Sistemas bioinspirados aplicados no estudo de interação e sensoriamento de derivados xantênicos

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Estudo da formação de domínios em membranas modelos induzidos por peptídeos antimicrobianos e sua ação interfacial

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Estudo da formação de domínios em membranas modelos induzidos por peptídeos antimicrobianos e sua ação interfacial

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Mechanics and dynamics of liposomes and cells studied by QCM and ECIS

The present thesis introduces a novel sensitive technique based on TSM resonators that provides quantitative information about the dynamic properties of biological cells and artificial lipid systems. In order to support and complement results obtained by this method supplementary measurements based on ECIS technique were carried out. The first part (chapters 3 and 4) deals with artificial lipid systems. In chapter 3 ECIS measurements were used to monitor the adsorption of giant unilamellar vesicles as well as their thermal fluctuations. From dynamic Monte Carlo Simulations the rate constant of vesicle adsorption was determined. Furthermore, analysis of fluctuation measurements reveals Brownian motion reflecting membrane undulations of the adherent liposomes. In chapter 4 QCM-based fluctuation measurements were applied to quantify nanoscopically small deformations of giant unilamellar vesicles with an external electrical field applied simultaneously. The response of liposomes to an external voltage with shape changes was monitored as a function of cholesterol content and adhesion force. In the second part (chapters 5 - 8) attention was given to cell motility. It was shown for the first time, that QCM can be applied to monitor the dynamics of living adherent cells in real time. QCM turned out to be a highly sensitive tool to detect the vertical motility of adherent cells with a time resolution in the millisecond regime. The response of cells to environmental changes such as temperature or osmotic stress could be quantified. Furthermore, the impact of cytochalasin D (inhibits actin polymerization) and taxol (facilitate polymerization of microtubules) as well as nocodazole (depolymerizes microtubules) on the dynamic properties of cells was scrutinized. Each drug provoked a significant reduction of the monitored cell shape fluctuations as expected from their biochemical potential. However, not only the abolition of fluctuations was observed but also an increase of motility due to integrin-induced transmembrane signals. These signals were activated by peptides containing the RGD sequence, which is known to be an integrin recognition motif. Ultimately, two pancreatic carcinoma cell lines, derived from the same original tumor, but known to possess different metastatic potential were studied. Different dynamic behavior of the two cell lines was observed which was attributed to cell-cell as well as cell-substrate interactions rather than motility. Thus one may envision that it might be possible to characterize the motility of different cell types as a function of many variables by this new highly sensitive technique based on TSM resonators. Finally the origin of the broad cell resonance was investigated. Improvement of the time resolution reveals the "real" frequency of cell shape fluctuations. Several broad resonances around 3-5 Hz, 15-17 Hz and 25-29 Hz were observed and that could unequivocally be assigned to biological activity of living cells. However, the kind of biological process that provokes this synchronized collective and periodic behavior of the cells remains to be elucidated.

Microscopy imaging of liposomes:from coverslips to environmental SEM

A plethora of techniques for the imaging of liposomes and other bilayer vesicles are available. However, sample preparation and the technique chosen should be carefully considered in conjunction with the information required. For example, larger vesicles such as multilamellar and giant unilamellar vesicles can be viewed using light microscopy and whilst vesicle confirmation and size prior to additional physical characterisations or more detailed microscopy can be undertaken, the technique is limited in terms of resolution. To consider the options available for visualising liposome-based systems, a wide range of microscopy techniques are described and discussed here: these include light, fluorescence and confocal microscopy and various electron microscopy techniques such as transmission, cryo, freeze fracture and environmental scanning electron microscopy. Their application, advantages and disadvantages are reviewed with regard to their use in analysis of lipid vesicles.

Desenvolvimento e estudo em microcanais de fluidos inovadores análogos ao sangue baseados em vesículas unilamelares gigantes

O escoamento sanguíneo é um dos temas de grande interesse para a comunidade científica. Assim, a busca de fluidos que sejam análogos ao sangue bem como o estudo do seu escoamento em microcanais, tal como acontece com o sangue nos capilares, continua a ser alvo de investigação. Numa primeira fase deste trabalho, procedeu-se ao desenvolvimento de um modelo inovador para produzir glóbulos vermelhos artificiais, constituído por Vesículas Unilamelares Gigantes, vulgarmente designadas Giant Unilamellar Vesicles (GUVs), com três concentrações diferentes. Pretende-se que estas vesículas tenham um comportamento reológico idêntico ao escoamento dos glóbulos vermelhos (GVs) em microcanais, permitindo assim proceder a vários estudos hemodinâmicos. No desenvolvimento destas vesículas, foi verificado que as mais adequadas são constituídas por uma mistura natural de lípidos e lecitina de soja. Foi realizado um estudo relativamente à sua concentração, onde se verificou que, com o aumento da quantidade da lecitina de soja nas soluções, a concentração de GUVs tende a aumentar. Foi também realizado um estudo relativo aos diâmetros dos GUVs para verificar se estes se aproximavam em termos de tamanho dos GVs, onde foi verificado que a maioria dos GUVs possuem diâmetros com dimensões entre os 5 e 7 μm, tal como os GVs. Foi ainda verificado que a solução com a menor concentração de lecitina de soja possui uma maior quantidade de GUVs com diâmetros entre os 5 e 7 μm. Na segunda fase, foi estudado experimentalmente o escoamento das três soluções de GUVs em microcanais hiperbólicos, com três caudais diferentes, com o objetivo de visualizar a Camada Livre de Células (CLC), determinar a deformação e estudar as velocidades destes. Foi verificado que existe a formação de CLC em todas as concentrações e que aumenta com o aumento do caudal. Relativamente à deformação, esta é bastante mais evidente na contração do microcanal onde a taxa deformação é máxima. Para o caso da velocidade, foi observado um aumento bastante significativo e linear da velocidade na região da contração do microcanal hiperbólico e uma velocidade baixa e aproximadamente constante a montante e jusante da contração. vi Por fim, foi também realizado o estudo reológico dos GUVs, de forma a investigar se estes têm uma viscosidade próxima do sangue. Foi verificado que os GUVs apresentam uma viscosidade inferior à do sangue total e que existe um ligeiro aumento da viscosidade dos GUVs com o aumento da sua concentração. Por último, também foi efetuada uma comparação da viscosidade da solução de GUVs com uma solução de 5% de Hematócrito (Hct) em soro fisiológico, onde foi verificado que ambas as viscosidades são muito próximas.

Interaction of octaethylene glycol n-dodecyl monoether with dioctadecyldimethylammonium bromide and chloride vesicles

Several different methods were used to investigate the vesicle-to-micelle transition induced by the addition of the nonionic surfactant octaethylene glycol n-dodecyl monoether (C12E8) to spontaneously formed vesicle dispersions of dioctadecyldimethylammonium bromide and chloride (DODAX, X = Cl- and Br-). Dynamic light scattering reveals that fast mode micelles are formed upon addition of C12E6. The micellar mode becomes progressively dominant as the C12E8/DODAX molar ratio (R) is increased until the vesicle-to-micelle transition is complete. Turbidity, calorimetry, fluorescence quantum yield, and anisotropy measurements indicate two critical compositions: the first, R-sat, when the vesicle bilayer is saturated with C12E8 and the second, R-sol, which corresponds to the complete vesicle-to-micelle transition. Below R-sat the vesicles swell due to incorporation of the surfactant into the vesicle bilayer, and above R-sat mixed micelles and bilayer structures coexist, the determined R-sat and R-sol range from 0 to 1 and 4 to 6, respectively, depending on the surfactant counterion and the experimental method used. Cryo-transmission electron microscopy micrographs show that when R approximate to 4, micelles coexist with extended bilayer fragments. In pure DODAX (1.0 mM) dispersions, unilamellar vesicles are observed. According to the DSC results, C12E8 lowers the gel-to-liquid crystalline transition temperature, T-m, of DODAX and broadens the main transition peak which disappears around R approximate to 5 and 6 for DODAC and DODAB, respectively.

The role of counterion on the thermotropic phase behavior of DODAB and DODAC vesicles

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Dioctadecyldimethylammonium bromide vesicles prepared by the surfactant removal method

Dioctadecyldimethylammonium bromide (DODAB) is a double chain vesicle-forming cationic surfactant, whereas octa-ethyleneglycol mono-n-dodecyl ether (C12E8) is a single chain micelle-forming nonionic surfactant. At room temperature (ca. 22 degrees C) C12E8 molecules self-assemble in water as micelles while DODAB is insoluble. A mixture of DODAB and C12E8, however, can be soluble in water at room temperature depending on the relative amount of the compounds. We report the formation of small unilamellar vesicles (SUVs) by dialyzing at room temperature a mixture of 1.0 mM DODAB with 10 mM C12E8 in water. Extended bilayers are formed as well in equilibrium with vesicles. Such structures are viewed by a cryogenic transmission electron microscopy (cryo-TEM) image. (c) 2006 Elsevier B.V. All rights reserved.

Effect of the aspartic acid D2 on the affinity of Polybia-MP1 to anionic lipid vesicles

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Interaction of the meso-tetrakis (4-N-methylpyridyl) porphyrin with gel and liquid state phospholipid vesicles

The interaction of the cationic meso-tetrakis 4-N-methylpyridyl porphyrin (TMPyP) with large unilamellar vesicles (LUVs) was investigated in the present study. LUVs were formed by mixtures of the zwitterionic 1,2-dipalmitoyl-sn-glycero-phosphatidylcholine (DPPC) and anionic 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG) phospholipids, at different DPPG molar percentages. All investigations were carried out above (50 degrees C) and below (25 degrees C) the main phase transition temperature of the LUVs (similar to 41 degrees C). The binding constant values, K-b, estimated from the time-resolved fluorescence study, showed a significant increase of the porphyrin affinity at higher mol% DPPG. This affinity is markedly increased when the LUVs are in the liquid crystalline state. For both situations, the increase of the K-b value was also followed by a higher porphyrin fraction bound to the LUVs. The displacement of the vesicle-bound porphyrins toward the aqueous medium, upon titration with the salt potassium chloride (KCl), was also studied. Altogether, our steady-state and frequency-domain fluorescence quenching data results indicate that the TMPyP is preferentially located at the LUVs Stern layer. This is supported by the zeta potential studies, where a partial neutralization of the LUVs surface charge, upon porphyrin titration, was observed. Dynamic light scattering (DLS) results showed that, for some phospholipid systems, this partial neutralization leads to the LUVs flocculation. (C) 2012 Elsevier Inc. All rights reserved.

Effect of Altered Polar Headgroup of Phosphatidylethanolamines on Transbilayer Aminophospholipid Distribution in Sonicated Vesicles

The transbilayer aminophospholipid distributions in small unilamellar vesicles comprising of phosphatidylethanolamine or its analogs (bearing modifications in the polar headgroup) and egg hosphatidylcholine were ascertained using trinitrobenzenesulfonic acid as external membrane probe. These vesicles, containing 10-30 mol% phosphatidylethanolamine or its analogs, were formed by sonication and fractionated by centrifugation. Phosphatidylethanolamine at low concentrations (10 mol%) preferentially localized in the outer monolayer. This preference appeared to be reversed at higher phosphatidylethanolamine concentrations (30 mol%). Unlike this finding, phosphatidylethanolamine bearing ethyl, phenyl and benzyl substituents at the carbon atom adjacent to the amino group distributed mainly in the outer surface irrespective of their concentrations. Similar results were obtained when the phosphate and amino groups were separated by three methylene residues. These observations suggest that the effective polar headgroup volume and/or hydrogen-bonding capacity of phospholipids are the important factors that determine their distribution in small unilamellar vesicles.

The origins of stability of spontaneous vesicles

Equilibrium unilamellar vesicles are stabilized by one of two distinct mechanisms depending on the value of the bending constant. Helfrich undulations ensure that the interbilayer potential is always repulsive when the bending constant, K, is of order kBT. When K ≫ kBT, unilamellar vesicles are stabilized by the spontaneous curvature that picks out a particular vesicle radius; other radii are disfavored energetically. We present measurements of the bilayer elastic constant and the spontaneous curvature, Ro, for three different systems of equilibrium vesicles by an analysis of the vesicle size distribution determined by cryo-transmission electron microscopy and small-angle neutron scattering. For cetyltrimethylammonium bromide (CTAB)/sodium octyl sulfonate catanionic vesicles, K = .7 kBT, suggesting that the unilamellar vesicles are stabilized by Helfrich-undulation repulsions. However, for CTAB and sodium perfluorooctanoate (FC7) vesicles, K = 6 kBT, suggesting stabilization by the energetic costs of deviations from the spontaneous curvature. Adding electrolyte to the sodium perfluorooctanoate/CTAB vesicles leads to vesicles with two bilayers; the attractive interactions between the bilayers can overcome the cost of small deviations from the spontaneous curvature to form two-layer vesicles, but larger deviations to form three and more layer vesicles are prohibited. Vesicles with a discrete numbers of bilayers at equilibrium are possible only for bilayers with a large bending modulus coupled with a spontaneous curvature.

Supra-assembly of siliceous vesicles

We report a new approach to produce macroporous (110 nm in diameter) ordered siliceous foams (MOSF) by using block copolymers as templates in the absence of any organic cosolvent. The fine three-dimensional honeycomb structure of MOSF was determined by electron tomography. A formation mechanism of MOSF that spans from the atomic to macroscopic scale is proposed, which involves the cooperative self-assembly of unilamellar vesicles followed by the supra-assembly of vesicles. The fusion of soft vesicles finally leads to MOSF with well-ordered and defined honeycomb structures.

Vaccine entrapment in liposomes

The use of liposomes as carriers of peptide, protein, and DNA vaccines requires simple, easy-to-scale-up technology capable of high-yield vaccine entrapment. Work from this laboratory has led to the development of techniques that can generate liposomes of various sizes, containing soluble antigens such as proteins and particulate antigens (e.g., killed or attenuated bacteria or viruses), as well as antigen-encoding DNA vaccines. Entrapment of vaccines is carried out by the dehydration-rehydration procedure which entails freeze-drying of a mixture of "empty" small unilamellar vesicles and free vaccines. On rehydration, the large multilamellar vesicles formed incorporate up to 90% or more of the vaccine used. When such liposomes are microfluidized in the presence of nonentrapped material, their size is reduced to about 100 nm in diameter, with much of the originally entrapped vaccine still associated with the vesicles. A similar technique applied for the entrapment of particulate antigens (e.g., Bacillus subtilis spores) consists of freeze-drying giant vesicles (4-5 microm in diameter) in the presence of spores. On rehydration and sucrose gradient fractionation of the suspension, up to 30% or more of the spores used are associated with generated giant liposomes of similar mean size.