Marité Cárdenas

Transparent Films Based on PLA and Montmorillonite with Tunable Oxygen Barrier Properties

Polylactide (PLA) is viewed as a potential material to replace synthetic plastics (e.g., poly(ethylene terephthalate) (PET)) in food packaging, and there have been a number of developments in this direction. However, for PLA to be competitive in more demanding uses such as the packaging of oxygen-sensitive foods, the oxygen permeability coefficient (OP) needs to be reduced by a factor of ~10. To achieve this, a layer-by-layer (Lbl) approach was used to assemble alternating layers of montmorillonite clay and chitosan on extruded PLA film surfaces. When 70 bilayers were applied, the OP was reduced by 99 and 96%, respectively, at 20 and 50% RH. These are, to our knowledge, the best improvements in oxygen barrier properties ever reported for a PLA/clay-based film. The process of assembling such multilayer structures was characterized using a quartz crystal microbalance with dissipation monitoring. Transmission electron microscopy revealed a well-ordered laminar structure in the deposited multilayer coatings, and light transmittance results demonstrated the high optical clarity of the coated PLA films.

Monitoring Shifts in the Conformation Equilibrium of the Membrane Protein Cytochrome P450 Reductase (POR) in Nanodiscs

Background: Investigating the mechanism of NADPH-dependent conformational changes of POR in nanodiscs. Results: The conformational equilibrium of compact and extended POR, shifts toward the compact form (from 30 to 60%) upon reduction by NADPH. Conclusion: The NADPH-dependent conformational changes follow the “swinging model.” Significance: This is the first time that the action of a membrane protein located in a lipid bilayer environment is probed by neutron reflectivity. Nanodiscs are self-assembled ∼50-nm2 patches of lipid bilayers stabilized by amphipathic belt proteins. We demonstrate that a well ordered dense film of nanodiscs serves for non-destructive, label-free studies of isolated membrane proteins in a native like environment using neutron reflectometry (NR). This method exceeds studies of membrane proteins in vesicle or supported lipid bilayer because membrane proteins can be selectively adsorbed with controlled orientation. As a proof of concept, the mechanism of action of the membrane-anchored cytochrome P450 reductase (POR) is studied here. This enzyme is responsible for catalyzing the transfer of electrons from NADPH to cytochrome P450s and thus is a key enzyme in the biosynthesis of numerous primary and secondary metabolites in plants. Neutron reflectometry shows a coexistence of two different POR conformations, a compact and an extended form with a thickness of 44 and 79 Å, respectively. Upon complete reduction by NADPH, the conformational equilibrium shifts toward the compact form protecting the reduced FMN cofactor from engaging in unspecific electron transfer reaction.

Unraveling Dendrimer Translocation Across Cell Membrane Mimics

Poly(amidoamine) (PAMAM) dendrimers are promising candidates in several applications within the medical field. However, it is still to date not fully understood whether they are able to passively translocate across lipid bilayers. Recently, we used fluorescence microscopy to show that PAMAM dendrimers induced changes in the permeability of lipid membranes but the dendrimers themselves could not translocate to be released into the vesicle lumen. Because of the lack of resolution, these experiments could not assess whether the dendrimers were able to translocate but remained attached to the membrane. Using quartz crystal microbalance with dissipation monitoring and neutron reflectivity, a structural investigation was performed to determine how dendrimers interact with zwitterionic and negatively charged lipid bilayers. We hereby show that dendrimers adsorb on top of lipid bilayers without significant dendrimer translocation, regardless of the lipid membrane surface charge. Thus, most likely dendrimers are actively transported through cell membranes by protein-mediated endocytosis in agreement with previous cell studies. Finally, the higher activity of PAMAM dendrimers for phosphoglycerol-containing membranes is in line with their high antimicrobial activity against Gram-negative bacteria.

The protein corona of dendrimers: PAMAM binds and activates complement proteins in human plasma in a generation dependent manner

Dendrimers are polymers with a strong role in nanomedicine. In the current work we have developed a platform for mapping out the biomolecule corona for polyamidoamine (PAMAM) dendrimers. Complement proteins including C3 and C4b were found for the high generation dendrimers, suggesting high affinities to the dendrimers and most importantly complement activation.

Composition and structure of mixed phospholipid supported bilayers formed by POPC and DPPC

In this paper we present a systematic study of the morphology and composition of supported lipid bilayers (SLBs) formed by vesicle fusion using a wide variety of surface sensitive techniques that give information about the lateral as well as vertical structure and bilayer fluidity. SLBs of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) mixtures at five different bulk vesicle compositions were formed in such a way that the phase separation boundaries were crossed. For all compositions studied, the SLBs were systematically enriched with POPC compared to the nominal vesicle composition. Nevertheless, gel-fluid domain coexistence was observed for SLB compositions in which phase separation was expected based on the bulk phase diagram. The probable causes for the compositional difference in the SLBs are discussed in terms of the phase behaviour of the mixture and its effect on the membrane formation process by vesicle fusion.

Formation of supported lipid bilayers by vesicle fusion: effect of deposition temperature.

We have investigated the effect of deposition temperature on supported lipid bilayer formation via vesicle fusion. By using several complementary surface-sensitive techniques, we demonstrate that despite contradicting literature on the subject, high-quality bilayers can be formed below the main phase-transition temperature of the lipid. We have carefully studied the formation mechanism of supported DPPC bilayers below and above the lipid melting temperature (Tm) by quartz crystal microbalance and atomic force microscopy under continuous flow conditions. We also measured the structure of lipid bilayers formed below or above Tm by neutron reflection and investigated the effect of subsequent cooling to below the Tm. Our results clearly show that a continuous supported bilayer can be formed with high surface coverage below the lipid Tm. We also demonstrate that the high dissipation responses observed during the deposition process by QCM-D correspond to vesicles absorbed on top of a continuous bilayer and not to a surface-supported vesicular layer as previously reported.

Model cell membranes: discerning lipid and protein contributions in shaping the cell.

The high complexity of biological membranes has motivated the development and application of a wide range of model membrane systems to study biochemical and biophysical aspects of membranes in situ under well defined conditions. The aim is to provide fundamental understanding of processes controlled by membrane structure, permeability and curvature as well as membrane proteins by using a wide range of biochemical, biophysical and microscopic techniques. This review gives an overview of some currently used model biomembrane systems. We will also discuss some key membrane protein properties that are relevant for protein-membrane interactions in terms of protein structure and how it is affected by membrane composition, phase behavior and curvature.

Continuous Flow Atomic Force Microscopy Imaging Reveals Fluidity and Time-Dependent Interactions of Antimicrobial Dendrimer with Model Lipid Membranes

In this paper, an amphiphilic peptide dendrimer with potential applications against multi-resistant bacteria such as Staphylococcus aureus was synthesized and studied on model cell membranes. The combination of quartz crystal microbalance and atomic force microscopy imaging during continuous flow allowed for in situ monitoring of the very initial interaction processes and membrane transformations on longer time scales. We used three different membrane compositions of low and high melting temperature phospholipids to vary the membrane properties from a single fluid phase to a pure gel phase, while crossing the phase coexistence boundaries at room temperature. The interaction mechanism of the dendrimer was found to be time-dependent and to vary remarkably with the fluidity and coexistence of liquid-solid phases in the membrane. Spherical micelle-like dendrimer-lipid aggregates were formed in the fluid-phase bilayer and led to partial solubilization of the membrane, while in gel-phase membranes, the dendrimers caused areas of local depressions followed by redeposition of flexible lipid patches. Domain coexistence led to a sequence of events initiated by the formation of a ribbon-like network and followed by membrane solubilization via spherical aggregates from the edges of bilayer patches. Our results show that the dendrimer molecules were able to destroy the membrane integrity through different mechanisms depending on the lipid phase and morphology and shed light on their antimicrobial activity. These findings could have an impact on the efficacy of the dendrimers since lipid membranes in certain bacteria have transition temperatures very close to the host body temperature.

Cellulose-nanofiber/polygalacturonic acid coatings with high oxygen barrier and targeted release properties

A bio-inspired coating consisting of pectin (polygalacturonic acid) and cationic cellulose nanofibers were successfully produced by the layer-by-layer method. The build-up and the morphology of the resulting coatings were studied with spectroscopic ellipsometry and atomic force microscopy, respectively. The coating was able to survive the exposure of a simulated gastric fluid, but was partially degraded upon exposure to pectinase enzyme, which simulate the action of the microbial symbionts present in the human colon. Prior to exposure, the oxygen permeability coefficient of the coating (0.033 ml(STP)mmm(-2)day(-1)atm(-1) at 23°C and 20% RH) was in the same order of magnitude as for ethylene vinyl alcohol films (0.001-0.01 ml(STP)mmm(-2)day(-1)atm(-1)). However, after exposure to the mimicked gastrointestinal (GI) tract conditions, the contribution of coating to the overall barrier properties was not measurable.

Induced dye leakage by PAMAM G6 does not imply dendrimer entry into vesicle lumen

Dendrimers are polymers with unique properties that make them promising in a variety of applications such as potential drug and gene delivery systems. Polyamidoamine (PAMAM) dendrimers, in particular, have been widely investigated since they enter rapidly into cells. The entry mechanism, however, is still not yet fully clarified as both passive and active uptake have been proposed. In this work we focus on understanding passive uptake, for which simple cell model systems are used in order to ensure that only dendrimer–lipid interactions are probed. We developed protocols for investigating independently the effect of the dendrimer on lipid bilayer integrity, in terms of permeability of small dyes and effective dendrimer translocation. This was achieved by the use of membrane labeled giant unilamellar vesicles (GUVs) either containing Alexa 488 hydrazide in the vesicle lumen or FITC-labeled PAMAM G6 dendrimers. Vesicle integrity and dendrimer–membrane binding were then assessed by fluorescence microscopy. The importance of membrane fluidity and charge was investigated using GUVs composed of various lipid compositions. A quartz crystal microbalance with dissipation was used to probe the effect of dendrimers on the rigidity of vesicle layers. The results indicate that PAMAM dendrimers can locally alter the membrane properties. An increased bilayer permeability towards soluble small dyes but no effective translocation, where PAMAM dendrimers could dissociate from the lipid membrane into the vesicle lumen, was observed. To our knowledge this is the first time it is shown that PAMAM G6 dendrimer does not effectively translocate the lipid bilayer although it readily interacts with the model membrane, regardless of lipid membrane properties. However, bilayer charge and fluidity modulate the dendrimer interaction in agreement with previous reports. The results clearly highlight the importance of the choice of the model system when investigating nanoparticles interaction with lipid membranes.