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-2oleoyl-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.

Allosteric regulation of enzymatic activity forms the basis for controlling a plethora of vital cellular processes. While the mechanism underlying regulation of multimeric enzymes is generally well understood and proposed to primarily operate via conformational selection, the mechanism underlying allosteric regulation of monomeric enzymes is poorly understood. Here we monitored for the first time allosteric regulation of enzymatic activity at the single molecule level. We measured single stochastic catalytic turnovers of a monomeric metabolic enzyme (Thermomyces lanuginosus Lipase) while titrating its proximity to a lipid membrane that acts as an allosteric effector. The single molecule measurements revealed the existence of discrete binary functional states that could not be identified in macroscopic measurements due to ensemble averaging. The discrete functional states correlate with the enzyme's major conformational states and are redistributed in the presence of the regulatory effector. Thus, our data support allosteric regulation of monomeric enzymes to operate via selection of preexisting functional states and not via induction of new ones.

There is increasing evidence that certain nanoparticles (NPs) can overcome the placental barrier, raising concerns on potential adverse effects on the growing fetus. But even in the absence of placental transfer, NPs may pose a risk to proper fetal development if they interfere with the viability and functionality of the placental tissue. The effects of NPs on the human placenta are not well studied or understood, and predictive in vitro placenta models to achieve mechanistic insights on NP-placenta interactions are essentially lacking. Using the scaffold-free hanging drop technology, we developed a well-organized and highly reproducible 3D co-culture microtissue (MT) model consisting of a core of placental fibroblasts surrounded by a trophoblast cell layer, which resembles the structure of the in vivo placental tissue. We could show that secretion levels of human chorionic gonadotropin (hCG) were significantly higher in 3D than in 2D cell cultures, which indicates an enhanced differentiation of trophoblasts grown on 3D MTs. NP toxicity assessment revealed that cadmium telluride (CdTe) and copper oxide (CuO) NPs but not titanium dioxide (TiO) NPs decreased MT viability and reduced the release of hCG. NP acute toxicity was significantly reduced in 3D co-culture MTs compared to 2D monocultures. Taken together, 3D placental MTs provide a new and promising model for the fast generation of tissue-relevant acute NP toxicity data, which are indispensable for the safe development of NPs for industrial, commercial and medical applications.

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.