ABSTRACT
Lipid coating is a method highly used to improve the biocompatibility of nanoparticles (NPs), even though its effect on the NP properties is still object of investigation. Herein, silica NPs containing methylene blue, which is a photosensitizer used in a variety of biomedical applications, were coated with a phospholipid bilayer. Regarding the photophysical properties, lipid-coating did not cause significant changes since bare and lipid-coated NPs presented very similar absorption spectra and generated singlet oxygen with similar efficiencies. However, NP interaction with cells and membrane mimics was totally different for bare and lipid-coated NPs. Lipid-coated NPs were distributed through the cell cytoplasm whereas bare NPs were detected only in some vacuolar regions within the cells. Since cellular uptake and cytolocalization are influenced by NP adsorption on cell membranes, the interaction of lipid-coated and bare NPs were studied on a membrane mimic, i.e., Hybrid Bilayer Membranes (HBMs) made of different compositions of negatively charged and neutral lipids. Interactions of bare and lipid-coated NPs with HBMs were analyzed by Surface Plasmon Resonance Imaging. Bare NPs presented high adsorption and aggregation on HBMs independently of the surface charge. Conversely, lipid-coated NPs presented less aggregation on the membrane surface and the adsorption was dependent on the charges of the NPs and of the HBMs. Our results indicated that NPs aggregation on the membrane surface can be modulated by lipid coating, which affects the cytosolic distribution of the NPs.
Subject(s)
Cell Membrane/drug effects , Coated Materials, Biocompatible/pharmacokinetics , Nanoparticles/chemistry , Phosphatidylcholines/pharmacology , Phosphatidylglycerols/pharmacology , Silicon Dioxide/chemistry , Adsorption , Animals , Cell Membrane/metabolism , Cells, Cultured , Coated Materials, Biocompatible/chemistry , Hydrophobic and Hydrophilic Interactions/drug effects , Lipid Bilayers/chemistry , Lipid Bilayers/metabolism , Materials Testing , Membranes, Artificial , Mice , Phosphatidylcholines/chemistry , Phosphatidylglycerols/chemistry , Surface Properties/drug effectsABSTRACT
We aim in this study to characterize the effect of cations and polycations on the formation of hybrid bilayer membranes (HBMs), especially those that mimic the inner mitochondrial membrane (IMM), with a proper composition of phosphatidylcholine (PC), phosphatidylethanolamine (PE) and cardiolipin (CL) adsorbed on an alkanethiol monolayer. HBMs are versatile membrane mimetics that show promising results in sensor technology. Its formation depends on the fusion of vesicles on hydrophobic surfaces, a process that is not well understood at the molecular level. Our results showed to which extend and in which condition the presence of cations and polycations facilitate the formation of HBMs. The required time for lipid layer formation was reduced several times and the lipid layer reaches the expected thickness of 19.5±1.8 Ǻ, in contrast to only 2±1.5 Ǻ usually observed in the absence of cations. In the presence of specific concentrations of spermine and Ca(2+) the amount of adsorbed phospholipids on the thiol layer increased nearly 70% compared to that observed when Na(+) was used at concentrations 10 times higher. Divalent cations and polycations adsorb specifically on the lipid headgroups destabilizing the hydration forces, facilitating the process of vesicle fusion and formation of lipid monolayers. The concepts and conditions described in the manuscript will certainly help the development of the field of membrane biosensors.
Subject(s)
Cations , Intracellular Membranes , Lipid Bilayers , Mitochondria , Adsorption , Kinetics , Microscopy, Atomic Force , Molecular MimicryABSTRACT
We constructed multi-channel hybrid bilayer membrane (HBM) biochips and characterized them by surface plasmon resonance imaging. Each channel in the biochip was prepared using vesicles with different proportions of negative, neutral, and positive lipids. The HBM surfaces were tested by interaction with two globular proteins that recognize surfaces covered with opposite charges. Spots modified with the same HBM show responses within a relative standard deviation of 10% or smaller. These devices were also used to study in detail the interaction between cytochrome-c (cyt-c) and HBMs. Cooperative binding between cyt-c and negative HBMs was demonstrated. Using an adaptation of the Hill model, we calculated a Hill coefficient of 5 and a 10-fold increase in the binding constant with the increase in cyt-c concentration. We propose that this treatment can be used to evaluate the cooperative binding of surface proteins to membranes.
Subject(s)
Cytochromes c/chemistry , Lipid Bilayers/chemistry , Models, Chemical , Protein Array Analysis , Protein BindingABSTRACT
The detection of aromatic compounds from pesticides and industrial wastewater has become of great interest, since these compounds withstand chemical oxidation and biological degradation, accumulating in the environment. In this work, a highly sensitive biosensor for detecting catechol was obtained with the immobilization of Cl-catechol 1,2-dioxygenase (CCD) in nanostructured films. CCD layers were alternated with poly(amidoamine) generation 4 (PAMAM G4) dendrimer using the electrostatic layer-by-layer (LbL) technique. Circular dichroism (CD) measurements indicated that the immobilized CCD preserved the same conformation as in solution. The thickness of the very first CCD layers in the LbL films was estimated at ca. 3.6 nm, as revealed by surface plasmon resonance (SPR). PAMAM/CCD 10-bilayer films were employed in detecting diluted catechol solutions using either an optical or electrical approach. Due to the mild immobilization conditions employed, especially regarding the pH and ionic strength of the dipping solutions, CCD remained active in the films for periods longer than 3 weeks. The optical detection comprised absorption experiments in which the formation of cis-cis muconic acid, resulting from the reaction between CCD and catechol, was monitored by measuring the absorbance at 260 nm after film immersion in catechol solutions. The electrical detection was carried out using LbL films deposited onto gold-interdigitated electrodes immersed in aqueous solutions at different catechol concentrations. Using impedance spectroscopy in a broad frequency range (1Hz-1kHz), we could detect catechol in solutions at concentrations as low as 10(-10) M.
Subject(s)
Biosensing Techniques/instrumentation , Catechol 1,2-Dioxygenase/chemistry , Catechols/analysis , Chlorine/chemistry , Electrochemistry/instrumentation , Membranes, Artificial , Nanostructures/chemistry , Adsorption , Biosensing Techniques/methods , Catechols/chemistry , Crystallization/methods , Electrochemistry/methods , Electrodes , Enzymes, Immobilized/chemistry , Equipment Design , Equipment Failure Analysis , Nanostructures/analysis , Nanotechnology/instrumentation , Nanotechnology/methods , Protein Binding , Surface PropertiesABSTRACT
An automated method for dn/dc determination using a surface plasmon resonance instrument in tandem with a flow injection gradient system (FIG-SPR) is proposed. dn/dc determinations of small molecule and biomolecule, surfactant, polymer, and biopolymer solutions with precision around 1-2% and good accuracy were performed using the new method. dn/dc measurements were also carried out manually on a conventional SPR equipment with similar accuracy and precision. The FIG-SPR instrument is inexpensive and could be easily coupled to commercially available SPR and liquid chromatography instruments to obtain several properties of the solutions, which are based on measurements of refractive index.
