Silica nanoparticles in oral peptide delivery for diabetes Mellitus control and treatment
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2014
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O presente trabalho visou o desenvolvimento de nanopartículas de sílica (SiNP, do inglês “silica nanoparticles”) com a finalidade de explorar o potencial destes sistemas para a administração oral de péptidos, usando a insulina como fármaco modelo.
As SiNP foram produzidas a partir da hidrólise e condensação do silicato de tetraetila (TEOS) pelo método sol-gel e otimizadas usando um desenho fatorial 22. O tamanho médio (Z-Ave) e o índice de polidispersão (PI) das nanopartículas foram influenciados pela concentração de TEOS e pela velocidade de agitação durante a síntese das mesmas. Foram obtidas nanopartículas com 0,43 mol.L-1 de TEOS e uma velocidade de homogeneização de 5000 rpm, revelando um Z-Ave de 256,6 nm e um PI de 0,218. Trealose, manitol e sorbitol foram utilizados como crioprotetores a fim de melhorar a estabilidade das nanopartículas durante a liofilização. Calorimetria diferencial de varrimento (DSC) e raio-X revelaram que as SiNP na presença dos crioprotetores apresentaram uma forma cristalina. No entanto, a trealose foi a mais adequada para a manutenção da integridade das nanopartículas após a sua reconstituição em água, uma vez que este dissacarídeo oferece maior resistência das camadas em torno das nanopartículas, a qual promove maior interação com os grupos SiOH presentes na superfície da sílica.
O revestimento das SiNP com quitosano, alginato de sódio, PEG 6000 e PEG 20000 foi realizado para desenvolver uma formulação destinada à administração oral de insulina por meio da combinação das vantagens das SiNP e das propriedades mucoadesivas desses polímeros. A associação da insulina às nanopartículas foi realizada por adsorção após a produção das mesmas. No caso das nanopartículas revestidas, a insulina foi previamente dissolvida na solução polimérica e posteriormente adicionada às SiNP.
Estudos de DSC revelaram que os picos endotérmicos e exotérmicos dos polímeros puros foram deslocados para temperaturas mais elevadas nas SiNP revestidas, provavelmente devido à interação de ligação de hidrogénio entre os grupos de SiOH das SiNP e os grupos funcionais específicos do PEG (OH), do quitosano (NH) ou do alginato de sódio (COOH). Esta alteração resultou em maior estabilidade das nanopartículas. A difração de raio-X mostrou que as SiNP revestidas exibiram uma estrutura menos ordenada comparado com os polímeros puros. A associação da insulina às nanoparticulas resultou numa estrutura mais cristalina, provavelmente devido à solubilização da insulina nas soluções poliméricas originando a cristalização das nanoparticulas durante o armazenamento. A análise por infravermelho por transformada de Fourier (FTIR) também mostrou a eficiência do revestimento das nanopartículas, além das bandas de absorção de amida que são características essenciais dos espectros de proteínas. Análises por dicroísmo circular (CD) demonstraram que a estrutura da insulina foi ligeiramente afetada pelo revestimento durante a síntese das nanopartículas.
Termogramas obtidos por nano DSC mostraram que as SiNP não revestidas podem promover melhor interação com os grupos polares dos liposomas. Além disso, as SiNP e o PEG 6000 foram mais eficazes em proteger a insulina contra a desnaturação térmica.
A mucoadesividade das nanopartículas foi avaliada in vitro pela interação com mucina a pH gástrico e intestinal. SiNP revestidas com quitosano ou alginato apresentaram melhor capacidade de adsorver a mucina, em comparação com as SiNP não revestidas e as SiNP revestidas com PEG (SiNP-PEG 6000).
O revestimento das nanopartículas resultou numa libertação da insulina mais rápida tanto a pH 2,0 como a pH 6,8, comparando com as nanopartículas não revestidas, devido à baixa interação entre a insulina e os grupos SiOH da superfície da sílica. O perfil de libertação de insulina foi afetado apenas pelas SiNP não revestidas e pelas SiNP-PEG 6000 em relação à solução de insulina. Embora as SiNP não revestidas e as SiNP-PEG 6000 tenham provocado redução da libertação de insulina a pH gástrico, 60% da insulina foi libertada após 2 horas de incubação.
Os estudos in vitro de permeação da insulina foram realizados por meio do saco intestinal invertido de ratos, comparando as nanopartículas não revestidas e SiNP-PEG 6000 e SiNP-PEG 20000. No entanto, a presença do PEG não alterou significativamente o comportamento da permeação da insulina na mucosa intestinal de ratos.
As nanopartículas mostraram baixa toxicidade nas linhagens celulares testadas (HepG2 e Caco-2) nas concentrações de 50-500 μg/mL. A incorporação da insulina nas nanopartículas não afetou a viabilidade celular. Doses mais elevadas de SiNP-PEG 6000 apresentaram toxicidade nas linhas celulares selecionadas. Imagens obtidas por microscopia óptica revelaram a presença de vacúolos no interior das células Caco-2, indicando uma possível endocitose dessas nanopartículas.
As SiNP podem ser consideradas como sistemas promissores para a dministração oral da insulina aplicando uma tecnologia simples, de baixo-custo e sem danos ao ambiente.
The present work has been focused on the synthesis of silica nanoparticles (SiNP) intended to oral peptide delivery for Diabetes mellitus control and treatment, using insulin as model drug. SiNP were synthesized from the hydrolysis and condensation of tetraethyl orthosilicate (TEOS) by sol-gel technology. Nanoparticles were optimized using a 22factorial design approach. The mean particle size (Z-Ave) and polydispersity index (PI) were influenced by TEOS concentration and by stirring speed during nanoparticle synthesis. Optimized SiNP were obtained using 0.43 mol.L-1of TEOS and the homogenization speed of 5000 rpm, depicting a Z-Ave of 256.6 nm and a PI of 0.218. Trehalose, mannitol and sorbitol were used as cryoprotectants to improve SiNP stabilization during lyophilization. Differential scanning calorimetry (DSC) and X-ray studies demonstrated that SiNP in the presence of different cryoprotectans showed a crystalline behavior. However, trehalose was more suitable in maintaining particle integrity after reconstitution of lyophilized nanoparticles in water, due to the higher spatial resistance of its layers around the nanoparticles, leading to stronger interaction with SiOH groups in comparison to mannitol or sorbitol. After formulation optimization, the next step was to coat SiNP with chitosan, sodium alginate, PEG 6000 and PEG 20000 to develop a system for oral insulin administration by combining the SiNP advantages and the mucoadhesive properties of hydrophilic polymers. Insulin was associated to different nanoparticles by adsorption after nanoparticles production. In the case of coated nanoparticles, insulin was previously dissolved in the polymer solution and then added to SiNP. Interaction between insulin and nanoparticles was assessed by DSC, X-ray and Fourier-transform infrared (FTIR) studies. DSC showed that endothermic and exothermic peaks of pure polymers were shifted to higher temperatures in all coated SiNP, probably due to the interaction by hydrogen bounds between SiNP SiOH groups and specific functional groups of PEG (OH), chitosan (NH) or sodium alginate (COOH), resulting in more stable nanoparticles. X-ray diffraction showed that coated SiNP displayed less ordered structure compared with pure polymers. The association of insulin to nanoparticles resulted in more crystalline structure due to the insulin solubilization into the polymers solutions leading to the nanoparticles crystallization during storage. FTIR analysis showed the high efficiency nanoparticles coating, as well as amide absorption bands which are characteristic of protein spectra. Insulin secondary structure was assessed by Circular Dichroism (CD) after protein dissolution into polymer solutions during nanoparticle synthesis, showing that insulin structure was slightly affected by coating. Nano DSC was used to evaluate the interaction between nanoparticles and the biomembrane models (liposomes), and the thermal insulin stability dissolved in the different polymers. The interaction between nanoparticles and liposomes showed that uncoated SiNP could promote higher interaction with the polar head groups of liposomes. Also, uncoated SiNP were more effective in protecting insulin from thermal denaturation. The nanoparticles mucoadhesive properties were assessed by in vitro interaction with mucin at gastric and intestinal pH. SiNP coated with chitosan or alginate showed better ability in adsorbing to mucin in comparison to uncoated SiNP and PEG-coated SiNP. It was observed that nanoparticles coated with mucoadhesive polymers resulted in faster insulin release at pH 2.0 and 6.8 in comparison to uncoated nanoparticles due to the low interaction between insulin and SiOH present onto the silica surface. Insulin release profile was only significantly affected by uncoated SiNP and PEG 6000-coated SiNP in comparison to insulin solution. Although, uncoated and PEG-coated SiNP reduced insulin release at gastric pH, 60% of insulin was released at pH 2.0 after 2 h. In vitro insulin permeation studies were conducted through everted rat intestine comparing uncoated and PEG-coated SiNP. However, the presence of PEG onto the silica surface did not significantly change the permeation behavior of insulin through the intestinal mucosa. In general, all nanoparticles showed to be biocompatible, revealing low toxicity in different human cancer cell lines (HepG2 and Caco-2) at tested concentrations (50-500 μg/mL). The presence of insulin did not affect the cell viability significantly. Higher toxicities were observed for PEG 6000-coated SiNP at high concentrations. Microscope images revealed the formation of vacuoles in the Caco-2 cell body, indicating a possible endocytosis of these nanoparticles. The present work allows the conclusion that coating of silica nanoparticles with mucoadhesive polymers influences their physicochemical properties, insulin release, as well as the cell viability. These studies and findings show the feasibility of applying silica nanoparticles for oral insulin delivery using a simple, cheap and environmentally friendly technology.
The present work has been focused on the synthesis of silica nanoparticles (SiNP) intended to oral peptide delivery for Diabetes mellitus control and treatment, using insulin as model drug. SiNP were synthesized from the hydrolysis and condensation of tetraethyl orthosilicate (TEOS) by sol-gel technology. Nanoparticles were optimized using a 22factorial design approach. The mean particle size (Z-Ave) and polydispersity index (PI) were influenced by TEOS concentration and by stirring speed during nanoparticle synthesis. Optimized SiNP were obtained using 0.43 mol.L-1of TEOS and the homogenization speed of 5000 rpm, depicting a Z-Ave of 256.6 nm and a PI of 0.218. Trehalose, mannitol and sorbitol were used as cryoprotectants to improve SiNP stabilization during lyophilization. Differential scanning calorimetry (DSC) and X-ray studies demonstrated that SiNP in the presence of different cryoprotectans showed a crystalline behavior. However, trehalose was more suitable in maintaining particle integrity after reconstitution of lyophilized nanoparticles in water, due to the higher spatial resistance of its layers around the nanoparticles, leading to stronger interaction with SiOH groups in comparison to mannitol or sorbitol. After formulation optimization, the next step was to coat SiNP with chitosan, sodium alginate, PEG 6000 and PEG 20000 to develop a system for oral insulin administration by combining the SiNP advantages and the mucoadhesive properties of hydrophilic polymers. Insulin was associated to different nanoparticles by adsorption after nanoparticles production. In the case of coated nanoparticles, insulin was previously dissolved in the polymer solution and then added to SiNP. Interaction between insulin and nanoparticles was assessed by DSC, X-ray and Fourier-transform infrared (FTIR) studies. DSC showed that endothermic and exothermic peaks of pure polymers were shifted to higher temperatures in all coated SiNP, probably due to the interaction by hydrogen bounds between SiNP SiOH groups and specific functional groups of PEG (OH), chitosan (NH) or sodium alginate (COOH), resulting in more stable nanoparticles. X-ray diffraction showed that coated SiNP displayed less ordered structure compared with pure polymers. The association of insulin to nanoparticles resulted in more crystalline structure due to the insulin solubilization into the polymers solutions leading to the nanoparticles crystallization during storage. FTIR analysis showed the high efficiency nanoparticles coating, as well as amide absorption bands which are characteristic of protein spectra. Insulin secondary structure was assessed by Circular Dichroism (CD) after protein dissolution into polymer solutions during nanoparticle synthesis, showing that insulin structure was slightly affected by coating. Nano DSC was used to evaluate the interaction between nanoparticles and the biomembrane models (liposomes), and the thermal insulin stability dissolved in the different polymers. The interaction between nanoparticles and liposomes showed that uncoated SiNP could promote higher interaction with the polar head groups of liposomes. Also, uncoated SiNP were more effective in protecting insulin from thermal denaturation. The nanoparticles mucoadhesive properties were assessed by in vitro interaction with mucin at gastric and intestinal pH. SiNP coated with chitosan or alginate showed better ability in adsorbing to mucin in comparison to uncoated SiNP and PEG-coated SiNP. It was observed that nanoparticles coated with mucoadhesive polymers resulted in faster insulin release at pH 2.0 and 6.8 in comparison to uncoated nanoparticles due to the low interaction between insulin and SiOH present onto the silica surface. Insulin release profile was only significantly affected by uncoated SiNP and PEG 6000-coated SiNP in comparison to insulin solution. Although, uncoated and PEG-coated SiNP reduced insulin release at gastric pH, 60% of insulin was released at pH 2.0 after 2 h. In vitro insulin permeation studies were conducted through everted rat intestine comparing uncoated and PEG-coated SiNP. However, the presence of PEG onto the silica surface did not significantly change the permeation behavior of insulin through the intestinal mucosa. In general, all nanoparticles showed to be biocompatible, revealing low toxicity in different human cancer cell lines (HepG2 and Caco-2) at tested concentrations (50-500 μg/mL). The presence of insulin did not affect the cell viability significantly. Higher toxicities were observed for PEG 6000-coated SiNP at high concentrations. Microscope images revealed the formation of vacuoles in the Caco-2 cell body, indicating a possible endocytosis of these nanoparticles. The present work allows the conclusion that coating of silica nanoparticles with mucoadhesive polymers influences their physicochemical properties, insulin release, as well as the cell viability. These studies and findings show the feasibility of applying silica nanoparticles for oral insulin delivery using a simple, cheap and environmentally friendly technology.
Descrição
Tese de Doutoramento em Ciências Químicas e Biológicas
Palavras-chave
Nanopartículas de sílica , Insulina , Citotoxicidade , Polímeros mucoadesivos , Administração oral