New insights on the mechanism of polyethylenimine transfection and their implications on gene therapy and DNA vaccines.

Polyethylenimine (PEI) has been demonstrated as an efficient DNA delivery vehicle both in vitro and in vivo. There is a consensus that PEI-DNA complexes enter the cells by endocytosis and escape from endosomes by the so-called "proton sponge" effect. However, little is known on how and where the polyplexes are de-complexed for DNA transcription and replication to occur inside the cell nucleus. To better understand this issue, we (i) tracked the cell internalization of PEI upon transfection to human epithelial cells and (ii) studied the interaction of PEI with phospholipidic layers mimicking nuclear membranes. Both the biological and physicochemical experiments provided evidence of a strong binding affinity between PEI and the lipidic bilayer. Firstly, confocal microscopy revealed that PEI alone could not penetrate the cell nucleus; instead, it arranged throughout the cytoplasm and formed a sort of aureole surrounding the nuclei periphery. Secondly, surface tension measurements, fluorescence dye leakage assays, and differential scanning calorimetry demonstrated that a combination of hydrophobic and electrostatic interactions between PEI and the phospholipidic monolayers/bilayers led to the formation of stable defects along the model membranes, allowing the intercalation of PEI through the monolayer/bilayer structure. Results are also supported by molecular dynamics simulation of the pore formation in PEI-lipidic bilayers. As discussed throughout the text, these results might shed light on a the mechanism in which the interaction between PEI and the nucleus membrane might play an active role on the DNA release: on the one hand, the PEI-membrane interaction is anticipated to facilitate the DNA disassembly from the polyplex by establishing a competition with DNA for the PEI binding and on the other hand, the forming defects are expected to serve as channels for the entrance of de-complexed DNA into the cell nucleus. A better understanding of the mechanism of transfection of cationic polymers opens paths to development of more efficiency vectors to improve gene therapy treatment and the new generation of DNA vaccines.

Interactions of lauryl gallate with phospholipid components of biological membranes.

The effect of different amounts of lauryl gallate (LG) on properties of the model membranes of phosphatidylcholines (PC), differing in the presence of double bonds in the hydrocarbon chains, and phosphatidylglycerol (PG) was described in terms of phase behaviour of mixtures, interactions between both components, monolayers stability and their organization. The Langmuir monolayer technique was used to monitor the surface thermodynamics (i.e. the excess area and excess Gibbs energy of mixing) on the basis of surface pressure-area per molecule (π-A) isotherms. Simultaneously, morphology of the studied monolayers was visualized by the Brewster angle microscopy (BAM). This allowed evaluating the kind and magnitude of interactions which influence on the phase behaviour and structural properties of the monolayers. The obtained results can be helpful to reveal the mechanism of phospholipid antioxidant protection and important pharmacological (antimicrobial) role of lauryl gallate for production of effective therapeutic substances.

Effect of ionization on the behavior of n-eicosanephosphonic acid monolayers at the air/water interface. Experimental determinations and molecular dynamics simulations.

Monolayers of n-eicosanephosphonic acid, EPA, were studied using a Langmuir balance and a Brewster angle microscope at different subphase pH values to change the charge of the polar headgroups (Zav) from 0 to -2. Molecular dynamics simulations (MDS) results for |Zav| = 0, 1, and 2 were compared with the experimental ones. EPA monolayers behave as mixtures of mutually miscible species (C20H41-PO3H2, C20H41-PO3H(-), and C20H41-PO3(2-), depending on the subphase pH). The order and compactness of the monolayers decrease when increasing |Zav|, while go from strongly interconnected by phosphonic-phosphonic hydrogen bonds (|Zav| = 0-0.03) through an equilibrium between the total cohesive energy and the electrostatic repulsion between the charged polar groups (0.03 < |Zav| < 1.6) to an entirely ionic monolayer (|Zav| ≈ 2). MDS reveal for |Zav| = 0 that the chains form spiralled nearly rounded structures induced by the hydrogen-bonded network. When |Zav| ≈ 1 fingering domains were identified. When Z ≈ 2, the headgroups are more disordered and distanced, not only in the xy plane but also in the z direction, forming a rough layer and responding to compression with a large plateau in the isotherm. The monolayers collapse behavior is consistent with the structures and domains founds in the different ionization states and their consequent in-plane rigidity: there is a transition from a solid-like response at low pH subphases to a fluid-like response at high pH subphases. The film area in the close-packed state increases relatively slow when the polar headgroups are able to form hydrogen bonds but increases to near twice that this value when |Zav| ≈ 2. Other nanoscopic properties of monolayers were also determined by MDS. The computational results confirm the experimental findings and offer a nanoscopic perspective on the structure and interactions in the phosphonate monolayers.

Interaction of human serum albumin with monofluorinated phospholipid monolayers.

In this work the interaction between human serum albumin (HSA) and a monofluorinated phospholipid, 1-palmitoyl-2-[16-fluoropalmitoyl-phosphatidylcholine] (F-DPPC), was studied by using Langmuir monolayer and Brewster angle microscopy (BAM) techniques. Different amounts of F-DPPC were spread on a previously formed HSA monolayer located at the air/water interface at 25°C and the mixed monolayers thus obtained showed the existence of a liquid expanded-liquid condensed (LE-LC) phase transition (at 14 mN/m), attributed to the pure F-DPPC monolayer, coexisting with a second transition (at 22-24 mN/m) corresponding to the protein conformational change from an unfolded state to another in "loops" configuration. Relative thickness measurements recorded during the compression of the mixed monolayers showed the existence of an "exclusion" surface pressure (π(exc)), above which the protein is squeezed out the interface, but not totally. BAM images reveal that some protein molecules in a packed "loops" configuration remain at the interface at surface pressures higher than the "exclusion" surface pressure. The application of the Defay-Crisp phase rule to the phase diagram of the F-DPPC/HSA system can explain the existence of certain regions of surface pressure in which the mixed monolayer components are miscible, as well as those others that they are immiscible.

Monolayer and Brewster angle microscopy study of human serum albumin-dipalmitoyl phosphatidyl choline mixtures at the air-water interface.

The aim of this study is to deepen the understanding of the behavior of human serum albumin (HSA) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) mixed monolayers. For this purpose, different amounts of DPPC were spread at 25°C on the water surface containing a monolayer of HSA. Surface film balance and Brewster angle microscopy techniques have been used to analyze the structural and energetic characteristics (structure, topography, thickness, miscibility and interactions) of these mixtures. HSA/DPPC mixed monolayers exhibit two phase transitions evidenced by two discontinuities in the corresponding π-A isotherms and by two minimum values in the compressional modulus (C(s)(-1))-surface pressure (π) curves. The plot of the molecular areas occupied by the mixed monolayers as function of the mass fraction of DPPC shows the absence of deviations from linearity, a typical behavior for ideal or inmiscible system. This result was confirmed from the values calculated for the free energy of excess (ΔG(exc)), which are practically zero whatever the composition of the mixtures and the surface pressures at which ΔG(exc) values were calculated. In addition, relative thickness values of HSA/DPPC mixed monolayers showed the existence of an exclusion surface pressure (π(exc)), below which the monolayer is composed of a mixture of both components, while above π(exc) the HSA molecules are squeezed out the interface, but not totally. In fact, although in this region DPPC domains predominate at the interface, the existence of protein molecules in a packing "loops" configuration can be observed in BAM images. Moreover, relative thickness measurements confirm this hypothesis.

Monolayer properties of uronic acid bicatenary derivatives at the air-water interface: effect of hydroxyl group stereochemistry evidenced by experimental and computational approaches.

By screening uronic acid-based surfactant interfacial properties, the effect of the hydroxyl group stereochemistry (OH-4) on the conformation of bicatenary (disubstituted) derivatives at the air-water interface has been evidenced by experimental and computational approaches. Physical and optical properties of a monolayer characterized by Langmuir film balance, Brewster angle microscopy, and ellipsometry at 20 °C reveal that the derivative of glucuronate (C(14/14)-GlcA) forms a more expanded monolayer, and shows a transition state under compression, in the opposite to that of galacturonate (C(14/14)-GalA). Both films are very mechanically resistant (compression modulus > 300 mN m(-1)) and stable (collapse pressure exceeding 60 mN m(-1)), while that of C(14/14)-GalA exhibits a very high compression modulus up to 600 mN m(-1) like films in the solid state. Computational approaches provide single and assembly molecular models that corroborate the molecule expansion degree and interactions data from experimental results. Differences in the molecular conformation and film behaviours of uronic acid bicatenary derivatives at the air-water interface are attributed to the intra-H-bonding formation, which is more favourable with an OH-4 in the axial (C(14/14)-GalA) than in the equatorial position (C(14/14)-GlcA).

A comparative study of F-DPPC/DPPC mixed monolayers. Influence of subphase temperature on F-DPPC and DPPC monolayers.

The surface behavior of two zwitterionic phospholipids: 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1-palmitoyl-2-[16-fluoropalmitoyl-phosphatidylcholine] (F-DPPC), has been investigated at the air-water interface at the temperature range from 10 to 30 °C. Surface pressure-area isotherms, BAM images and thickness-time curves were obtained for monolayers made from these pure phospholipids and from their mixtures.The comparative study of the behavior of both phospholipid monolayers with temperature showed some differences as the disappearance of the liquid expanded (LE)-liquid condensed (LC) phase transition at low temperatures for the DPPC but not for F-DPPC, because the F-DPPC monolayer is more expanded and more resistant to changes of temperature. On the other hand, film elasticity (C(s)(-1)) values calculated for both phospholipids show that the film condensation diminishes when the temperature increases, in accordance with the results obtained from surface pressure measurements.BAM images for F-DPPC monolayers recorded at different surface pressures and temperatures show the existence of numerous ovoid-like domains when the LE-LC phase transition is reached. However, in the LE and LC phases, homogeneous images were obtained. Time evolution of relative thickness along the compression of F-DPPC and DPPC monolayers shows similar behavior of both phospholipids, except at low temperatures.For DPPC/F-DPPC mixed systems, the plots of the mean molecular area as a function of F-DPPC mole fraction (X(F-DPPC)) indicated that, whatever the surface pressure, the experimental results match the theoretical values calculated from the additivity rule, a typical behavior for ideal mixed monolayers made of miscible components. This conclusion is confirmed from the values calculated for the free energy of excess (ΔG(exc)) of this system, which are practically zero, whatever the composition of the mixtures and the surface pressure at which ΔG(exc) values were calculated.

Monolayer and Brewster angle microscopy studies of poly(methyl methacrylate)-monopalmitin mixed systems at the air-water interface.

Mixed monolayers of poly(methyl metacrylate) (PMMA) and monopalmitin (Mp) were used for the study of their interactions. A thorough analysis of surface pressure (pi)-area (A) isotherms with the Langmuir monolayer technique, complemented with Brewster angle microscopy (BAM) images was performed. Mixed films show two phase transitions at a surface pressure of 14.5 mN/m and at 20-21 mN/m, respectively. Moreover, mixed monolayers show two well-defined collapses: one, corresponding to the lipid (at surface pressures of 50-51 mN/m) and the another one, ascribed to the PMMA, at surface pressure values of 57-58 mN/m. When the mean molecular areas of the mixed films (A(1,2)) were plotted versus film composition (X(1) or X(2)), positive deviations from the ideal behavior were observed at surface pressures below 15 mN/m, which were mainly attributed to a change in the conformation of the PMMA molecules at the surface. However, at higher surface pressures, the areas per monomer unit of the mixed monolayers obey the additivity rule, attributed to the fact that the film components form an immiscible system in these conditions.

Interactions between the ganglioside GM1 and hexadecylphosphocholine (miltefosine) in monolayers at the air/water interface.

The ganglioside, GM1, was studied as Langmuir monolayers at the air/water interface with surface pressure-area measurements in addition to Brewster angle microscopy. A characteristic plateau transition, observed on aqueous subphases of pH 2 and 6, 20 degrees C, at the surface pressure of ca. 20 mN/m, was attributed to the reorientation of GM1 polar group upon film compression. This transition was found to disappear at alkaline subphases (pH 10) due to the hydration of fully ionized polar group, hindering its reorientation. The interactions between GM1 and hexadecylphosphocholine (miltefosine) were investigated in mixed monolayers and analyzed with the mean molecular areas, excess areas of mixing and the excess free energy of mixing versus film composition plots. The monolayers stability, quantified by the collapse pressure values, as well as the strength of interaction was found to diminish in the following order: pH 6>pH 2>pH 10. The strongest interaction occurs for mixed films of miltefosine molar fraction, XM=0.7-0.8, especially at low pressure region, and are explained as being due to the surface complex formation of 3:1 or 4:1 (miltefosine:ganglioside) stoichiometry (XM=0.75 or 0.8, respectively).