Towards controlled mesomorphic phase behaviour of lipid bilayers
Ever since the invention of the microscope in the 17th century, scientists have studied the structure and behaviour of living cells. The knowledge acquired since then has led to many applications and subsequently improved the quality of our lives immensely, from an improved personal hygiene to healthcare and the availability of many medicines. It should be clear however, that the cell still holds many secrets. In this thesis we focus on one of those secrets, namely how the cell controls the mesomorphic phase behaviour of its lipid membranes. In other words, how come biomembranes of the cell and its organelles come in so many different shapes and forms? As we explain in the introduction of this thesis (chapter 1) the answer ultimately lies in the composition of the lipid bilayer, which is the universal component of all biological membranes. However, how the constituents of the lipid bilayer effect its mesomorphic phase behaviour remains unclear. The main goal of this thesis is to provide the tools and insight necessary to obtain control on the mesomorphic phase behaviour of lipid bilayers. To do so, we have chosen to combine experimental work with theoretical modelling.In chapters 2 and 3 we describe our experimental efforts to bring bilayers together in close proximity using limited vesicle aggregation. This is important as inducing a mesomorphic phase change in the bilayer such as the formation of membrane handles or inducing vesicle fusion, bringing membranes together is the very first step. In chapter 2 we present three successful strategies that lead to stable finite-sized vesicles aggregates. We show that by tuning various experimental parameters it is possible to limit the aggregation of the vesicles to the vesicle-pair level following a process we dub ‘self-limiting aggregation’ and provide a detailed analysis on the process of this aggregation under various physico-chemical conditions. Amongst the successful strategies, the one involving a thermo-sensitive surfactant (C18-pNIPAm) as a linker molecule shows an additional feature: the limited aggregation of vesicles is completely and repeatedly reversible using temperature as the trigger.In chapter 3 we build on the work of chapter 2 and combine the biotin/streptavidin linker system with the thermo-sensitive C18-pNIPAm surfactants. In doing so further control over the aggregation of the vesicles is obtained. Due to the presence of C18-pNIPAM no aggregation occurs below the LCST of C18-pNIPAm (32oC), and self-limiting aggregation occurs above this temperature. However, the presence of the biotin/streptavidin linkers makes it that the aggregation is no longer reversible. Instead, repeated temperature cycles cause a step-wise aggregation process that under well-chosen experimental conditions allows for the formation of small vesicle aggregates of predetermined sizes.In chapters 4 and 5 we discuss our results on Scheutjens and Fleer self-consistent field (SF-SCF) calculations on lipid bilayers. Our contribution in chapter 4 is two-fold. Firstly, we introduce a lattice refinement to the existing SF-SCF framework. Secondly, we apply the lattice-refined SF-SCF machinery to systematically study bilayer self-assembly of phospholipids in a selective solvent (water). With the lattice refinement implemented it is possible to extract mechanical parameters that govern the mesomorphic phase behaviour of lipid bilayers, such as the mean (κ) and Gaussian (κ ̅) bending moduli, as well as the spontaneous curvature of the monolayer (J_0^m) following a grand canonical ensemble route. We predict the abovementioned mechanical parameters in addition to various structural parameters as a function of several lipid properties. In general we find that increasing the hydrophobicity of the phospholipid leads to larger values for κ ̅, and lower ones for J_0^m. This indicates that, amongst others, the lipophilic to hydrophilic ratio is an important mechanism to control the mesomorphic phase behaviour of lipid bilayer membranes.Chapter 5 expands on the work of chapter 4 where we introduce various additives in the system to stimulate changes in the self-assembled phase behaviour of monoolein (MO) and 1,2-dioleoyl-phosphatidylcholine (DOPC) lipids in water. More specifically, we obtain trends in the mechanical parameters (κ, κ ̅ and J_0^m) of equilibrium bilayers as a function of increasing additive content and compare these with known experimental phase behaviour if available. The trends in the mechanical parameters are subsequently correlated to various mesomorphic phase changes in the system. In particular, the phase transition from a lamellar bilayer topology to a saddle-shaped topology is verified to correlate with a sign-switch of κ ̅. After the establishment of multiple trends in the mechanical parameters, various groups of additives are subsequently identified that potentially induce mesomorphic phase changes. In agreement with our results in chapter 4, we find that the inclusion of primarily hydrophobic additives that partition in the bilayer core, drives the bilayer towards a topological state of increased negative interfacial curvature and saddle-shape configurations. Micelle-forming surfactants do the opposite. The inclusion of small additives, both hydrophilic (i.e. ‘solvents’) or hydrophobic, generally decreases the mean bending modulus of the bilayers and thus makes bilayers more flexible. This may take a lipid system from a bicontinuous cubic phase closer to a sponge phase or decrease the persistence length of lamellar bilayers and increase the undulation repulsion.In the general discussion of this thesis (chapter 6) I put the results obtained in the previous chapters in a wider context and discuss aspects and results that have not yet been addressed. In particular, we report on the successful preparation of a supported double lipid bilayer (SDLB) using biotin/streptavidin linkers. The general discussion additionally includes an overview of the challenges that still remain and a discussion on the direction future research may take. Most prominently, I envision this should involve inducing a mesomorphic phase change in the small vesicle aggregates causing the vesicles to either fuse together or to form stable membrane handles between them. This can be realized through the addition of additives that cause κ ̅ to switch sign from negative to positive as described in chapter 5, thus using primarily hydrophobic additives such as long-tailed fatty acids or the lipid monoolein. SCF calculations can further guide and support such experiments by modeling an actual stable membrane handle
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| Format: | Doctoral thesis biblioteca |
| Language: | English |
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Wageningen University
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| Subjects: | Life Science, |
| Online Access: | https://research.wur.nl/en/publications/towards-controlled-mesomorphic-phase-behaviour-of-lipid-bilayers |
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