Desenvolvimento e caracterização de nanopartículas com propriedades mucoadesivas baseadas em ácido hialurônico para liberação cólon específica de metotrexato
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Data
2017-09-29
Autores
Boni, Fernanda Isadora [UNESP]
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Universidade Estadual Paulista (Unesp)
Resumo
A nanotecnologia farmacêutica é uma relevante ferramenta tecnológica para o desenvolvimento de novos sistemas para a veiculação de fármacos, pois devido às suas dimensões reduzidas são capazes de circular por capilares que irrigam os tecidos, escapar da fagocitose por células do sistema imunológico e permear passivamente células e epitélios. Além disso, esses sistemas possibilitam a encapsulação de fármacos de baixa estabilidade, protegendo-os contra degradação prematura e/ou permitindo a modulação de suas propriedades físico-químicas, bem como a interação biológica na biointerface. O metotrexato (MTX) é um dos fármacos mais utilizados no tratamento de tumores sólidos e doenças inflamatórias intestinais, no entanto, por ser altamente citotóxico e não seletivo promove diversos efeitos colaterais. Sua eficácia terapêutica é comprometida devido à resistência adquirida pelas células tumorais e por sua baixa permeabilidade intestinal, principalmente por meio do mecanismo de efluxo da forma livre. Nesse trabalho, nanopartículas poliméricas (NPs) orais compostas por quitosana (QS), ácido hialurônico (AH) e ftalato de hidroxipropilmetilcelulose (HP), polímeros que possuem propriedades como solubilidade pH dependente, mucoadesividade e de funcionalização, foram desenvolvidas como plataforma tecnológica para a vetorização do MTX para o cólon, visando o tratamento local de patologias intestinais. As análises de peso molecular e coeficiente viral demostraram a adequação dos polímeros e solventes para a obtenção das nanoestruturas pelo método de complexação polieletrolítica. A avaliação da influência do pH no potencial zeta dos polímeros permitiu a seleção do valor ótimo (pH 5,5) para obtenção das nanoestruturas. O diâmetro médio das NPs sem fármaco, contendo ou não HP variou de 451,73-574,95 nm e a incorporação do MTX reduziu significativamente esses valores para a faixa de 216,83-345,03 nm (p<0,05). A adição do HP, bem como do MTX promoveu a redução do potencial zeta das NPs (p<0,05). O índice de polidispersão das NPs contendo ou não MTX variou de 0,21-0,39 a 0,26-0,33, respectivamente, indicando a homogeneidade de tamanho. A forma esférica das partículas foi também foi demonstrada. Os espectros de absorção na região IV indicaram a formação dos complexos polieletrolíticos e as análises de DRX a formação de estruturas cristalinas. A eficiência de incorporação do MTX variou de 20,06% a 36,18%, sendo as NHP0,5-1 e NHP0,2-5 as que apresentaram as maiores porcentagens (32,21% e 36,18%, respectivamente). Embora o MTX tenha promovido a redução dos valores de mucoadesão (p<0,05), a elevada mucoadesividade (71,22% a 91,53%) de todas as NPs foi evidenciada. De acordo com as isotermas de adsorção, os dados de interação com a mucina apresentaram correlação com o modelo de Freundlich, que indica a adsorção reversível em multicamadas. Em pH 6,8, a interação da mucina com as NPs promoveu a redução do potencial zeta, o que pode estar relacionado a maior capacidade de adsorção nesse valor de pH. A incorporação do MTX nas NPs permitiu a redução da liberação em meio ácido em até 40%, em relação ao fármaco livre, enquanto a adição de HP à matriz das NPs reduziu a liberação em até 10%, neste pH. O mecanismo de liberação do fármaco foi avaliado por diferentes modelos matemáticos. A viabilidade nas linhagens Caco-2 e HT29-MTX para os polímeros e NPs (0,01 - 1 μg.mL-1) foi demonstrada. Para as NPs contendo MTX, nas concentrações de 10 μg.mL-1 e 100 μg.mL- 1, a viabilidade na linhagem Caco-2 após 24 h de incubação foi de aproximadamente 50%. Nos estudos de permeabilidade in vitro, as NPs sem adição de HP permearam aproximadamente 3 vezes mais as monocamadas de Caco-2 e de co-cultura tripla de células, em comparação às amostras contendo HP e ao fármaco livre. A maior associação celular para o fármaco incorporado às NPs, em comparação ao MTX livre, demonstrou a capacidade do sistema em modular a interação biológica.
The pharmaceutical nanotechnology is a relevant technological tool for the development of new drug delivery systems, because of their small size they are able to circulate through capillaries that irrigate tissues, escape from phagocytosis by the immune system cells and passively permeate cells and epithelia. In addition, these systems enable the encapsulation of low stability drugs, protecting them against premature degradation and/or allowing the modulation of their physico-chemical properties, as well as the biological interaction in the biointerface. Methotrexate (MTX) is one of the most used drug in the treatment of solid tumors and inflammatory bowel diseases, however, because MTX highly cytotoxic and nonselectivity promotes several side effects. Its therapeutic efficacy is compromised due to the resistance acquired by tumor cells and it low intestinal permeability, mainly through the efflux mechanism of the free form. In this work, oral nanoparticles composed of chitosan (CS), hyaluronic acid (HA) and hydroxypropylmethylcellulose (HP) phthalate, polymers that have properties such as pH-dependent solubility, mucoadhesiveness and functionalization have been developed as a technological platform for the target of MTX to the colon, aiming the treatment of local pathologies. Molecular weight and second viral coefficient analyzes demonstrated the suitability of polymers and solvents to obtain nanostructures by the polyelectrolyte complexation method. The evaluation of the pH influence on the zeta potential of the polymers allowed to the selection of the optimum value (pH 5.5) to obtain the nanostructures. The mean diameter of the MTX free nanoparticles, containing or not HP ranged from 451.73-574.95 nm and the incorporation of MTX significantly reduced these values for the range of 216.83-345.03 nm (p <0.05). The addition of HP as well as MTX promoted reduction of zeta potential of nanoparticles (p <0.05). The polydispersity index of the nanoparticles containing or not MTX ranged from 0.21-0.39 to 0.26-0.33, respectively, indicating homogeneity of size. The spherical shape of the particles has been demonstrated. The absorption spectra in the IV region indicated the formation of the polyelectrolyte complexes and the DRX analyzes, the formation of crystalline structures. The efficiency of drug incorporation ranged from 20.06% to 36.18%, with the NHP0.5-1 and NHP0.2-5 samples having the highest percentages (32.21% and 36.18%, respectively). Although MTX promoted the reduction of mucoadhesion values (p<0.05), the high mucoadhesiveness (71.22% to 91.53%) of all nanoparticles was evidenced. According to the adsorption isotherms, the interaction data with the mucin presented a correlation with the Freundlich model, which indicates the reversible adsorption in multilayers. At pH 6.8, the interaction of mucin with the nanoparticles promoted the reduction of the zeta potential, which may be related to the higher adsorption capacity at this pH value. The incorporation of MTX in the nanoparticles allowed to the reduction of MTX release in acidic media by up to 40% in relation to the free drug and the addition of HP to the nanoparticle matrix reduced the release by up to 10% at pH 1.2. The mechanism of drug release was evaluated by different mathematical models. The viability of Caco-2 and HT29-MTX lines for polymers and NPs (0.01-1 μg.mL-1) was demonstrated. For nanoparticles containing MTX, at concentrations of 10 μg.mL-1 and 100 μg.mL-1 , the viability of Caco-2 after 24 h incubation was approximately 50%. In the in vitro permeability studies, samples without HP permeated approximately 3- fold more monolayers of Caco-2 and triple co-culture compared to samples containing HP and the free drug. The higher cellular association for the drug incorporated into the nanoparticles, compared to free MTX, demonstrated the ability of the system to modulate the biological interaction
The pharmaceutical nanotechnology is a relevant technological tool for the development of new drug delivery systems, because of their small size they are able to circulate through capillaries that irrigate tissues, escape from phagocytosis by the immune system cells and passively permeate cells and epithelia. In addition, these systems enable the encapsulation of low stability drugs, protecting them against premature degradation and/or allowing the modulation of their physico-chemical properties, as well as the biological interaction in the biointerface. Methotrexate (MTX) is one of the most used drug in the treatment of solid tumors and inflammatory bowel diseases, however, because MTX highly cytotoxic and nonselectivity promotes several side effects. Its therapeutic efficacy is compromised due to the resistance acquired by tumor cells and it low intestinal permeability, mainly through the efflux mechanism of the free form. In this work, oral nanoparticles composed of chitosan (CS), hyaluronic acid (HA) and hydroxypropylmethylcellulose (HP) phthalate, polymers that have properties such as pH-dependent solubility, mucoadhesiveness and functionalization have been developed as a technological platform for the target of MTX to the colon, aiming the treatment of local pathologies. Molecular weight and second viral coefficient analyzes demonstrated the suitability of polymers and solvents to obtain nanostructures by the polyelectrolyte complexation method. The evaluation of the pH influence on the zeta potential of the polymers allowed to the selection of the optimum value (pH 5.5) to obtain the nanostructures. The mean diameter of the MTX free nanoparticles, containing or not HP ranged from 451.73-574.95 nm and the incorporation of MTX significantly reduced these values for the range of 216.83-345.03 nm (p <0.05). The addition of HP as well as MTX promoted reduction of zeta potential of nanoparticles (p <0.05). The polydispersity index of the nanoparticles containing or not MTX ranged from 0.21-0.39 to 0.26-0.33, respectively, indicating homogeneity of size. The spherical shape of the particles has been demonstrated. The absorption spectra in the IV region indicated the formation of the polyelectrolyte complexes and the DRX analyzes, the formation of crystalline structures. The efficiency of drug incorporation ranged from 20.06% to 36.18%, with the NHP0.5-1 and NHP0.2-5 samples having the highest percentages (32.21% and 36.18%, respectively). Although MTX promoted the reduction of mucoadhesion values (p<0.05), the high mucoadhesiveness (71.22% to 91.53%) of all nanoparticles was evidenced. According to the adsorption isotherms, the interaction data with the mucin presented a correlation with the Freundlich model, which indicates the reversible adsorption in multilayers. At pH 6.8, the interaction of mucin with the nanoparticles promoted the reduction of the zeta potential, which may be related to the higher adsorption capacity at this pH value. The incorporation of MTX in the nanoparticles allowed to the reduction of MTX release in acidic media by up to 40% in relation to the free drug and the addition of HP to the nanoparticle matrix reduced the release by up to 10% at pH 1.2. The mechanism of drug release was evaluated by different mathematical models. The viability of Caco-2 and HT29-MTX lines for polymers and NPs (0.01-1 μg.mL-1) was demonstrated. For nanoparticles containing MTX, at concentrations of 10 μg.mL-1 and 100 μg.mL-1 , the viability of Caco-2 after 24 h incubation was approximately 50%. In the in vitro permeability studies, samples without HP permeated approximately 3- fold more monolayers of Caco-2 and triple co-culture compared to samples containing HP and the free drug. The higher cellular association for the drug incorporated into the nanoparticles, compared to free MTX, demonstrated the ability of the system to modulate the biological interaction
Descrição
Palavras-chave
Nanopartículas poliméricas, Quitosana, Complexação polieletrolítica, Mucoadesão, Permeabilidade, Células Caco-2, Co-cultura tripla de células