Our study of pBAE polyplexes unveil their insight distribution and peptide-dependent properties. This analysis makes the gap from bench to bedside closer due to the possibility to select the most appropriate oligopeptide combination depending on the application.
Liposomes are widely used as drug delivery carriers and as cell model systems. Here, we measure the dielectric properties of individual liposomes adsorbed on a metal electrode by in-liquid scanning dielectric microscopy in force detection mode. From the measurements the lamellarity of the liposomes, the separation between the lamellae and the specific capacitance of the lipid bilayer can be obtained. As application we considered the case of non-extruded DOPC liposomes with radii in the range ~ 100–800 nm. Uni-, bi- and tri-lamellar liposomes have been identified, with the largest population corresponding to bi-lamellar liposomes. The interlamellar separation in the bi-lamellar liposomes is found to be below ~ 10 nm in most instances. The specific capacitance of the DOPC lipid bilayer is found to be ~ 0.75 µF/cm2 in excellent agreement with the value determined on solid supported planar lipid bilayers. The lamellarity of the DOPC liposomes shows the usual correlation with the liposome's size. No correlation is found, instead, with the shape of the adsorbed liposomes. The proposed approach offers a powerful label-free and non-invasive method to determine the lamellarity and dielectric properties of single liposomes. [Figure not available: see fulltext.].
In this study, we show that dry saturated phospholipid layers prepared by the spin-coating technique could present thinner regions associated to interdigitated phases under some conditions. The morphological characteristics of lipid layers of saturated phosphocholines, such as dilauroylphosphatidylcholine (DLPC), dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC) and distearoylphosphatidylcholine (DSPC), have been measured by Atomic Force Microscopy and revealed that the presence of interdigitated regions is not induced by the same parameters that induce them in hydrated samples. To achieve these results the effect of the lipid hidrocabonated chain length, the presence of alcohol in the coating solution, the spinning velocity and the presence of cholesterol were tested. We showed that DPPC and DSPC bilayers, on the one side, can show structures with similar height than interdigitated regions observed in hydrated samples, while, on the other side, DLPC and DMPC tend to show no evidence of interdigitation. Results indicate that the presence of interdigitated areas is due to the presence of lateral tensions and, hence, that they can be eliminated by releasing these tensions by, for instance, the addition of cholesterol. These results demonstrate that interdigitation in lipid layers is a rather general phenomena and can be observed in lipid bilayers in dry conditions.
We measured and quantified the local electric polarization properties of ultrathin (~ 5 nm) biolayers on mm-thick mica substrates. We achieved it by scanning a sharp conductive tip (< 10 nm radius) of an electrostatic force microscope over the biolayers and quantifying sub-picoNewton electric polarization forces with a sharp-tip model implemented using finite-element numerical calculations. We obtained relative dielectric constants ?r = 3.3, 2.4 and 1.9 for bacteriorhodopsin, dioleoylphosphatidylcholine (DOPC) and cholesterol layers, chosen as representative of the main cell membrane components, with an error below 10% and a spatial resolution down to ~ 50 nm. The ability of using insulating substrates common in biophysics research, such as mica or glass, instead of metallic substrates, offers both a general platform to determine the dielectric properties of biolayers and a wider compatibility with other characterization techniques, such as optical microscopy. This opens up new possibilities for biolayer research at the nanoscale, including nanoscale label-free composition mapping.
We investigate the effects of Cholesterol (Chol) in the structural and nanomechanical properties of binary and ternary spin-coated single lipid bilayers made of Dioleoylphosphatidylcholine (DOPC) and Sphingomyelin (SM) in dry conditions. We show that for the DOPC/Chol bilayers, Chol induces an initial increase of the bilayer thickness, followed by decrease for concentrations above 30% Chol. The mechanical properties, instead, appear practically insensitive to the Chol content. For the SM/Chol bilayers we have observed both the thinning of the bilayer and the decrease of the force necessary to break it for Chol content above 40. mol%. In both binary mixtures phase separation is not observed. For ternary single bilayers of DOPC/SM/Chol, Chol induces phase segregation and the formation of domains resembling lipid rafts. The domains show a thickness and mechanical response clearly distinct from the surrounding phase and dependent on the relative Chol content. Based on the results obtained for the binary mixtures, DOPC- and SM-enriched domains can be identified. We highlight that many of the effects of Chol reported here for the dry multicomponent single lipid bilayers resemble closely those observed in hydrated bilayers, thus offering an additional insight into their properties.
We present what is, to our knowledge, the first experimental demonstration of dielectric constant measurement and quantification of supported lipid bilayers in electrolyte solutions with nanoscale spatial resolution. The dielectric constant was quantitatively reconstructed with finite element calculations by combining thickness information and local polarization forces which were measured using an electrostatic force microscope adapted to work in a liquid environment. Measurements of submicrometric dipalmitoylphosphatidylcholine lipid bilayer patches gave dielectric constants of εr ∼ 3, which are higher than the values typically reported for the hydrophobic part of lipid membranes (εr ∼ 2) and suggest a large contribution of the polar headgroup region to the dielectric response of the lipid bilayer. This work opens apparently new possibilities in the study of biomembrane electrostatics and other bioelectric phenomena.
We show that optical visualization of ultrathin mica flakes on metallic substrates is viable using semitransparent gold as substrates. This enables to easily localize mica flakes and rapidly estimate their thickness directly on gold substrates by conventional optical reflection microscopy. We experimentally demonstrate it by comparing optical images with atomic force microscopy images of mica flakes on semitransparent gold. Present results open the possibility for simple and rapid characterization of thin mica flakes as well as other thin sheets directly on metallic substrates.
Atomic force microscopy (AFM) has been used to study the structural and mechanical properties of low concentrated spin-coated dioleoylphosphatidylcholine (DOPC) layers in dry environment (RH approximate to 0%) at the nanoscale. It is shown that for concentrations in the 0.1-1 mM range the structure of the DOPC spin-coated samples consists of an homogeneous lipid monolayer similar to 1.3 nm thick covering the whole substrate on top of which lipid bilayer (or multilayer) micro- and nanometric patches and rims are formed. The thickness of the bilayer structures is found to be similar to 4.5 nm (or multiples of this value for multilayer structures), while the lateral dimensions range from micrometers to tens of nanometer depending on the lipid concentration. The force required to break a bilayer (breakthrough force) is found to be similar to 0.24 nN. No dependence of the mechanical values on the lateral dimensions of the bilayer structures is evidenced. Remarkably, the thickness and breakthrough force values of the bilayers measured in dry environment are very similar to values reported in the literature for supported DOPC bilayers in pure water.
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Navalon-Lopez, M, Dols-Perez, A, Grijalvo, S, Fornaguera, C, Borros, S, (2023). Unravelling the role of individual components in pBAE/polynucleotide polyplexes in the synthesis of tailored carriers for specific applications: on the road to rational formulations Nanoscale Advances 5, 1611-1623 
Biophysical Journal , 104, (6), 1257-1262