Acanthamoeba culbertsoni (A. culbertsoni) is an amphizoic amoeba that is the causative agent of Granulomatous Amoebic Encephalitis (GAE), an often fatal central nervous system infection that is seen most frequently in severely immunocompromised patients and is characterized by hemorrhagic and necrotic lesions of the brain as well as varying degrees of granuloma formation. A.culbertsoni isolates have also been identified in a few cases of Amoebic Keratitis, a painful, sight-threatening corneal infection that disproportionately affects contact lens users irrespective of immune status. Common features of both infections include amoebic interaction with host extracellular matrix (ECM) components as requisites for both attachment to, and subsequent invasion of, host tissues to facilitate disease establishment. Previous studies have demonstrated that pathogenic species of Acanthamoeba , such as A.culbertsoni, bind to the ECM proteins Laminin-1 and Collagen I to a greater extent than non-pathogenic species. It has also been documented in the literature that secreted Acanthamoeba proteases have the ability to degrade components of the extracellular matrix. The role of amoebic proteases in mediating the attachment and invasion processes is not entirely understood. Initial experiments conducted in the present study revealed secretion of approximately 150 and 55-kDa serine proteases during attachment as well as invasion of the ECM by A. culbertsoni. However, inhibition of these serine proteases using phenylmethylsulfonyl fluoride (PMSF) did not diminish the ability of amoebae to attach or invade. It was demonstrated that secretion of the observed proteases occurred in a constitutive rather than substrate-induced manner and that amoebae secrete these proteases under a number of different conditions. Additionally, a 140-kDa membrane-associated serine protease was identified which may prove to play a role in focal proteolytic degradation. Collectively, our results suggest that attachment to extracellular matrix components is mediated through non-protease-dependent mechanisms. We also suggest that ECM invasion by A.culbertsoni is predominately a mechanical process that may be supplemented or enhanced by focal proteolytic degradation of extracellular matrix components by membrane-associated proteases.

Free-living amoebae (AVL), belonging to the genus Acanthamoeba are widely distributed in nature, being found in various types of soil and water, in the air, in air conditioning and sewage, and other environments. These amoebae are able to cause severe disease in human beings, highlighting the granulomatous amoebic encephalitis and keratitis. The amoebic keratitis is an infectious disease that affects the cornea and it can lead to blindness, with symptoms such as blurred vision, intense pain, photophobia and increased eye pressure. Users of contact lenses are more amenable to this type of infection because they may present microtraumatisms in the cornea, which makes the environment favorable action of these amoebae. There is no effective treatment for this disease and most used drugs suffers resistance from the parasite and others such as corticosteroids, they may give an initial improvement, but then the worsening of the disease. The solutions for cleaning contact lenses are also not efficient in eliminating this parasite, since the ISO 14729 and FDA (Food and Drug Administration, USA), which regulate the microbiological requirements and test methods for the production of hygiene products for contact lenses do not require that such solutions are tested against Acanthamoeba, which makes users more amenable to this type of lenses infection. In view of this, the objective of this study was to develop a microemulsion with the essential oil of L. gracilis and check its amoebicide potential front of trophozoites of Acanthamoeba castellanii. The formulations were prepared by using the phase diagram, using Tween 80 and propylene glycol as surfactant and co-surfactant of the system. The physico-chemical characterization of the formulations was performed by analysis of polarized light microscopy, rheology, light low angle scattering (SAXS), electrical conductivity, particle size and polydispersity. Amoebicide front activity assays for trophozoite A. castellanii formulation in question and all its separate excipients were performed. The obtained samples had transparent and thermodynamically stable to droplet size and characteristic polydispersity index microemulsion. The formulations presented are isotropic in polarized light microscopy and, through SAXS curves, it was observed that the formulations showed characteristics bicontinuous microemulsion. These formulations showed low electrical conductivity which can be associated with high amount of nonionic surfactant. The rheological properties of the system have shown that they possess newtonian behavior characteristic of microemulsion and that the viscosity increases when the amount of oil decreases and hence the droplet size. In bioassays, the surfactants showed no significant activity against the trophozoites and the essential oil of L. gracilis showed an IC50 of 9,52μg / ml. When the test was conducted with the microemulsion containing the same oil, this IC 50 fell to 2.55 μg / ml and using a formulation with smaller droplet tamnho this value fell to 0.65μg/ml, thus proving the efficacy of nanossistema. / Amebas de vida livre (AVL), pertencentes ao gênero Acanthamoeba, estão amplamente distribuídas na natureza, sendo encontradas em diversos tipos de solo e água, no ar, em ar-condicionado e esgotos, entre outros ambientes. Essas amebas são capazes de ocasionar graves doenças nos seres humanos, destacando a encefalite granulomatosa e a ceratite amebiana. A ceratite amebiana é uma doença infecciosa que acomete a córnea e que pode levar a cegueira, apresentando sintomas como visão borrada, dor intensa, fotofobia e aumento da pressão ocular. Os usuários de lentes de contato são os mais propícios a este tipo de infecção, pois podem apresentar microtraumatismos na córnea, o que torna o ambiente favorável a ação dessas amebas. Ainda não existe um tratamento eficaz para esta doença e a maioria dos medicamentos utilizados sofre resistência por parte do parasito e outros, como os corticosteróides, podem apresentar uma melhora inicial, mas em seguida o agravamento da doença. As soluções para limpeza das lentes de contato também não são tão eficientes em eliminar este parasita, uma vez que a ISO 14729 e a FDA (Food and Drug Administration, Estados Unidos), que regulamentam os requisitos microbiológicos e os métodos de ensaio para a produção de produtos de higiene para lentes de contato, não obrigam que essas soluções sejam testadas contra Acanthamoeba, o que torna os usuários de lentes mais propícios a este tipo de infecção. Diante disto, o objetivo deste trabalho foi desenvolver e caracterizar sistemas microemulsonados, contendo o óleo essencial de Lippia gracilis como fase oleosa e analisar sua potencial ação amebicida frente a trofozoítos de Acanthamoeba castellanii. As formulações foram preparadas através do uso do diagrama de fase, utilizando o Tween 80 e o propilenoglicol como tensoativo e cotensoativo, respectivamente, do sistema. A caracterização físico-química das formulações foi realizada por meio de análises de microscopia de luz polarizada, reologia, espalhamento de luz a baixo ângulo (SAXS), condutividade elétrica, tamanho de partícula e índice de polidispersão. Foram realizados ensaios de atividade amebicida frente à trofozoítos de A. castellanii da formulação em questão e de todos os seus excipientes separados. As amostras obtidas apresentaram-se transparentes e termodinamicamente estáveis com tamanho de gotícula e índice de polidispersão característicos de microemulsão. As formulações apresentaram-se isotrópicas na microscopia de luz polarizada e, através das curvas de SAXS, foi possível observar que as formulações apresentaram características de microemulsão bicontínua. Estas formulações mostraram baixa condutividade elétrica a qual pode estar associada a alta quantidade de tensoativo não iônico. As propriedades reológicas do sistema demonstraram que estes possuem comportamento newtoniano característico de microemulsão e que a viscosidade aumenta quando diminui a quantidade de óleo e, consequentemente, o tamanho da gotícula. Nos ensaios biológicos, os tensoativos não apresentaram atividade significativa frente aos trofozoítos e o óleo essencial de L. gracilis apresentou uma IC50 de 9,52μg/ml. Quando o teste foi realizado com a microemulsão contendo o mesmo óleo, essa IC50 caiu para 2,55 μg/ml e, ao utilizar uma formulação com tamanho de gotícula menor, esse valor caiu para 0,65 μg/ml, comprovando, assim, a eficácia do nanossistema. / São Cristóvão, SE