Microfluidic methods for the controlled preparation of soft self-assembled nanocarriers for drug delivery

Dissertação de mestrado em Biophysics and BionanosystemsCubosomes are dispersions of lipid bicontinuous cubic phases in water. These particles consist of an interior continuous matrix of lipid bilayers arranged in a 3D lattice of cubic symmetry that separates two independent continuous networks of water channels. This coexistence of lipidic and aqueous domains makes cubosomes promising candidates for the encapsulation and delivery of both hydrophobic and hydrophilic drugs. Cubosomes are typically prepared either by fragmenting the cubic liquid crystal in excess water using high energy input (e.g. ultra-sonication), or using solvent-shifting approaches, in which the lipid is first dissolved in a water-miscible solvent (typically ethanol), and later mixed with water and polymer stabilizer. In both cases, poor experimental control at the micron- and nanoscales (e.g. poor control on concentration and heat gradients), limits the fine tuning of the particle properties and results in cubosomes with broad size distributions. In this work, we employ the solvent-shifting method to the monoolein-ethanol-water system and form cubosomes. For this, a microfluidic device capable of mixing the fluids in a rapid and controlled way at the micron-scale, and obtaining cubosomes of tuneable size and low polydispersity, is used. The micron-sized channels in microfluidics lead to laminar flow regimes and enhanced experimental control. In this regime, hydrodynamic focusing can be used to narrow down the length that solvent molecules have to travel to mix, thus decreasing the mixing time. An ethanol-lipid solution is flowed in a central inlet, which is squeezed by two side streams of water with stabilizer. As the lipid-ethanol solution narrows, ethanol and water are mixed in a controlled way by diffusion, leading to formation of cubosomes. By manipulating the flow rate ratio between the two solutions we manipulate the width in which the hydrodynamic focusing occurs, influencing the assembly time in a homogeneous way. This way, by manipulating the flow rate ratio, we were able to tune the size of the cubosome nanoparticles, achieving smaller sizes when increasing the extent of the hydrodynamic focusing (i.e. increasing the flow rate ratio). In addition, the final particle size was also found to be influenced by the composition of the initial solutions. At lipid concentrations above 1 wt% in the ethanolic solution, control over the final particle size is lost. Likewise, inclusion of dioctadecyldimethylammonium bromide (a cationic surfactant) into the formulation also led to large and size-uncontrolled particles. On the other hand, including 29% of water in the initial ethanolic solution leads to smaller particle sizes in the end, also in a controlled way also dependent on the flow rate ratio. In contrast, using a stabilizer to lipid ratio of 1:1 or 3:1 did not show a significant change in the final sizes obtained. Altogether, these results suggest that cubosome size is directly dependent on the exchange rate of solvent molecules until a critical water concentration that induces particle formation is achieved. By allowing a precise control over the mixing and exchange between the two solvents, microfluidics promises to be a promising approach to further tailor the structure and efficiency of drug delivery systems. Nanoparticle size is a key parameter in the carriage of pharmaceuticals. Controlling cubosome size is therefore a relevant step towards the design of new and more efficient formulations.Cubossomas são dispersões lipídicas de fases cúbicas bicontínuas em água. Estas partículas possuem no seu interior uma matriz de bicamadas lipídicas arranjadas numa rede tridimensional contínua de simetria cúbica que separa duas redes continuas de canais de água. Tal coexistência de domínios lipídicos e aquosos faz dos cubossomas excelentes candidatos para a encapsulação e entrega de compostos hidrofóbicos e hidrofílicos. Os cubossomas são geralmente preparados quer por fragmentação da fase bicontínua cúbica em excesso de água ao introduzir uma grande quantidade de energia (como por exemplo ultra-sonicação), ou então por uma troca de solventes, no qual o lípido é primeiramente dissolvido num solvente miscível em água (tipicamente o etanol), e mais tarde dissolvido em água e um polímero estabilizante. Em ambos os casos, um fraco controlo experimental à micro e nano-escala (e.g. fraco controlo nos gradientes de concentração e temperatura), limita a manipulação das propriedades das partículas e resulta em cubossomas com amplas distribuições de tamanho. Neste trabalho, o método da troca de solventes é aplicado ao sistema monooleína-etanol-água para formar cubossomas. Para este fim, um dispositivo de microfluídica capaz de misturar os solventes rápida e controladamente à microescala, e obter cubossomas com tamanhos manipuláveis e baixa polidispersão, foi usado. Os canais à microescala usados nos dispositivos de microfluídica fazem com que o regime de fluxo seja laminar e reforce o controlo experimental. Neste regime, a focagem hidrodinâmica pode ser usada para diminuir a distância que as moléculas têm de atravessar para se misturarem, o que leva a uma diminuição no tempo de mistura. Uma solução de lípido-etanol é inserida pela entrada central, onde será de seguida espremida por dois fluidos laterais de água com estabilizante. À medida que a solução de lípido-etanol é espremida, o etanol e a água vão sendo misturados de uma forma controlada por difusão, o que leva à formação dos cubossomas. Ao manipular o rácio dos diferentes caudais entre as diferentes soluções, a largura à qual a focagem hidrodinâmica se dá é ajustada, influenciando o tempo de associação entre as moléculas lipídicas de forma homogénea. Desta forma, ao manipular o rácio dos caudais, foi possível manipular o tamanho dos cubossomas, atingindo menores tamanhos quando a extensão da focagem hidrodinâmica foi aumentada (aumentando o rácio dos caudais). Conjuntamente, também foi observado que a composição das soluções iniciais também influência o tamanho final das partículas. A concentrações superiores a 1 %(m/m) de lípido na solução de etanol, o controlo sobre o tamanho das partículas é reduzido. Assim como a inclusão do surfactante catiónico brometo de dioctadecil dimetil amónio (DODAB), que na formulação também gerou partículas de grandes tamanhos de forma descontrolada. Por outro lado, a inclusão de 29 %(m/m) de água na solução de lípido-etanol levou a que se formassem partículas com tamanhos reduzidos, também de uma forma controlada e dependente do rácio entre os caudais dos fluidos. A contrastar, quando utilizado um rácio de estabilizante para lípido de 1:1 ou 3:1, os tamanhos finais obtidos não demonstraram diferenças significativas entre ambos os rácios. Resumindo, estes resultados sugerem que o tamanho dos cubossomas está diretamente dependente do ritmo ao qual as moléculas de solvente vão sendo trocadas até atingirem uma concentração crítica de água que induz a formação das partículas. Ao permitir um controlo preciso sobre a mistura e troca entre ambos os solventes, as técnicas de microfluídica assumem-se como uma forma promissora de manipular a estrutura e aumentar a eficiência dos sistemas de entrega de fármacos. O tamanho das nanopartículas é um fator chave na entrega de fármacos e a possibilidade de controlar o tamanho dos cubossomas é, portanto, um passo na direção do desenvolvimento de novas e mais eficientes formulações

Risk of cardiovascular disease in soccer referees: a cross sectional study

Introducrion: although the mortality rate among soccer players is low, it causes much impact on the entire community and is a fascinating conundrum. Objetive: to evaluate the cardiovascular risk of soccer referees, to describe the frequency of cardiovascular risk considering age groups younger and older than 35 years old and to relate the Framingham and PROCAM indices for the comparison of cardiovascular risk in soccer referees. Methods: We analyzed 50 referees of Paulista Soccer Federation subjected to clinical and laboratory based evaluations at the Center for Sports Health at the Faculty of Medicine of ABC, and were divided into two groups: lower and higher than 35 years of age. For analysis involving all variables of the study it was initially carried out as a descriptive evaluation of the entire group, and then the Framingham and PROCAM methods were applied in a two-dimensional way. Finally, the logistic regression analysis was applied. Anthropometric statistics, blood pressure, laboratory tests and ergospirometric values were within normal limits. Results: referees older than 35 years old presented significant increased values of waist circumference, body mass index, glucose blood levels, lower VO2max and higher cardiovascular risk according to Framingham and PROCAM. Conclusion: the risk for coronary heart disease was higher in referees older than 35 years old

Conflicts of interests on the issue of Timber Exploitation within the Chico Mendes Extractive Reserve, Acre, Brazil

This paper analyzes the problematic situation of commercial timber exploitation as a land use option in the community of Rio Branco, located in the Chico Mendes Extractive Reserve, Acre, Brazil, and examines the controversial implementation of the Community Forest Management Project (CFMP). Interviews with extractivists and representatives from nine government and non-government organizations resulted in a list of eight desired land use practices. Each of the interviewees was asked to rank the land uses according to his/her individual priorities. Through the use of the Indicator of Sustainability in Systems of Interests (ISSI), the degree of convergence was calculated between the interest (rank of priorities) of each extractivist and the collective interest, as well as between the interests of the extractivists and other institutions. Non-Timber Forest Products (NTFPs) management and shifting cultivation are the preferred land uses noted by extractivists who are ‘non-participants’ in the CFMP, who attribute minimum priority to timber exploitation. Those who participate in CFMP rank timber exploitation as their top priority. Members of government institutions and NGOs indicated a preference for land uses that cause less impact on the forest, choices that are closer to the group of ‘non-participant’ extractivists than to the ‘participant’ group. In a second round of ranking, CFMP ‘participants’ significantly changed their previous choices, this time electing management of NTFPs and shifting cultivation as their top priorities. The results revealed that the issue of timber exploitation in the Chico Mendes Extractive Reserve constitutes a complex system of interests and that some extractivists do not feel confident in relation to the consequences of the implementation of the project, i.e., the project was not sufficiently mature to be implemented

Propofol infusion syndrome and Brugada syndrome electrocardiographic phenocopy

This anesthetic drug may cause a rare condition named propofol infusion syndrome, characterized by unexplained lactic acidosis, lipemia, rhabdomyolysis, cardiovascular collapse and Brugada-like electrocardiographic pattern or Brugada electrocardiographic phenocopy changes following high-dose propofol infusion over prolonged periods of time. Several articles have contributed to our understanding of the cause of the syndrome, and the growing number of case reports has made it possible to identify several risk factors. Uncertainty remains as to whether a genetic susceptibility exists. The favorable recovery profile associated with propofol offers advantages over traditional anesthetics in clinical situations in which rapid recovery is important. Propofol is a safe anesthetic agent, but propofol infusion syndrome is a rare lethal complication. (Cardiol J 2010; 17, 2: 130-135

CFD Approach to Evaluate Heat Transfer in Reciprocating Compressors

How to deal with the heat transfer inside compressors is one of the main challenges on its design nowadays. The heating of the gas entering the compressors decreases its density reducing directly the mass flow and compressor efficiency, so should be avoid. To study this topic it was developed a full three-dimensional model of the gas and the solid parts inside a compressor. Prescribed steady state mass flow rate is imposed at suction and discharge tubes and heat sources due electrical and mechanical inefficiencies of the compressor are imposed. The portion of the suction and cavity gas are separated from the portion of the discharge gas and an isentropic compression from the suction muffler outlet temperature is assumed to define the temperature of the gas flowing through the discharge port. The main advantage of this approach is the directly calculation of the heat transfer coefficients from the solid parts to the fluid, however with a high computational cost. Comparison with experimental data shows good agreement is some parts, but some disagreement in order, probably due poor mesh resolution affecting negatively the heat exchange prediction and also due the non-consideration of the oil flowing inside the compressor, which are topics for future investigations

Predicting the Suction Gas Superheating in Reciprocating Compressors

A considerable portion of the cooling capacity losses on a household compressor occurs due to heating of the gas entering through the suction tube, which is called as superheating. A better understanding of the flow and heat transfer phenomena involved is crucial to improve compressor efficiency. In this work, it is presented a numerical simulation study using a commercial code to analyze a transient three-dimensional, compressible and non-isothermal flow, which has been used for modeling the problem. The computational domain considered the suction tube, a portion of the compressor cavity and the suction muffler. Prescribed transient mass flow rates obtained by simpler 0D/1D models have been used as boundary conditions while the cavity gas and wall temperatures were prescribed based on experimental methods. The results showed that the mean temperature of the gas entering into the compressor was 40°C, while the mean temperature of the gas leaving the suction muffler was 63°C, presenting 23°C of superheating

CFD Approach to Evaluate Heat Transfer in Reciprocating Compressors

How to deal with the heat transfer inside compressors is one of the main challenges on its design nowadays. The heating of the gas entering the compressors decreases its density reducing directly the mass flow and compressor efficiency, so should be avoid. To study this topic it was developed a full three-dimensional model of the gas and the solid parts inside a compressor. Prescribed steady state mass flow rate is imposed at suction and discharge tubes and heat sources due electrical and mechanical inefficiencies of the compressor are imposed. The portion of the suction and cavity gas are separated from the portion of the discharge gas and an isentropic compression from the suction muffler outlet temperature is assumed to define the temperature of the gas flowing through the discharge port. The main advantage of this approach is the directly calculation of the heat transfer coefficients from the solid parts to the fluid, however with a high computational cost. Comparison with experimental data shows good agreement is some parts, but some disagreement in order, probably due poor mesh resolution affecting negatively the heat exchange prediction and also due the non-consideration of the oil flowing inside the compressor, which are topics for future investigations

Efeitos da fluoxetina sobre a ultraestrutura mitocondrial no ventrículo direito de ratos expostos ao estresse pelo frio

OBJECTIVE: To assess fluoxetine effects on mitochondrial structure of the right ventricle in rats exposed to cold stress. METHODS: The experimental study procedures were performed in 250-300g male EPM-Wistar rats. Rats (n=40) were divided into four groups: 1) Control group (CON); 2) Fluoxetine (FLU); 3) Induced hypothermia (IH) and; 4) Induced hypothermia treated with fluoxetine (IHF). Animals of FLU group were treated by the administration of gavages containing 0.75 mg/kg/day fluoxetine during 40 days. The induced hypothermia was obtained by maintaining the groups 3 and 4 in a freezer at -8ºC for 4 hours. The animals were sacrificed and fragments of the right ventricle (RV) were removed and processed prior to performing electron microscopic analysis. RESULTS: The ultrastructural changes in cardiomyocytes were quantified through the number of mitochondrial cristae pattern (cristolysis). The CON (3.85%), FLU (4.47%) and IHF (8.4%) groups showed a normal cellular structure aspect with preserved cardiomyocytes cytoarchitecture and continuous sarcoplasmic membrane integrity. On the other hand, the IH (34.4%) group showed mitochondrial edema and lysis in cristae. CONCLUSION: The ultrastructural analysis revealed that fluoxetine strongly prevents mitochondrial cristolysis in rat heart, suggesting a protector effect under cold stress condition.OBJETIVO: Analisar os efeitos da fluoxetina sobre a estrutura mitocondrial do ventrículo direito de ratos expostos ao estresse pelo frio. MÉTODOS: Os procedimentos do estudo foram realizados em ratos Wistar-EPM (250-300g) machos. Os ratos (n=40) foram divididos em quatro grupos: 1) Controle (CON); 2) Fluoxetina (FLU); 3) Induzidos à hipotermia (IH) e; 4) Induzidos à hipotermia tratados com fluoxetina (IHF). O grupo FLU foi tratado com gavagem contendo 0,75 mg/kg/dia de fluoxetina durante 40 dias. O estresse induzido pelo frio foi realizado mantendo os grupos 3 e 4 em um freezer (-8ºC) por quatro horas. Os animais foram sacrificados e fragmentos do ventrículo direito (VD) foram removidos e processados antes de serem conduzidos para a microscopia eletrônica. RESULTADOS: As alterações ultraestruturais dos cardiomiócitos foram quantificadas pelo número padrão de cristas mitocondriais (cristólises). Os grupos CON (3,85%), FLU (4,47%) e IHF (8,4%) mostraram aspecto normal de suas estruturas celulares com a citoarquitetura dos cardiomiócitos preservada com integridade sarcoplasmática contínua. Por outro lado, o grupo IH (34,4%) apresentou edema mitocondrial e lise nas cristas. CONCLUSÃO: A análise ultraestrutural revelou que a fluoxetina previne fortemente cristólises mitocondriais em miocárdio de ratos, sugerindo possível efeito protetor na condição de estresse induzido pelo frio.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Universidade Federal de São Paulo (UNIFESP)Faculdade de Medicina do ABCFaculdade de Medicina do ABC Departamento de Morfologia e FisiologiaUniversidade Federal de São Paulo (UNIFESP) Departamento de MedicinaUNIFESP, Depto. de MedicinaSciEL

Evaluación de la función barorrefleja en ratas jóvenes espontáneamente hipertensas

BACKGROUND: The literature describes contradictory data regarding the onset of the baroreflex reduction in spontaneously hypertensive rats. OBJECTIVE:This investigation was undertaken to evaluate the baroreflex function in 13-week-old spontaneously hypertensive rats. METHODS:Male Wistar Kyoto (n=15) and spontaneously hypertensive rats (n=15) aged 13 weeks were studied. Cannulas were inserted in the abdominal aortic artery through the right femoral artery to measure mean arterial pressure and heart rate. Baroreflex function was calculated as the derivative of the variation of HR in function of the MAP variation (Δheart rate/Δmean arterial pressure) tested with a depressor dose of sodium nitroprusside (50µg/kg) and with a pressor dose of phenylephrine (8µg/kg) in the right femoral venous approach through an inserted cannula in awake spontaneously hypertensive rats and Wistar-Kyoto. Differences with p values < 0.05 were considered statistically significant. RESULTS:Spontaneously hypertensive rats: Δmean arterial pressure=43.5mmHg±5.2, Δheart rate=-59.7ppm±17.9 and Δheart rate/Δmean arterial pressure=1.3ppm/mmHg±0.1 tested with phenylephrine; Wistar Kyoto: Δmean arterial pressure=&56mmHg±3, Δheart rate=*-114.9ppm±11.3 and Δheart rate/Δmean arterial pressure=#1.9ppm/mmHg±0.3 tested with phenylephrine; spontaneously hypertensive rats: Δmean arterial pressure=-45.6mmHg±8.1, Δheart rate=40.1ppm±11.6 and Δheart rate/Δmean arterial pressure=0.9ppm/mmHg±0.5 tested with sodium nitroprusside; Wistar Kyoto: Δmean arterial pressure=-39.8mmHg±6.2, Δheart rate=51.9ppm±21.8 and Δheart rate/Δmean arterial pressure=1.4ppm/mmHg±0.7 tested with sodium nitroprusside (*p<0.05; #p<0.01; &p<0.001). CONCLUSION: Our results showed that 13-week-old spontaneously hypertensive rats presented reduced baroreflex function when tested with phenylephrine.FUNDAMENTO: La literatura ha descrito datos contradictorios con relación al inicio de la disminución de la función barorrefleja en ratas espontáneamente hipertensas. OBJETIVO: Se realizó este estudio con el objetivo de evaluar la función barorrefleja en ratas jóvenes de 13 semanas, espontáneamente hipertensas. MÉTODOS: Se estudiaron ratas machos Wistar Kyoto (WKY) (n=15) y ratas espontáneamente hipertensas (REH) de 13 semanas de edad (n=15). Se insertaron cánulas en la arteria aorta abdominal -a través de la arteria femoral derecha- para medir la presión arterial media (PAM) y la frecuencia cardiaca (FC). Se calculó la función barorrefleja como la derivada de la variación de la FC en función de la variación de la PAM (ΔFC/ΔPAM). Dicho cálculo se efectuó tras prueba con una dosificación depresora de nitroprusiato de sodio (50µg/kg) y también con una dosificación para presión arterial de fenilefrina (8µg/kg) a través de una cánula insertada en la vena femoral derecha tanto de ratas espontáneamente hipertensas como de WKY. Se consideraron estadísticamente significantes diferencias con un valor de p < 0.05. RESULTADOS: Ratas espontáneamente hipertensas sometidas a prueba con fenilefrina: ΔPAM=43,5 mmHg±5,2, ΔFC=-59,7 ppm±17,9 y ΔFC/ΔPAM=1,3 ppm/mmHg±0,1; Wistar Kyoto probadas con fenilefrina: ΔPAM=&56mmHg±3, ΔFC=*-114,9ppm±11,3 y ΔFC /ΔPAM =#1,9ppm/mmHg±0,3. Ratas espontáneamente hipertensas probadas con nitroprusiato de sodio: ΔPAM=-45,6mmHg±8,1, ΔFC=40,1ppm±11,6 y ΔFC/ΔPAM=0,9ppm/mmHg±0,5; Wistar Kyoto sometidas a prueba con nitroprusiato de sodio: ΔPAM =-39,8mmHg±6,2, ΔFC=51,9ppm±21,8 y ΔFC /ΔPAM =1,4ppm/mmHg±0,7 (*p<0,05; #p<0,01; &p<0,001). CONCLUSIÓN: Nuestros resultados muestran que ratas espontáneamente hipertensas de 13 semanas de edad presentaron reducción de la función barorrefleja cuando probadas con fenilefrina.FUNDAMENTO: A literatura tem descrito dados contraditórios em relação ao início da diminuição da função barorreflexa em ratos espontaneamente hipertensos. OBJETIVO:Este estudo foi realizado para avaliar a função barorreflexa em ratos jovens de 13 semanas espontaneamente hipertensos. MÉTODOS:Foram estudados ratos machos Wistar Kyoto (WKY) (n=15) e ratos espontaneamente hipertensos (REH) de 13 semanas (n=15). Cânulas foram inseridas na artéria aorta abdominal através da artéria femoral direita para medir a pressão arterial média (PAM) e a freqüência cardíaca (FC). A função barorreflexa foi calculada como a derivada da variação da FC em função da variação da PAM (ΔFC/ΔPAM), quando submetida a teste com uma dose depressora de nitroprussiato de sódio (50µg/kg) e com uma dose pressora de fenilefrina (8µg/kg) através de cânula inserida na veia femoral direita em ratos espontaneamente hipertensos e WKY. Diferenças com um valor de p < 0.05 foram consideradas estatisticamente significantes. RESULTADOS:Ratos espontaneamente hipertensos: ΔPAM=43,5 mmHg±5,2, ΔFC=-59,7 ppm±17,9 e ΔFC/ΔPAM=1,3 ppm/mmHg±0,1 testados com fenilefrina; Wistar Kyoto: ΔPAM=&56mmHg±3, ΔFC=*-114,9ppm±11,3 e ΔFC /ΔPAM =#1,9ppm/mmHg±0,3 testados com fenilefrina; ratos espontaneamente hipertensos: ΔPAM=-45,6mmHg±8,1, ΔFC=40,1ppm±11,6 e ΔFC/ΔPAM=0,9ppm/mmHg±0,5 testados com nitroprussiato de sódio; Wistar Kyoto: ΔPAM=-39,8mmHg±6,2, ΔFC=51,9ppm±21,8 e ΔFC/ΔPAM=1,4ppm/mmHg±0,7 testados com nitroprussiato de sódio (*p<0,05; #p<0,01; &p<0,001). CONCLUSÃO: Nossos resultados mostram que ratos espontaneamente hipertensos de 13 semanas apresentaram redução da função barorreflexa quando testados com fenilefrina.Universidade Federal de São Paulo (UNIFESP)Faculdade de Medicina do ABCUNIFESPSciEL