RESUMO
Polyelectrolyte multilayers serve as effective reservoirs for bioactive molecules which are stored and released from the multilayers for cellular applications. However, control over the release without significantly affecting the multilayers and biomolecules is still a challenge. On the other hand, externally stimulated release would make the multilayers promising for the development of stimuli-sensitive planar carriers with release performance switched on demand. In this study soft composite films are designed by coating hyaluronic acid/poly-l-lysine (HA/PLL) multilayers with temperature responsive poly(N-isopropylacrylamide) (PNIPAM) microgels. Microgels are flattened and immersed into the multilayers to maximize the number of contacts with the surrounding polyelectrolytes (HA and PLL). The microgel coating serves as an efficient switchable barrier for the PLL transport into the multilayers. PLL diffusion into the film is significantly hindered at room temperature but is dramatically enhanced at 40 °C above the volume phase transition temperature (VPTT) of PNIPAM at 32 °C associated with microgel shrinkage. Scanning force microscopy micrographs show that the mechanism of volume phase transition on soft surfaces cannot be directly deduced from the processes taking place at solid substrates.
Assuntos
Resinas Acrílicas/química , Temperatura , Adsorção , Difusão , Corantes Fluorescentes/química , GéisRESUMO
A new simple concept for a miniaturized monolayer trough is described. The overall monolayer area in the expanded state is approximately 150 mm2 and can be reduced by a factor of 2. The surface area is a function of the shape of the meniscus formed by the subphase and is controlled by the amount of water in the monolayer trough. The controlled compression of monolayers to a desired area per molecule with simultaneous observation of the lateral distribution of fluorescently labeled molecules is shown. A biological reaction between a specific antibody and lipid anchored peptide demonstrates the feasibility of monolayer experiments, which require only very small quantities of substance (in the pmol range). This trough might also be a valuable tool for the 2D crystallization of proteins at lipid layers via specific binding sites such as metal chelators.
Assuntos
Lipídeos de Membrana/química , 1,2-Dipalmitoilfosfatidilcolina/química , 4-Cloro-7-nitrobenzofurazano/química , Capsídeo/química , Proteínas do Capsídeo , Lipoproteínas/química , Microscopia de Fluorescência , Politetrafluoretileno/química , Propriedades de Superfície , Proteínas Virais/químicaRESUMO
Therapeutic proteins are an integral part of today's pharmaceutical practice, but they still present challenges from the drug delivery point of view. In this work, a new approach is studied based on hard templating for fabrication of microparticles composed of pure insulin, which may enable effective delivery, for instance pulmonary delivery. The approach is both simple and versatile: the protein particles are prepared by selective precipitation into porous CaCO3 microtemplates, followed by full decomposition of the template at the isoelectric point of the protein (pH 5.2). Control over the main material parameters (mechanical properties, porosity, morphology and stability at physiological conditions) are critical for the envisioned application in drug delivery. It is demonstrated that these critical parameters can be significantly tuned by a slight final pH variation around the isoelectric point (pH range 4-6) and by the denaturation degree of insulin. Electrostatic interactions and inter-protein crosslinking in the protein particles as well as their internal structure are considered, to explain the variation in the particle properties. The particle property parameters are explored using atomic force microscopy, optical microscopy and circular dichroism spectra. Finally, phagocytic clearance of the protein particles in vitro was studied to explore possible enhancements in particle fabrication to improve the efficiency of insulin delivery by inhalation.
Assuntos
Carbonato de Cálcio/metabolismo , Endocitose , Insulina/metabolismo , Microesferas , Animais , Bovinos , Dicroísmo Circular , Módulo de Elasticidade , Concentração de Íons de Hidrogênio , Tamanho da Partícula , Fagocitose , Desnaturação Proteica , Espectrofotometria Ultravioleta , Eletricidade EstáticaRESUMO
We present an electrohydrodynamic device for filtration of nanometre-sized particles from suspensions. A high-frequency electric field is locally generated through the action of mutually parallel microelectrodes integrated into a microfluidic channel. Due to the mechanism of ohmic heating, a thermal gradient arises above these electrodes. In conjunction with temperature-sensitive properties of the fluid, an eddy flow behaviour emerges in the laminar environment. This acts as an adjustable filter. For quantification of the filtration efficiency, we tested a wide range of particle concentrations at different electric field strengths and overall external flow velocities. Particles with a diameter of 200 nm were retained in this manner at rates of up to 100%. Numerical simulations of a model taking into account the hydrodynamic as well as electric conditions, but no interactions between the point-shaped particles, yield results that are similar to the experiment in both the flow trajectories and the particle accumulation. Our easy technique could become a valuable tool that complements conventional filtration methods for handling nanometre-scaled particles in medicine and biotechnology, e.g. bacteria and viruses.
Assuntos
Filtração/instrumentação , Técnicas Analíticas Microfluídicas/instrumentação , Nanopartículas/química , Hidrodinâmica , Modelos Teóricos , SuspensõesRESUMO
We demonstrate that the combined application of Langmuir-Blodgett and self-assembly techniques allows the fabrication of patterns with contrasting surface properties on gold substrates. The process is monitored using fluorescence microscopy and surface plasmon spectroscopy and microscopy. These structures are suitable for the investigation of biochemical processes at surfaces and in ultrathin films. Two examples of such processes are shown. In the first example, the structures are addressed through the binding of a monoclonal antibody to a peptide. This demonstrates the formation of self-assembled monolayers by cysteine-bearing peptides on gold, and the directed binding of proteins to the structured layers. A high contrast between specific and unspecific binding of proteins is observed by the patterned presentation of antigens. Such films possess considerable potential for the design of multichannel sensor devices. In the second example, a structured phospholipid layer is produced by controlled self-assembly from vesicle solution. The structures created--areas of phospholipid bilayer, surrounded by a matrix of phospholipid monolayer--allow formation of a supported bilayer which is robust and strongly bound to the gold support, with small areas of free-standing bilayer which very closely resemble a phospholipid cell membrane.
Assuntos
Membranas Artificiais , Sequência de Aminoácidos , Fenômenos Biofísicos , Biofísica , Técnicas In Vitro , Bicamadas Lipídicas/química , Proteínas de Membrana/química , Microscopia de Fluorescência , Modelos Químicos , Dados de Sequência Molecular , Ácido Palmítico , Ácidos Palmíticos/química , Peptídeos/química , Enxofre/química , Propriedades de SuperfícieRESUMO
The formation of self-assembled monolayers (SAMs) on gold surfaces containing an antigenic peptide (NANP)6 and HS(CH2)11OH, and the specific binding of a monoclonal antibody to these layers were investigated by surface plasmon resonance (SPR). Peptides were synthesized by solid-state phase synthesis and were linked either to cysteine or to an alkyl-thiol to allow covalent attachment to gold. The content of the peptide in the SAMs was systematically varied, and the binding properties of the monoclonal antibody were compared with those measured by microcalorimetry in solution. At a critical peptide concentration in the SAM an optimal antibody binding and complete surface coverage was attained. At lower peptide concentrations, the amount of adsorbed antibody decreased; at higher peptide concentrations, the binding constant decreased. These effects can be explained if the accessibility of the antigenic epitopes depends on the peptide density. Addition of free antigen induced the desorption of bound antibodies and allowed accurate measurements of the dissociation rate constant. Binding constants obtained from steady-state measurements and from measurements of the kinetic rate constants were compared.
Assuntos
Apresentação de Antígeno , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais , Azidas/química , Azidas/imunologia , Sítios de Ligação , Engenharia Biomédica , Fenômenos Biofísicos , Biofísica , Calorimetria , Ouro , Técnicas In Vitro , Cinética , Ligantes , Dados de Sequência Molecular , Fosfato de Piridoxal/análogos & derivados , Fosfato de Piridoxal/química , Fosfato de Piridoxal/imunologia , Propriedades de SuperfícieRESUMO
Recent observations of charged colloidal particles trapped at the air-water interface revealed long-range interparticle attractive forces, not accounted for by the standard theories of colloidal interactions. We propose a mechanism for attraction which is based on nonuniform wetting causing an irregular shape of the particle meniscus. The excess water surface area created by these distortions can be minimized when two adjacent particles assume an optimum relative orientation and distance. Typically, for spheres with diameter of 1 &mgr;m at an interparticle distance of 2 &mgr;m, deviations from the ideal contact line by as little as 50 nm result in an interaction energy of the order of 10(4)kT. Roughness-induced capillarity explains the experimental findings, including the cluster dissolution caused by addition of detergent to the subphase and the formation of linear aggregates. This kind of interaction should also be of importance in particle-stabilized foams and emulsions.
RESUMO
Shear force mapping on thiolipid Langmuir-Blodgett monolayers probed with Al-coated tips leads to a contrast highly dependent on the monolayer molecular organization, which does not correspond to the topographical relief of the sample. The use of functionalized surface probes offers the possibility to better control the probe-to-sample interaction. In addition, hydrophilic surface probes are totally insensitive to alkyl chain arrangements in the monolayer. Hydrophobic probes, instead, can be chosen to map shear force on soft samples in liquid environment, since their mechanical properties are not influenced by the surrounding liquid.
Assuntos
Fosfolipídeos/química , Compostos de Sulfidrila/química , Microscopia de Fluorescência/métodos , Estresse MecânicoRESUMO
We describe a simple method for patterning biomolecular films on surfaces with high resolution. A conventional polymeric resist is structured by electron-beam lithography. The exposed and developed patterns are then used for the directed self-assembly (SA) of a first molecule from solution. Removal of the remaining resist allows the SA of a second species. We illustrate the potential of the approach by assembling on gold (Au) substrates two alkanethiols of contrasting terminal functionality. The patterns have dimensions from the micrometer range down to 40 nm and an edge resolution of 3.5 nm.
RESUMO
Lateral force microscopy in the wearless regime was used to study the friction behavior of a lipid monolayer on mica. In the monolayer, condensed domains with long-range orientational order of the lipid molecules were present. The domains revealed unexpectedly strong friction anisotropies and non-negligible friction asymmetries. The angular dependency of these effects correlated well with the tilt direction of the alkyl chains of the monolayer, as determined by electron diffraction and Brewster angle microscopy. The molecular tilt causing these frictional effects was less than 15 degrees, demonstrating that even small molecular tilts can make a major contribution to friction.