months after preparation, under accelerated degradation conditions – 40 ºC and 75% of relative humidity). Moreover, an increased water content was observed along the three time points. Further, a focus group and a sensory analysis study with a naïve panel allowed to understand the suitability of the flavours but also the tolerability to acidity and bitterness by the consumer. A slight tendency to the acceptance of mint flavour and some tolerance to bitterness and acidity was verified when mint was used in the formulation. Developed and optimized oral delivery systems in the scope of the present thesis induced significant improvements on the in vitro pharmacokinetic behaviour of carried bioactive molecules. Indeed, the association of oral films with micro- and nanoparticles may represent conceptually new delivery systems that offer higher effectivity and consumer/patient compliance.
Description
Keywords
Alginate beads Antihypertensive peptide Buccal delivery Bioactive molecules Bioavailability Caffeine Carboxymethylcellulose Drug delivery Experimental design Gelatin type A Guar gum films Nutritional Oral delivery Oral films PLGA nanoparticles Relaxing peptide Slow release Whey protein