Lipossomas funcionalizados com peptídeos de transdução de membrana para administração intranasal de insulina no tratamento do diabetes mellitus
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Data
2019-08-29
Autores
Von Zuben, Eliete de Souza
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Editor
Universidade Estadual Paulista (Unesp)
Resumo
O diabetes mellitus (DM) é uma síndrome metabólica caracterizada por deficiência na produção/secreção pancreática de insulina e/ou resistência à ação do hormônio nos tecidos alvo, resultando em hiperglicemia. Diversas pesquisas têm desencadeado o desenvolvimento de novos sistemas de administração de insulina que possibilitem a utilização de vias alternativas à parenteral, com destaque à administração de insulina por via nasal. Esta via tem-se mostrado promissora, pois pode promover uma rápida absorção do fármaco e aumentar a sua biodisponibilidade. Entretanto, existem mecanismos de depuração mucociliar que limitam a administração de fármacos, além da baixa permeabilidade do epitélio nasal, o qual dificulta a absorção de fármacos com alto massa molar. Uma estratégia para vencer tais barreiras é a utilização de sistemas nanoestruturados (lipossomas), pois são amplamente utilizados para o aperfeiçoamento da potencialização da ação terapêutica de fármacos. Além disso estes lipossomas foram funcionalizados com peptídeos de transdução de membrana (CPPs), tais como os peptídeos TAT e Penetratin (PNT), que atuam como promotores da penetração e absorção do fármaco, com posterior dispersão em hidrogel de hidroxietilcelulose. O objetivo deste trabalho foi desenvolver e caracterizar lipossomas contendo solução de insulina, funcionalizados com CPPs (TAT e PNT) e dispersos em hidrogel, avaliar o potencial pela via nasal, in vivo, para a melhora dos níveis séricos e efeito hipoglicemiante da insulina. Os resultados obtidos revelaram que as formulações apresentaram valores médios de diâmetro hidrodinâmico na escala nanométrica, índice de polidispersão e de potencial zeta estáveis, durante o período de armazenamento de 90 dias. A funcionalização realizada com os CPPs na concentração de 0,1mM e demonstraram que houve um decréscimo nos valores de potencial zeta de -53,60 ± 3,02 mV para os lipossomas encapsulados com insulina para -10,90± 0,47 mV quando funcionalizados com TAT e -9,05± 0,60 mV quando funcionalizados com PNT, indicativo de interação eletrostática, devido à carga altamente positiva dos CPPs. As imagens obtidas por microscopia eletrônica de transmissão mostraram que os lipossomas com e sem fármaco e funcionalizados com CPPs apresentam uma morfologia multilamelares e unilamelares pequenas e em escalas nanométricas (80 a 150 nm). Os espectros de dicroismo circular realizados nas formulações recém-preparadas com e sem os CPPs e nas que foram submetidas ao ensaio de liberação in vitro demonstraram que a integridade e a estabilidade da insulina foram preservadas, quando comparada com a insulina em solução. O método analítico para quantificação da insulina foi validado e utilizado nos ensaios de eficiência de encapsulação (EE%), liberação in vitro e permeação em mucosa nasal suína. Optou-se pelo método de preparo de hidratação do filme lipídico por apresentar os resultados mais elevados em relação a EE%. O ensaio de força mucoadesiva nas duas membranas modelos foram muito similares e da ordem de 0.06 N e não apresentaram diferença significativa (valor de p > 0,05). O ensaio de seringabilidade mostrou-se favorável, no que diz respeito à facilidade de aplicação deste tipo de formulação pelo paciente na via nasal. As análises reológicas evidenciaram um comportamento do tipo pseudoplástico com caráter mais viscoso do que elástico (G” > G’) para todas as formulações. Os ensaios de liberação in vitro demonstraram que as formulações estudadas apresentam cinética de liberação, conforme modelo matemático de Weibull, que relaciona a quantidade de fármaco acumulado em solução em função do tempo. Nos ensaios de permeação em mucosa nasal suína observou-se que o fluxo (J) da formulação Hidrogel+Lipossomas+Insulina (HLI) foi quase duas vezes maior em relação às formulações Hidrogel+Lipossomas+Insulina+TAT(HLITAT) e Hidrogel+Lipossomas+Insulina+PNT (HLIPNT) e a mesma relação foi observada para os resultados de coeficiente de permeabilidade (Kp). Enquanto que o J da formulação Hidrogel +Insulina (HI) foi 1,5 vezes maior em relação à formulação HLI, sendo que a mesma relação foi observada para o resultado de Kp. Os ensaios in vivo evidenciaram que as formulações HLITAT, HLIPNT, HI e HLI demostraram resultados satisfatórios, entretanto, as formulações HLITAT e HLIPNT possuem potencial para serem melhoradas quando comparadas as formulações HI e HLI.
Diabetes mellitus (DM) is a metabolic syndrome characterized by deficiency in pancreatic insulin production / secretion and / or resistance to hormone action in target tissues, resulting in hyperglycemia. Several researches have triggered the development of new insulin delivery systems that allow the use of alternative parenteral routes, especially the nasal administration of insulin. This route has been promising as it can promote rapid absorption of the drug and increase its bioavailability. However, there are mucociliary clearance mechanisms that limit drug administration, in addition to the low permeability of the nasal epithelium, which hinders the absorption of drugs with high molar mass. One strategy to overcome such barriers is the use of nanostructured systems (liposomes), as they are widely used to enhance the potentiation of therapeutic action of drugs. In addition, these liposomes were functionalized with membrane transduction peptides (CPPs), such as TAT and Penetratin (PNT) peptides, which act as penetration and absorption promoters of the drug, with later dispersion in hydroxyethylcellulose hydrogel. The objective of this work was to develop and characterize insulin-containing liposomes, functionalized with CPPs (TAT and PNT) and dispersed in hydrogel, to evaluate the nasal potential in vivo for the improvement of serum levels and hypoglycemic effect of insulin. The results showed that the formulations showed stable values of hydrodynamic diameter on the nanometer scale, stable polydispersion index and zeta potential during the storage period of 90 days. Functionalization performed with CPPs at a concentration of 0.1 mM and demonstrated that there was a decrease in zeta potential values from -53.60 ± 3.02 mV for insulin-encapsulated liposomes to -10.90 ± 0.47 mV when functionalized with TAT and -9.05 ± 0.60 mV when functionalized with PNT, indicative of electrostatic interaction, due to the highly positive charge of the CPPs. Transmission electron microscopy images showed that drug-based and drug-free liposomes with CPPs have small multilamellar and unilamellar morphology at nanometer scales (80 to 150 nm). Circular dichroism spectra performed on freshly prepared formulations with and without CPPs and those subjected to the in vitro release assay demonstrated that insulin integrity and stability were preserved as compared to insulin in solution. The analytical method for insulin quantification was validated and used in the encapsulation efficiency (EE%), in vitro release and permeation tests in porcine nasal mucosa. The method of preparation of hydration of the lipid film was chosen because it presents the highest results in relation to EE%. The mucoadhesive strength test in the two model membranes were very similar and of the order of 0.06 N and showed no significant difference (p value> 0.05). The syringability test proved to be favorable, regarding the ease of application of this type of formulation by the patient in the nasal route. The rheological analyzes showed a pseudoplastic type behavior with a more viscous than elastic character (G ”> G ') for all formulations. In vitro release assays demonstrated that the formulations studied presented release kinetics, according to Weibull's mathematical model, which relates the amount of drug accumulated in solution as a function of time. In the porcine nasal mucosal permeation assays it was observed that the flow (J) of the formulation Hydrogel + Liposomes + Insulin (HLI) was almost twice higher than the formulations Hydrogel + Liposomes + Insulin + TAT (HLITAT) and Hydrogel + Liposomes + Insulin + PNT (HLIPNT) and the same relationship was observed for the permeability coefficient (Kp) results. While the J of the Hydrogel + Insulin (HI) formulation was 1.5 times higher than the HLI formulation, the same relationship was observed for the Kp result. In vivo tests showed that HLITAT, HLIPNT, HI and HLI formulations showed satisfactory results, however, HLITAT and HLIPNT formulations have potential to be improved when compared to HI and HLI formulations.
Diabetes mellitus (DM) is a metabolic syndrome characterized by deficiency in pancreatic insulin production / secretion and / or resistance to hormone action in target tissues, resulting in hyperglycemia. Several researches have triggered the development of new insulin delivery systems that allow the use of alternative parenteral routes, especially the nasal administration of insulin. This route has been promising as it can promote rapid absorption of the drug and increase its bioavailability. However, there are mucociliary clearance mechanisms that limit drug administration, in addition to the low permeability of the nasal epithelium, which hinders the absorption of drugs with high molar mass. One strategy to overcome such barriers is the use of nanostructured systems (liposomes), as they are widely used to enhance the potentiation of therapeutic action of drugs. In addition, these liposomes were functionalized with membrane transduction peptides (CPPs), such as TAT and Penetratin (PNT) peptides, which act as penetration and absorption promoters of the drug, with later dispersion in hydroxyethylcellulose hydrogel. The objective of this work was to develop and characterize insulin-containing liposomes, functionalized with CPPs (TAT and PNT) and dispersed in hydrogel, to evaluate the nasal potential in vivo for the improvement of serum levels and hypoglycemic effect of insulin. The results showed that the formulations showed stable values of hydrodynamic diameter on the nanometer scale, stable polydispersion index and zeta potential during the storage period of 90 days. Functionalization performed with CPPs at a concentration of 0.1 mM and demonstrated that there was a decrease in zeta potential values from -53.60 ± 3.02 mV for insulin-encapsulated liposomes to -10.90 ± 0.47 mV when functionalized with TAT and -9.05 ± 0.60 mV when functionalized with PNT, indicative of electrostatic interaction, due to the highly positive charge of the CPPs. Transmission electron microscopy images showed that drug-based and drug-free liposomes with CPPs have small multilamellar and unilamellar morphology at nanometer scales (80 to 150 nm). Circular dichroism spectra performed on freshly prepared formulations with and without CPPs and those subjected to the in vitro release assay demonstrated that insulin integrity and stability were preserved as compared to insulin in solution. The analytical method for insulin quantification was validated and used in the encapsulation efficiency (EE%), in vitro release and permeation tests in porcine nasal mucosa. The method of preparation of hydration of the lipid film was chosen because it presents the highest results in relation to EE%. The mucoadhesive strength test in the two model membranes were very similar and of the order of 0.06 N and showed no significant difference (p value> 0.05). The syringability test proved to be favorable, regarding the ease of application of this type of formulation by the patient in the nasal route. The rheological analyzes showed a pseudoplastic type behavior with a more viscous than elastic character (G ”> G ') for all formulations. In vitro release assays demonstrated that the formulations studied presented release kinetics, according to Weibull's mathematical model, which relates the amount of drug accumulated in solution as a function of time. In the porcine nasal mucosal permeation assays it was observed that the flow (J) of the formulation Hydrogel + Liposomes + Insulin (HLI) was almost twice higher than the formulations Hydrogel + Liposomes + Insulin + TAT (HLITAT) and Hydrogel + Liposomes + Insulin + PNT (HLIPNT) and the same relationship was observed for the permeability coefficient (Kp) results. While the J of the Hydrogel + Insulin (HI) formulation was 1.5 times higher than the HLI formulation, the same relationship was observed for the Kp result. In vivo tests showed that HLITAT, HLIPNT, HI and HLI formulations showed satisfactory results, however, HLITAT and HLIPNT formulations have potential to be improved when compared to HI and HLI formulations.
Descrição
Palavras-chave
Diabetes mellitus, Insulina, Lipossomas, Peptídeos de transdução de membrana, Sistemas nanoestruturados, Administração nasal, Insulin, Liposomes, Cell-penetrating peptides (CPPs), Protein transduction domain (PTDs), Nanostructured systems, Nasal administration