Development and characterization of niosomes as new drugs delivery systems

Trabalho Final de Mestrado Integrado, Ciências Farmacêuticas, 2020, Universidade de Lisboa, Faculdade de Farmácia.A utilização de niossomas para a veiculação de medicamentos, tem recebido muita atenção devido à potencial ação selectiva destas nanovesículas. Os niossomas são nanovesículas que se formam de forma espontânea, através da hidratação de tensioativos sintéticos. As propriedades dos niossomas são determinadas pelo seu tamanho, forma e pelas propriedades físico-químicas da sua superfície, sendo capazes de modificar a biodistribuição do medicamento e consequentemente a farmacocinética do fármaco por ele veículado. Este trabalho começa por rever a literatura atualmente disponível sobre estas nanovesículas, relativamente aos diferentes componentes que são utilizados no seu desenvolvimento, métodos de preparação, fatores que afetam a sua formação, bem como metodologias utilizadas para a sua caracterização e principais aplicações. O presente trabalho de investigação está integrado num projecto que tem como objectivo o desenvolvimento e caracterização de niossomas funcionalizados com receptores de manose, para a veiculação de fármacos anti-tumorais. O trabalho realizado corresponde apenas a um dos primeiros objetivos deste projecto, o qual se focou no desenvolvimento e caracterização de niosomas vazios, tendo em vista a avaliação do impacto dos diferentes métodos de produção, bem como das diferentes proporções dos componentes da formulação nas propiedades físico-químicas destes sistemas. Foram testados três métodos diferentes para a produção de niossomas (TLE-Paddle, TLE-Vortex e Reverse Phase) e 6 proporções dos componentes dos niossomas (Span®60, colesterol e Solulan). O tamanho, a dispersão de tamanhos (PDI), e o potencial dos niossomas foram determinados de forma a averiguar o impato das variáveis acima referidas. Os resultados apresentados permitem concluir que apenas o diferente rácio molar da formulação testada teve impacto nas características dos niossomas, uma vez que as propriedades dos niossomas não foram afetadas pelos métodos de produção. De entre as formulações de niossomas testadas, a proporção 50:25:25 de Span®60:Colesterol:Solulan permitiu produzir niossomas com as características desejadas, relativamente ao tamanho (inferior a 200 nm) e PDI (inferior a 0,2), sendo seguido pelo sistema produzido utilizando Span®60:Colesterol:Solulan na proporção 40:35:25. 4 Futuramente, os niossomas serão funcionalizados com o recetor de manose à superfície, será selecionado do fármaco antitumoral a veicular, far-se-á a preparação, produção e caracterização destes sistemas, bem como a avaliação da sua ação biológica. Os niossomas constituem sistemas promissores para a veiculação direcionada de substâncias ativas. No entanto, há algumas limitações associadas à sua utilização, nomeadamente a sua translação para uso clínico e o custo de produção.The use of niosomes for the delivery of medicines has received much attention due to the potential selective action of these nanovesicles. Niosomes are self-assembled vesicular nanocarriers obtained by hydration of synthetic surfactants. The properties of niosomes are determined by their size, shape and physico-chemical characteristics of their surface, being able to modify the drug's biodistribution and consequently the pharmacokinetics of the entrapped drug. As a background of the present work, a literature review was made on niosomes: their formulation components, methods of preparation, factors affecting their formation, physicochemical characterization and major applications. This research work is part of a project that aims to develop and characterize functionalized niosomes for mannose-targeted antitumoral drug delivery. The work carried out corresponds only to one of the first objectives of this project: development and characterization of empty niosomes, produced by different methods and using different proportions of the formulation. Three different methods have been explored for vesicle preparation (TLE-Paddle, TLE-Vortex and Reverse Phase method) and 6 proportions of the main excipients have been tested for niosomes formulation (Span®60, cholesterol and Solulan). Niosomes were characterized, in terms of particle size, homogeneity (polydispersity index PDI) and zeta potential. The results obtained indicate that only the molar ratio composition had an impact on the characteristics of niosomas, since the properties of niosomes were not affected by the production methods. Among the tested niosome formulations, the 50:25:25 ratio of Span®60:Cholesterol:Solulan allowed to produce niosomes with the targeted characteristics, namely size (less than 200nm) and PDI (less than 0.2), followed by niosomes produced using Span®60:Colesterol:Solulan in the ratio 40:35:25. In the future, the niosome surface will be functionalized with the mannose receptor and loaded with a selected antitumoral drug. The physicochemical properties of these vesicles will be characterized and their biological effect will be evaluated. Niosomes constitute promising targeted drug delivery systems. However, there are some limitations associated with their use, namely their translation for clinical use and the cost of production

Narraciones políticas y procesos de enmarcamiento del discurso de las asociaciones del Foro para la Integración Social de los Inmigrantes de España

En este artículo, se realiza un pormenorizado análisis en torno a los mensajes políticos construidos por las asociaciones de inmigrantes que forman parte del Foro para la Integración Social de los Inmigrantes de España. Para ello, se ha optado por el uso del marco teórico del «análisis de marcos» (McAdams, McCarthy y Zald, 1999; Snow, Benford y Hunt, 1993), categoría analítica proveniente del «interaccionismo simbólico» de Goffman (1974), y que se considera como un elemento central en la formación de la identidad de los actores sociales y en su «actuación social», así como en la definición de problemáticas, causas y soluciones de sus reivindicaciones. Secundariamente, se esbozan las resonancias de sus propuestas y de sus programas políticos y sociales en diferentes «audiencias», con el objetivo de evaluar el impacto político de sus discursos.This article carries out a detailed analysis of the political messages constructed by the immigrants associations which are part of the Forum for the Social Integration of the Spain Immigrants. The author has opted to make use of the «frame analysis» theoretical mark (McAdams, McCarthy and Zald, 1999; Snow, Benford and Hunt, 1993), analytic category coming from the «symbolic interactionism» of Goffman (1974), considered as a central element in the identity formation of the social actors and in their «social performance», as well as in the definition of problematic, causes and solutions of their demands. Secondarily, the author addresses the resonance of their proposals and their political and social programs in different «audiences» in order to evaluate the political impact of their speeches

Investigating Cytoskeletal Alterations as a Potential Marker of Retinal and Lens Drug-Related Toxicity

Actin filaments play a critical role in the normal physiology of lenticular and retinal cells in the eye. Disruption of the actin cytoskeleton has been associated with retinal pathology and lens cataract formation. Ocular toxicity is an infrequent observation in drug safety studies, yet its impact to the drug development process is significant. Recognizing compounds through screening with a potential ocular safety liability is one way to prioritize development candidates while reducing development attrition. Lens epithelial cells from human, dog, and rat origins and retinal pigmented epithelium cells from human, monkey, and rat origins were cultured and investigated with immunocytochemical techniques. Cells were treated using noncytotoxic doses of the compound, fixed, stained for actin with rhodamine phalloidin, and counterstained for nuclei with TOTO-3, followed by confocal imaging. Tamoxifen and several experimental compounds known to be in vivo lens and retinal toxicants caused a reduction in F-actin fluorescence at noncytotoxic concentrations in all cells tested as observed by confocal microscopy. Developing an assay that predicts ocular toxicity helps the development process by prioritizing compounds for further investigation. Drug-induced cytoskeletal alterations may be useful as a potential safety-screening marker of retinal and lens toxicity. The knowledge of actin molecular biology and the application of other mechanistic screens to toxicology are discussed. Reducing this work to a high-throughput platform will enable chemists to select compounds with a reduced risk of ocular toxicity.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/63132/1/adt.2006.4.695.pd

Posterior Segment Approach for Subretinal Transplantation or Injection in the Canine Model

A posterior segment approach for cell transplantation or injection into the subretinal space of the dog has been developed. Controlled penetration to the subretinal space was achieved using a 29-gauge injection cannula, either blunted or with a 30° sharpened bevel, and partially ensheathed with moveable plastic tubing. Depending on the injection volume used, the retina detached, and the fluid was reabsorbed within 1–3 weeks, although for smaller volumes the retina reattached within a matter of days. The optimal injection volume used was between 100 and 150 μl, or two injections of 55 μl each. By ophthalmoscopy following the surgery, it was possible to serially monitor the injection site and retinal bleb through fundus photography. Light microscopy demonstrates the distribution of stable, viable RPE cells in the subretinal space up to 6 months. The transplantation technique developed for the dog is atraumatic and free from any major surgical or clinical complications. It can be readily used to deliver cells or fluids to localized regions of the subretinal space

Prostitucão e confinamento: o putanheiro 2.0

En este artículo analizamos el comportamiento de los clientes de prostitución o “puteros” durante los meses de confinamiento derivados de la pandemia global que estamos padeciendo. Pese al riesgo para la vida y la salud, en estos meses la demanda de sexo comercial en España no ha cesado, tal y como se desprende de los testimonios de las mujeres en prostitución y de los contenidos de los chats de puteros. Este hecho contradictorio demuestra la fortaleza actual del mandato de masculinidad patriarcal en España – primer país europeo en consumo de prostitución – y la hegemonía de un modelo de masculinidad fallido en su humanidad, incompatible con el momento de vulnerabilidade total que estamos sufriendo como sociedadIn this article we analyze the behavior of prostitution clients, or “whorers”, during the months of confinement derived from the global pandemic that we are experiencing. Despite the risk to life and health, the demand for commercial sex in Spain has not ceased in recent months, as can be seen from the testimonies of women in prostitution and the contents of the chat rooms of prostitution clients. This contradictory fact demonstrates the current strength of the patriarchal masculinity mandate in Spain – the first European country in the consumption of prostitution – and the hegemony of a failed model of masculinity in its humanity, incompatible with the moment of total vulnerability that we are suffering as a societyNeste artigo analisamos o comportamento dos clientes de prostituição ou “puteros” durante os meses de confinamento da pandemia global que estamos a viver. Apesar do risco para a vida e a saúde, nestes meses a procura de sexo comercial em Espanha não cessou, como se pode ver pelos testemunhos de mulheres na prostituição e pelos conteúdos dos chats dos clientes de sexo pago. Este facto contraditório demonstra a força atual do mandato patriarcal da masculinidade em Espanha – o primeiro país europeu em consumo de prostituição – e a hegemonia de um modelo de masculinidade que falhou na sua humanidade, incompatível com o momento de vulnerabilidade total que estamos a sofrer como sociedadeS

El valor de una historia digital en el contexto europeo de aprendizaje integrado a través de lengua y contenido (CLIL)

This article presents the research conducted within the framework of the funded project called European CLIL Resource Centre for Web 2.0 Education (E-CLIL, 504671-LLP-1-2009-1-ES-COMENIUS-CMP). The mission of E-CLIL is to increase children's exposure to European languages and to improve the quality of teaching through the implementation of content and language integrated learning (CLIL) within the European demand for acquiring a mother tongue plus two foreign languages. The project is currently building multilingual interactive resources for the use of CLIL teachers. More specifically, the present study investigates the value a digital story may bring to a Primary education context which promotes CLIL and ICT as methodological procedure and resources. The central digital story, originally created for this project, entails three main objectives which involves exposing young learners to European languages, children literature and science content. Our main aim was to prove whether the universal narrative elements and structure present in this digital story may enhance 8 to 10 year-old students' learning involvement and CLIL achievement. In an initial piloting phase of the study, the story was presented to a total of 12 experienced English CLIL teachers working in three different state bilingual schools in the region of Madrid. In order to gather objective data that may prove our hypotheses these teachers completed initial and final questionnaires which were then analysed. The outcome gathered has been used to extend the piloting to other European countries participating in the project. This larger study will provide information on cross-cultural similarities or differences regarding the results already obtained here. These initial findings point towards the richness a central digital storyline may add to a CLIL learning context at linguistic, content, literary, cognitive, social and cultural levels.Este artículo se inscribe en el marco de la investigación llevada a cabo desde 2009 en el proyecto European CLIL Resource Centre for Web 2.0 Education (E-CLIL, 504671-LLP-1-2009-1-ES-COMENIUS-CMP), uno de cuyos objetivos principales es la investigación y desarrollo de recursos digitales para la enseñanza integrada de contenidos y lengua (CLIL) según las directrices de la Unión Europea. Es un proyecto multilingüe y multicultural apoyado en las posibilidades que ofrece la web 2.0 en cuanto a interactividad, creación y conocimientos compartidos y conectividad, en el sentido más amplio del término. En este marco general, la investigación se ha dirigido a una etapa de educación primaria, la que corresponde a niños y niñas de 8 a 10 años, y el trabajo que aquí presentamos recoge una parte del mencionado proyecto y algunos de sus resultados iniciales, puesto que se encuentra todavía en fase de desarrollo. En esta fase inicial del pilotaje en los colegios, se presentó el cuento digital a un total de 12 profesoras de inglés en contexto CLIL que desarrollan su actividad docente en tres colegios públicos bilingües de la zona norte de la Comunidad de Madrid. Con el fin de compilar datos objetivos que probasen nuestra hipótesis inicial, estas profesoras completaron cuestionarios iniciales y finales para su posterior análisis. El resultado obtenido se tendrá en cuenta en el pilotaje ulterior que se extenderá a otros países europeos que participan en este proyecto de manera inminente. El aprendizaje simultáneo de los contenidos del currículum y una segunda o tercera lengua es una de las líneas prioritarias de la investigación educativa en el contexto europeo, y así lo recoge el proyecto que presentamos

El método del caso en docencia: aplicación y evaluación

El método del caso como estrategia docente ha sido ampliamente utilizado en otros países. En España, con la introducción de Espacio Europeo de Enseñanza Superior (EEES), se plantea como un recurso útil, sobre todo, en los últimos cursos de grados y, fundamentalmente, en los post-grados. Estas bondades del método del caso se acentúan considerablemente si su redacción se aborda desde una perspectiva multidisciplinar. En este trabajo proponemos una metodología propia a seguir para la utilización del método del caso con los alumnos y proponemos un mecanismo de evaluación de la experiencia.The case method like educational strategy has been widely used in other countries. In Spain, with the introduction of European Space of Higher education (EEES), it appears as a useful resource, especially, in the last courses of degrees and, fundamentally, in the post-degrees. This kindness of the case method accents considerably if his draft is approached from a multidisciplinary perspective. In this work we propose an own methodology to continuing for the utilization of the method of the case and propose a mechanism of evaluation of the experience

Sox-2 Positive Neural Progenitors in the Primate Striatum Undergo Dynamic Changes after Dopamine Denervation.

The existence of endogenous neural progenitors in the nigrostriatal system could represent a powerful tool for restorative therapies in Parkinson's disease. Sox-2 is a transcription factor expressed in pluripotent and adult stem cells, including neural progenitors. In the adult brain Sox-2 is expressed in the neurogenic niches. There is also widespread expression of Sox-2 in other brain regions, although the neurogenic potential outside the niches is uncertain. Here, we analyzed the presence of Sox-2+ cells in the adult primate (Macaca fascicularis) brain in naïve animals (N = 3) and in animals exposed to systemic administration of 1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine to render them parkinsonian (N = 8). Animals received bromodeoxyuridine (100 mg/kg once a day during five consecutive days) to label proliferating cells and their progeny. Using confocal and electron microscopy we analyzed the Sox-2+ cell population in the nigrostriatal system and investigated changes in the number, proliferation and neurogenic potential of Sox-2+ cells, in control conditions and at two time points after MPTP administration. We found Sox-2+ cells with self-renewal capacity in both the striatum and the substantia nigra. Importantly, only in the striatum Sox-2+ was expressed in some calretinin+ neurons. MPTP administration led to an increase in the proliferation of striatal Sox-2+ cells and to an acute, concomitant decrease in the percentage of Sox-2+/calretinin+ neurons, which recovered by 18 months. Given their potential capacity to differentiate into neurons and their responsiveness to dopamine neurotoxic insults, striatal Sox-2+ cells represent good candidates to harness endogenous repair mechanisms for regenerative approaches in Parkinson's disease

Wnt-Dependent Oligodendroglial-Endothelial Interactions Regulate White Matter Vascularization and Attenuate Injury.

Recent studies have indicated oligodendroglial-vascular crosstalk during brain development, but the underlying mechanisms are incompletely understood. We report that oligodendrocyte precursor cells (OPCs) contact sprouting endothelial tip cells in mouse, ferret, and human neonatal white matter. Using transgenic mice, we show that increased or decreased OPC density results in cognate changes in white matter vascular investment. Hypoxia induced increases in OPC numbers, vessel density and endothelial cell expression of the Wnt pathway targets Apcdd1 and Axin2 in white matter, suggesting paracrine OPC-endothelial signaling. Conditional knockout of OPC Wntless resulted in diminished white matter vascular growth in normoxia, whereas loss of Wnt7a/b function blunted the angiogenic response to hypoxia, resulting in severe white matter damage. These findings indicate that OPC-endothelial cell interactions regulate neonatal white matter vascular development in a Wnt-dependent manner and further suggest this mechanism is important in attenuating hypoxic injury.M.C. acknowledges fellowship awards from the American Heart Association and The Children’s Heart Foundation and funding support from a Career Development Grant awarded by Cerebral Palsy Alliance Research Foundation. J.M.G.V is funded by Red deTerapia Celular (TerCel-RD16/0011/0026) and the Valencian Council for Innovation, Universities Science and Digital Society (PROMETEO/2019/075). M.J.U.N was supported by a McDonald Fellowship from the Multiple Sclerosis International Federation. This work was supported by funding from the National Multiple Sclerosis Foundation (to D.H.R.), the Adelson Medical Research Foundation (D.H.R), the European Research Council (D.H.R.) and the National Institutes of Health, NINDS (1K99NS117804 to M.C; P01- NS083513 to D.H.R., E.J.H and P.S.M)

Climate relevant trace gases (N2O and CH4) in the Eurasian Basin (Arctic Ocean)

The concentration of greenhouse gases, including nitrous oxide (N2O), methane (CH4), and compounds such as total dimethylsulfoniopropionate (DMSPt), along with other oceanographic variables were measured in the icecovered Arctic Ocean within the Eurasian Basin (EAB). The EAB is affected by the perennial ice-pack and has seasonal microalgal blooms, which in turn may stimulate microbes involved in trace gas cycling. Data collection was carried out on board the LOMROG III cruise during the boreal summer of 2012. Water samples were collected from the surface to the bottom layer (reaching 4300 m depth) along a South-North transect (SNT), from 82.19°N, 8.75°E to 89.26°N, 58.84°W, crossing the EAB through the Nansen and Amundsen Basins. The Polar Mixed Layer and halocline waters along the SNT showed a heterogeneous distribution of N2O, CH4 and DMSPt, fluctuating between 42-111 and 27–649% saturation for N2O and CH4, respectively; and from 3.5 to 58.9 nmol L−1 for DMSPt. Spatial patterns revealed that while CH4 and DMSPt peaked in the Nansen Basin, N2O was higher in the Amundsen Basin. In the Atlantic Intermediate Water and Arctic Deep Water N2O and CH4 distributions were also heterogeneous with saturations between 52% and 106% and 28% and 340%, respectively. Remarkably, the Amundsen Basin contained less CH4 than the Nansen Basin and while both basins were mostly under-saturated in N2O. We propose that part of the CH4 and N2O may be microbiologically consumed via methanotrophy, denitrification, or even diazotrophy, as intermediate and deep waters move throughout EAB associated with the overturning water mass circulation. This study contributes to baseline information on gas distribution in a region that is increasingly subject to rapid environmental changes, and that has an important role on global ocean circulation and climate regulation