Estudo da eficácia de encapsulação da Cinarizina em cubossomos não-iônicos: caracterização estrutural e citotóxica / Study of the encapsulation efficacy of Cinnarizine in non-ionic cubosomes: structural and cytotoxic characterization

Os cubossomos são partículas nanoestruturadas em forma de bicamada lipídica, bicontínuas e altamente curvadas, as quais devem ser estabilizadas por um polímero não-iônico, neste caso o Pluronic® F-127. Podem ser compostos por alguns tipos de lipídios específicos que possuem a capacidade de se auto associar em estruturas cúbicas quando estão em excesso de água, como o fitantriol (PHY) e a monoleína (GMO). Devido a sua estrutura única, cubossomos possuem um grande potencial para serem considerados como sistemas drug delivery. Os sistemas drug delivery são amplamente utilizados na pesquisa farmacêutica e em contextos clínicos para aumentar a eficácia de compostos utilizados para diagnóstico e de fármacos. No caso da cinarizina (CNZ), fármaco já aprovado para o tratamento de náuseas, vômitos e vertigens causadas pela doença de Ménière, existem inúmeros efeitos colaterais associados a sua baixa solubilidade. Desta forma, a encapsulação em cubossomos se torna uma abordagem promissora para resolver os problemas de atividade farmacológica relacionados ao fármaco. Neste trabalho, realizamos uma caracterização biofísica da interação da CNZ em cubossomos (contendo PHY ou myverol, MYV, sendo este composto por 80% de GMO). As técnicas biofísicas utilizadas foram: espalhamento de raios-X em baixos ângulos (SAXS), espalhamento dinâmico de luz (DLS), microscopia eletrônica de transmissão (TEM), crio microscopia eletrônica de transmissão (Crio-TEM), análise de rastreamento de nanopartículas (NTA) e potencial zeta. A cromatografia líquida de alta eficiência (HPLC) foi realizada para verificar a porcentagem de eficiência de encapsulação (%EE) da CNZ nos cubossomos, enquanto que a citotoxicidade foi avaliada em eritrócitos através da análise da atividade hemolítica. Inicialmente, a influência de diferentes solventes (acetona, clorofórmio, etanol e octano) nas propriedades estruturais de cubossomos de PHY foi investigada, a fim de se minimizar os efeitos do solvente utilizados para a encapsulação da CNZ. Para amostras com acetona, descobriu-se que apenas altas concentrações tiveram influência na estrutura cristalográfica das nanopartículas, sendo o resultado foi totalmente reversível após 24h. O etanol fez com que o parâmetro de rede aumentasse de 10-15%. O clorofórmio e o octano tiveram efeitos diferentes sobre cubossomos de PHY em comparação com a acetona e o etanol; ambos induziram uma transição cúbico-hexagonal-micelar. Posteriormente, constatamos que as nanopartículas de PHY e MYV apresentaram diferentes estruturas cristalográficas, sendo elas Pn3m e Im3m, respectivamente. Devido a problemas com a baixa solubilidade de CNZ em PHY os estudos para esse lipídio foram suspensos. Nos testes para cubossomos de MYV ao incorporar a CNZ foi observado uma alteração da estrutura cúbica de Im3m para Pn3m e os valores dos parâmetros de rede se alteraram de acordo com a estrutura cristalina encontrada, porém os valores não apresentaram diferenças significativas de tamanho quando se trata da mesma estrutura, sugerindo que a CNZ não interferiu no parâmetro de rede. Os tamanhos das nanopartículas apresentaram uma população monodispersa com ~200 nm. DLS mostrou uma interferência da CNZ no tamanho dos cubossomos, variando de forma diretamente proporcional a concentração de CNZ na amostra, enquanto as técnicas de NTA e microscopia apresentaram nanopartículas de tamanhos bastante variados, mas independente da interferência da CNZ. A encapsulação de CNZ também foi dosada por HLPC em cubossomos de MYV, obtendo um limite superior de 0,6 mg/mL. A atividade citotóxica dos cubossomos foi testada em eritrócitos, revelando uma taxa de hemólise bastante inferior em cubossomos com CNZ em relação a cubossomos puros. Acreditamos que os cubossomos podem sim ser utilizados como sistemas carreadores de CNZ

Cubosomes are nanostructured particles in the form of a lipid bilayer, bicontinuous and highly curved, which must be stabilized by a non-ionic polymer, in this case Pluronic® F-127. They can be composed of some types of specific lipids that have the ability to self-associate in cubic structures when they are in excess of water, such as phytantriol (PHY) and monolein (GMO). Due to their unique structure, cubosomes have a great potential to be considered as drug delivery systems. Drug delivery systems are widely used in pharmaceutical research and clinical settings to increase the efficacy of compounds used for diagnostics and drugs. In the case of cinnarizine (CNZ), a drug already approved for the treatment of nausea, vomiting and vertigo caused by Ménière's disease, there are numerous side effects associated with its low solubility. Thus, cubosomal encapsulation becomes a promising approach to solve drug-related problems of pharmacological activity. In this work, we performed a biophysical characterization of the CNZ interaction in cubosomes (containing PHY or myverol, MYV, which is composed of 80% GMO). The biophysical techniques used were: low angle X-ray scattering (SAXS), dynamic light scattering (DLS), transmission electron microscopy (TEM), cryo transmission electron microscopy (Crio-TEM), nanoparticle tracking analysis (NTA) and zeta potential. High performance liquid chromatography (HPLC) was performed to verify the percentage of encapsulation efficiency (%EE) of CNZ in cubosomes, while cytotoxicity was evaluated in erythrocytes by analyzing the hemolytic activity. Initially, the influence of different solvents (acetone, chloroform, ethanol and octane) on the structural properties of PHY cubosomes was investigated in order to minimize the effects of the solvent used for the encapsulation of CNZ. For samples with acetone, it was found that only high concentrations had an influence on the crystallographic structure of the nanoparticles, with the result being fully reversible after 24h. Ethanol caused the network parameter to increase by 10-15%. Chloroform and octane had different effects on PHY cubosomes compared to acetone and ethanol; both induced a cubic-hexagonal-micellar transition. Later, we found that PHY and MYV nanoparticles presented different crystallographic structures, being Pn3m and Im3m, respectively. Due to problems with the low solubility of CNZ in PHY, studies for this lipid were suspended. In the tests for MYV cubosomes when incorporating CNZ, a change in the cubic structure from Im3m to Pn3m was observed and t he lattice parameters changed according to the crystal structure found, but the differences observed were not significant when it comes to the same structure, suggesting that the CNZ did not interfere with the network parameter. The nanoparticle sizes showed a monodisperse population with ~200 nm. DLS showed an interference of CNZ in the size of the cubosomes, varying directly proportionally to the concentration of CNZ in the sample, while NTA and microscopy techniques showed nanoparticles of widely varying sizes, but independent of CNZ interference. CNZ encapsulation was also dosed by HLPC in MYV cubosomes, obtaining an upper limit of 0.6 mg/ml. The cytotoxic activity of cubosomes was tested in erythrocytes, revealing a much lower rate of hemolysis in cubosomes with CNZ compared to pure cubosomes. We believe that cubosomes can indeed be used as CNZ carrier systems

Optimization of self-nanoemulsifying formulations for weakly basic lipophilic drugs: role of acidification and experimental design

ABSTRACT Formulators face great challenges in adopting systematic approaches for designing self-nanoemulsifying formulations (SNEFs) for different drug categories. In this study, we aimed to build-up an advanced SNEF development framework for weakly basic lipophilic drugs, such as cinnarizine (CN). First, the influence of formulation acidification on CN solubility was investigated. Second, formulation self-emulsification in media with different pH was assessed. Experimentally designed phase diagrams were also utilized for advanced optimization of CN-SNEF. Finally, the optimized formulation was examined using cross polarizing light microscopy for the presence of liquid crystals. CN solubility was significantly enhanced upon external and internal acidification. Among the various fatty acids, oleic acid-based formulations showed superior self-emulsification in all the tested media. Surprisingly, formulation turbidity and droplet size significantly decreased upon equilibration with CN. The design was validated using oleic acid/Imwitor308/Cremophor El (25/25/50), which showed excellent self-nanoemulsification, 43-nm droplet size (for CN-equilibrated formulations), and 88 mg/g CN solubility. In contrast to CN-free formulations, CN-loaded SNEF presented lamellar liquid crystals upon 50% aqueous dilution. These findings confirmed that CN-SNEF efficiency was greatly enhanced upon drug incorporation. The adopted strategy offers fast and accurate development of SNEFs and could be extrapolated for other weakly basic lipophilic drugs.