I would like to thank Carlos for being a wonderful supervisor, with a keen insight on the smallest detail. His mentorship in many aspects of soft matter physics and biophysics that I was lacking was only matched by his support on the many experimental idea I had, no matter how bizzarre.

André for giving me a deeper knowledge of the experimental part of my work, and always providing a practical side and feedback to all my idea and elucubrations, and always have time to discuss microscopy.

Fabrice for the patience during our discussion on theory and thermodynamics, and for its constant input in my experimental work via papers, ideas and knowledge.

And of course all the members of the Mcube team: Thierry, Pierre, Tatiana, Marc, Othmene, Tiago and Salvatore for being a great team and always being there to talk science or anything.

A great thank you goes to all the people I had the chance to meet at the ICS, who made for a wonderful environment: Arthur, Amer, Laure, Morgane, François, Marc, Rigoberto, Flavio, Marvin.

The rugby team of the CNRS campus, and in particular Arthur and Thierry, who made me discover and love this great sport.

The whole ITN SNAL network, a group of fantastic people who made not only for great colleagues and fellows PhD candidates, but also for great company and fun during any conference: Alex, Daniela, Anna Sofia, Shiqi, Chan Fei, Berardo and Adrien.

In particular I want to thank Monika, for helping throughout my work at ICS, performing complementary experiments and being such an invaluable friend.

Finally, I want to thank all my friends, both geographically far away and not, who have always been close to me: Gianmarco, Andrea, Laurence, Marco, Alessandro, Robert, Rita, Matteo, Simone, Eleonora, Francesco, Alessio, Gilles, Girolamo, Stefano, Francesco, Francesco, Tony, Salvatore, and all the guys from Quintet. You make me feel at home even when I am not there.

A last and important acknowledgement goes to my family, who has supported me all these years and pushed my desire to pursue a scientific career. All I have achieved so far it is also because and thanks to them.

Preface

The complexity of the cellular membrane has motivated the development of minimal model systems to investigate the role of lipids in cellular mechanisms. Over the years the role of phospholipids has been reevaluated by the scientific community as studies unraveled their crucial role in membrane signalling and functionality. In particular the phase behavior of lipid bilayers has been extensively investigated and allowed to understand the significance of composition and cholesterol in modulating membrane proteins activity and membrane fluidity. The hypothesis of membrane "rafts", domains with laterally different composition and properties, also came to prominence as these studies progressed. Therefore, the study of the effects of a molecule or nanobject on the phase behavior of lipid model membranes is significant in understanding the interplay between interactions and behaviors, and in assessing any possible hazardous effects on biological membranes. This thesis is the result of a three year project conducted at the Institut Charles Sadron (ICS) in Strasbourg, France, under the supervision of Dr. Carlos Marques and Dr. André Schroder, as a part of the european funded Initial Training Network Smart Nanoobjects Altering the Lipid bilayer (SNAL). The aim of the ITN consortium was to understand and investigate at different levels the interaction between nanoobjects and lipid membranes, with the aim of developing better drug delivery systems, by studying from the theoretical and experimental point of views both model and biological systems. The network developed by the ITN allowed fnot only for a fruitful collaboration and scientific discussion among members but as well for the participation in conferences and workshops to divulgate the results.

At ICS, the interaction between ITN members was also of great benefit for this work, as for instance for the joint work on Cryo-TEM and neutron scattering developed with Monika Kluzek and Marc Schmutz for the elucidation of membrane polystyrene interactions, or for the contribution and theoretical support provided xxii by Fabrice Thalmann for the chapter on membrane sucrose interaction, and by Jean Wolff for the binary phase diagrams of membranes containing polystyrene.

The manuscript falls in the field of membrane biophysics, and focuses on the alterations of the phase behavior of lipid bilayers induced by small molecules, and on the role of the different degrees of hydrophobicity/hydrophilicity on the interplay taking place.

Chapter 1 is an introduction to the lipid membrane and its physical-chemical properties, and to the changes in its phase behavior depending on composition and external factors.

In Chapter 2 a description of the techniques employed in the thesis is presented, with particular focus on data analysis and presentation of characteristic fluorescent probes sensitive to their surroundings.

Chapter 3 focuses on the changes in model membranes composed of unsaturated lipids upon incorporation of polystyrene oligomers.

Chapter 4 continues the investigation of polystyrene assessing its effects on more biologically relevant membranes, i.e. membrane displaying phase separation or containing cholesterol.

Chapter 5 is devoted to the effects of high concentration of sucrose on the phase transition of DPPC bilayers, motivated by the relevance of sugar in cryo-and biopreservation. The work is the result of a two months internship programme with a third year Bachelor Degree student, Matthieu Sommer, who performed part of the experiments under supervision.

Chapter 6 presents the preliminary results obtained in studying the effects of a cholesterol grafted amphiphilic polymer on phase separated lipid membranes. The polymer, synthesized and provided by the ITN SNAL member Alex Chen from the University of Cambridge, was developed as a possible lipid altering agent and provided an interesting and novel idea to pursue. Au fil des années, le rôle des phospholipides a été réévalué par la communauté scientifique alors que les études ont révélé leur rôle crucial dans la signalisation membranaire et la fonctionnalité. En particulier, le comportement en phase des bicouches lipidiques a été largement étudié et permis de comprendre l'importance de la composition et du cholestérol dans la modulation de l'activité des protéines membranaires et de la fluidité de la membrane. L'hypothèse de «radeaux» membranaires, domaines avec une composition et des propriétés latéralement différentes, est également apparue au fur et à mesure que ces études progressaient. Par conséquent, l'étude des effets d'une molécule ou d'un nanobjet sur le comportement de phase des membranes modèles lipidiques est significative pour comprendre l'interaction entre les interactions et les comportements, et pour évaluer les éventuels effets dangereux sur les membranes biologiques.

Accumulation des oligomères de polystyrene dans la bicouche lipidique

La quantité croissante de plastique présente dans les eaux océaniques est devenue un problème majeur ces dernières années, avec des préoccupations croissantes concernant les effets potentiellement dangereux qu'elle peut avoir sur les systèmes vivants. La production annuelle de plastique a atteint près de 300 millions de tonnes par an, dont on estime que 5 à 13 millions de tonnes atteindraient les eaux océaniques xxiv par différents moyens. Bien qu'initialement présent en grande taille, des facteurs environnementaux externes tels que la température, le rayonnement oxydant et la dégradation bactérienne peuvent réduire le plastique en fragments micrométriques et nanométriques. Les objets de cette échelle peuvent facilement entrer dans la chaîne alimentaire par digestion et il y a de plus en plus de preuves de micro-objets en plastique trouvés dans les formes de vie marine.

La présence de nano-objets n'a pas encore été signalée, mais le manque de preuves peut être associé à des difficultés à détecter de tels objets plutôt qu'à leur absence.

Outre la dégradation plastique, des particules de polymère de taille nanométrique sont également produites industriellement pour des applications spécifiques de recherche et de technologie, telles que l'imagerie, la détection et la préparation de nanocomposites. La présence de plastiques nanométriques dans les laboratoires de recherche, l'industrie et l'environnement soulève des questions sur leur toxicité potentielle.

La membrane cellulaire est la première barrière rencontrée par n'importe quel corps étranger entrant dans un organisme, et est donc le principal candidat d'investigation dans l'évaluation de la toxicité possible des nanoparticules de plastique. En particulier, le comportement de la phase membranaire joue un rôle crucial dans le maintien de la fonctionnalité cellulaire, et même de légères modifications des propriétés de la membrane peuvent entraîner une menace potentielle pour l'organisme.

Dans ce travail, nous effectuons une étude systématique sur les effets des oligomères de polystyrène (M n = 500 Da) sur la transition de phase des lipides non-saturés.

Nous avons étudié les changements de transition en utilisant la diffusion de neutrons aux petits angles, la calorimétrie différentiel et les spectres de fluorescence de Laurdan pour extraire des informations sur la structure et la thermodynamique. xxxiv Bien que préliminaires, nos données suggèrent que la greffe de molécules altérant la membrane sur l'épine dorsale des polymères est une approche viable dans le développement de stratégies thérapeutiques pour faire varier les propriétés des membranes cellulaires.

Conclusions 1

Introduction

The cell membrane is the gateway of the cell with the exterior, providing not only for compartmentalization, but also regulating its interactions and the signalling processes with the exterior.

In early stages of research on plasma membranes, proteins were the focus, being ascribed as responsible for the majority of recognition and functional processes. In later years, however, lipids have come under the spotlight [1,2], due to the hypothesis of lipid rafts -nano and micrometric domains which modulate several processes, including intracellular trafficking [3,4]. The existence of rafts strongly implies that the lipid bilayer is not just a matrix for saccharides and proteins, but a functional component whose phase behaviour and composition is vital for cellular activity. However, developing experimental studies on the biological membrane and on its interaction with other molecules is a difficult task, due to the high complexity of the membrane.

Model membranes provide an optimal platform to study phase behavior of lipids under different conditions, and many studies confirmed indeed that composition regulates the existence of domains in model lipids bilayers [5].

In this chapter we will outline the main processes involved in lipid bilayer' phase behaviour and the feature arising when one or two more component self-assemble into a membrane.

The plasma membrane

The cell membrane (or plasma membrane) envelops the cytoplasm, separating the interior of the cell and its components from the external environment. It has also the role, together with cytoskeleton, to provide the shape of the cell and by attaching to the extracellular matrix to form cell junctions. Being selectively permeable to ions

Membrane lipids

Lipids are amphiphilic molecules that contain a hydrophobic tail and a hydrophilic polar headgroup (Fig 1 .2), which readily interacts with water. The nature of the polar 1.1. The plasma membrane 3 head group (charged or uncharged) as well as the acyl chain (saturated or unsaturated and number of carbon atoms) will determine their localization and function in the body.

Lipids, as many amphiphiles, can self-assemble in aqueous medium into a variety of structures. The main driving force of self-assembly is the hydrophobic effect, which induces self-association of the hydrophobic domains to minimize the total surface that is in contact with water, resulting in an entropy-driven relaxation of water structure and an energy minimum for the self-associated molecular organization [7]. The polar domains of lipids interact with water and other head groups and are therefore energetically stable in an aqueous environment [START_REF] Vance | Biochemistry of Lipids, Lipoproteins and Membranes[END_REF]. Typical self-assemblies depend on the packing parameter. In case of an inverted cone geometry, internal assemblies like micelles or heaxagonal phases arise (B). When the geometry is conical (C), it self-assembles into an inverted structure. In the case of cylindrical packing (D), the assembly is lamellar.

The structure of the formed assemblies is dictated by the chemical nature of the amphipathic lipid as well by the self-assembling environment. One of the major parameters that determines the final conformation is a geometric factor referred to as the packing parameter, P . This dimensionless parameter is given as the ratio of the hydrocarbon volume (v) to the product of the area of the polar head group (a 0 ) and the critical acyl chain length (l) beyond which the chain can no longer be considered fluid.

P = v a 0 • l (1.1)
If the value of P is less than 1/3 then the amphipathic molecules are expected to form spherical micellar structures. If the value is above 1/3 but below 1/2 it may form non-spherical micellar structures while P values >1 favour inverted assemblies.

However, this prediction is applicable only when a single amphipathic molecule is involved in the self-assembly process. When more than one component is involved,

Chapter 1. Introduction due to the complex interactions involving electrostatic interactions, hydrogen bonding and/or van der Waals forces between the constituent molecules, deviations from the predicted structures are expected.

Lipids that can be found in mammalian plasma membranes fall into three main classes of lipids: i) glycerophospholipids (called phospholipids, PL), ii) sphingolipids (SLs) and iii) sterols (Figure 1.3).

Phospholipids have a chemical structure based on glycerol backbone with attached fatty acid hydrocarbon chains in adjacent positions. A phosphate linked to an alcohol(choline, ethalonamine or serine) occupies generally the sn3 position. There is also a large variability in the fatty chains in terms of chain length and degree of unsaturation, which contributes greatly to the lipid physico chemical properties. Cholesterol, which is present in the cell membrane plays an important role in membrane phase behaviour and, as first hypothized by Ikonen [START_REF] Simons | [END_REF] in 1997, the interaction with sphingolipids is important in the formation of rafts.

Membrane lipids

The lipid composition of the outer and inner monolayers or leaflets of the lipid bilayer are different. Specifically, the zwitterionic glycerophospholipid phosphatidylcholine (PC) and the zwitterionic phosphosphingolipid sphingomyelin (SM) are enriched in the outer monolayer, while the zwitterionic glycerophospholipid phosphatidylethanolamine (PE) and the anionic glycerophospholipid phosphatidylserine (PS) are enriched in or restricted to, the inner leaflet of the plasma membrane composition.

Model membranes

Many phenomena occurring in biological membranes can be extrapolated or reproduced using model lipid bilayers, which are membranes assembled from a known lipid composition. Lipid bilayers exhibit many interesting properties arising from the cooperative interaction between lipid molecules. Several self-assembled bilayers are therefore used as biomimetic models of cell membranes. Several structures have been developed for biophysical studies and the intrinsic properties of bilayer as well as their interaction with other molecules have been investigated. Depending on the length scale of the mechanism at study, different models exist, ranging from unilamellar and multilamellar vesicles, supported lipid bilayers, nanodiscs and others.

The advantage of working with model lipid bilayers is the ability to precisely control the composition and conditions of the membrane, which is of great utility when studying phase behaviour. Lipid vesicles, also known as liposomes, were first introduced by Bangham [10] in the 1960s and are structural homologues of biological membranes. Liposomes can be produced with a large range of sizes (from 30 nm to 100 um) and their size can be controlled with great precision. Liposomes have been used on spectroscopic and calorimetric studies to investigate phase behavior [11][12][13].

The development of giant unilamellar vesicles (GUVs) [START_REF] Angelova | Faraday Discuss[END_REF]15] has allowed the study of many macroscopic and mesoscopic lipid bilayer systems using optical and fluorescence microscopy techniques. These vesicles have sizes in the micrometric range Chapter 1. Introduction and are particularly suited for studying mechanical properties like viscosity, rigidity, and also phase separation and domain morphology in lipid membranes [16].

Moreover their size allows for studies of membrane shape deformation and membrane related processes of biological cells. Micropipette manipulation of GUVs has been successfully used to inject substances within the interior of the vesicle without damaging the membrane, probe the membrane permeability [17], bending rigidity [18], and incorporation of molecules within the lumen or within the hydrophobic core of the bilayer. In the study of phase behaviour of lipid membranes they have been useful in determining changes in domain morphology in response to changes in membrane curvature, tension, composition and presence of polymers or other molecules.

Phase behavior of lipid bilayer

A characteristic feature of the lipid bilayer is the changes in the mobility of individual lipid molecules with variation of temperature and lipid species. In eukaryotic cell membranes the hypothized rafts arise from changes in the lipid phase behavior and therefore the investigation of lipid phase behavior in single and multicomponent bilayers has been a focus in soft matter physics over the last decades.

Solid ordered phase Liquid crystalline phase

A) B) Generally, when considering a lipid bilayer, the membrane can exist in two possible states: a solid or a liquid phase [19]. The solid phase (also called "gel" phase, S o or L β ) is characterized by a tight packing of the lipid molecules. Hydrocarbon chains 1.2. Phase behavior of lipid bilayer 7 are in the fully extended all-trans conformation, the cross-sectional area of the phospholipid molecules is minimal, the thickness of the phospholipid bilayer is maximal, and both intra-and intermolecular motion are severely restricted [START_REF] Marsh | Handbook of lipid bilayers[END_REF][START_REF] Kučerka | [END_REF][22][23].

The fluid phase (also referred to as L α phase), on the other hand, has highly disordered chains, the phospholipid hydrocarbon chains contain a number of gauche rotational conformers, the cross-sectional area of the lipid molecules increases considerably, the phospholipid bilayer thins substantially [24], and relatively high rates of both intra-and intermolecular motion are present [START_REF] Marsh | Handbook of lipid bilayers[END_REF][25][26][27].

Many physical properties of the membrane change with respect to the phase, ranging from cross sectional area to bending rigidity and permeability [START_REF] Marsh | Handbook of lipid bilayers[END_REF].

The transition from gel to fluid, and vice versa, is a cooperative phenomenon that occurs when the temperature of the system goes above (or below) the characteristic temperature of the lipid [28,29]. The transition temperature T m , among other parameters, is strongly influenced by the length and degree of unsaturation of acyl chains, head group size and charge, etc [30,31]. Assuming a first order transition for the L β to L α phase change, it is possible to write:

P Lα (T m ) P So (T m ) = K • T m = e -∆G k B Tm = 1 (1.2)
where P Lα is the probability for a single lipid to be in the fluid phase and P So the probability of being in the gel phase, and ∆G is the Gibbs free energy difference. At the transition, it is possible to describe T m as:

∆G = ∆H -T m ∆S = 0 T m = ∆H ∆S (1.3)
where ∆H is the enthalpy and ∆S is the entropy of melting. Enthalpic and entropic contributions values strongly depend on the length and saturation of lipid tails [START_REF] Marsh | Handbook of lipid bilayers[END_REF],

and thus the value of the transition temperature. Other factors, however, such as cations (divalent ions) [32,33], membrane hydration [34,35], membrane curvature, and pH [33,[START_REF] Heimburg | Membrane Structure[END_REF] can affect the transition temperature and the Gibbs free energy.

Multicomponent bilayers

When the bilayer is composed of two or more lipid species, complex phase behavior arises due to the combination of each lipid transition. For a binary system composed of high melting lipid (like DPPC) and low temperature melting lipid (like DOPC), three distinct regions exist [START_REF] Koynova | [END_REF]38].

Chapter 1. Introduction

When the temperature is below both lipid T m , the bilayer is in a S o phase. Conversely, when temperature is above both T m the membrane is in a fluid phase.

In the intermediate region between the two lipid T m phase separation appears, and we observe a coexistence of S o and L α phase within the membrane. The relative amount of each phase is dependent on the temperature and molar ratio of the two components (Fig. 1.5). The behavior of phase separation can be affected by many factors, like temperature [33], charge [39], pH [33], interaction with molecules and types of lipids [40]. The phase separation process plays an important role in biological membranes due to the formation of compositionally distinct domains, implying the creation of regions of different bilayer hydrophobic thickness. It is reported that many membrane proteins partition preferentially in one of the domains and the activity of some membrane proteins depend on the hydrophobic mismatch created by phase separation [41].

Domains exist both at the nanoscopic and micrometric length scale. Nanoscopic domains have been studied using neutron scattering techniques and AFM imaging [START_REF] Mouritsen | Search of a New Biomembrane Model[END_REF], and have been reported to exist, together with percolative structures, in the two-phase coexistence region.

The properties and behavior of micrometric domains have been extensively investigated using two-photon microscopy [43] and confocal microscopy [44]. Solid ordered domains appear in many morphologies, as illustrated in Fig 1 .6 and several factors contribute to the typology of the domains observed, including membrane tension, temperature rate, and ionic strength [45]. Aside from liquid-solid coexistence, solid-solid coexistence has also been observed in systems composed of lipids with high chain length mismatch.

Cholesterol

Eukaryotic plasma membranes can contain up to 30% of cholesterol [46], therefore it is not surprising that the effect of the molecule on the membrane properties is very significant. Cholesterol has been identified as one of the main components of the cellular rafts, due to its ability to modulate diffusion and phase behaviour.

It was initially proposed that cholesterol's main role is to reduce the intrinsic phase separation in cellular membranes due to the lipid composition. Hao et al. [47] proved that indeed a cellular membrane depleted of cholesterol exhibits phase coexistence.

In model lipid bilayers the effects of cholesterol have been studied extensively using DSC [48], AFM, fluorescence microscopy [49,50], spectrometry [51,52], NMR [48,49,53] and X-ray scattering. Addition of cholesterol to the bilayer has been shown to reduce the enthalpy of the L α -S o transition in DPPC bilayers (eliminating it almost completely at 50% mol), alter the bilayer permeability [54,55] and the lipid lateral diffusion [56]. Moreover cholesterol-rich bilayers have smaller cross sectional area [57,58], larger membrane thickness than L α phase bilayers and different acyl chain order in both gel and liquid phase [59].

Chapter 1. Introduction

A A B

FIGURE 1.7: Structure of liquid ordered phase (L o ) of lipid bilayer. The sterol rings prevent the hydrocarbon chains of the lipid from strongly interacting together, but also block the total expansion of the lipid upon melting (A). The packing of the bilayer is almost as tight as in the S o phase, however there is no packing lattice and lipids have a higher diffusivity (B).

All the changes in the physical properties of the bilayer in presence of cholesterol point to an intermediate phase between gel and liquid. This state of the cholesterol rich bilayer, also called liquid ordered (L o ) phase [59], retains the tight packing and rigidity of the gel phase, but the fluidity of the liquid phase [46,60]. Conversely, fluid membrane in presence of cholesterol is called liquid disordered (L d ) phase.

Perhaps one of the most striking features of the cholesterol is the changes it induces in phase diagrams when incorporated in single or multi component membranes.

For single lipid bilayers, the presence of cholesterol at 7% already induces a phase separation between the S o and L o phases [61]. As the temperature rises, phase coexistence still remains, but between L d and L o phases [62,63]. Studies on micrometric liquid ordered phase have been performed on GUVs and planar membranes [65][66][67][68]. These domains are generally circular, due to line tension driving the phase domains toward the minimum perimeter, and coalesce upon collision to form larger domains.

Similar
Similarly for binary systems, evidence of nanometric liquid domains has been reported [69]. In some cases, submicron lipid organization is reported in vesicles that never exhibit large scale liquid immiscibility [48,70], and in other cases, the same vesicle system can produce both small and large-scale features, but at different compositions and temperatures [49,52,71].

Beyond ternary systems, Konyakhina et al. .

Concluding remarks

The possibility to replicate or probe biologically relevant phase behavior has made model lipid membranes an invaluable tool in biophysics. Indeed, many cellular mechanisms of interaction have been elucidated using lipid bilayers and despite being a minimal system, they have allowed an understanding of the mechanism of interaction between drugs, nanoparticles or biomolecules with cell membranes.

2

Materials and Methods

In this chapter the main techniques employed during this Ph.D project will be described. A brief description of the working principle of each methodology will be outlined, followed by a more detailed explanation of the data analysis used in the experimental work. The use of fluorescent probes sensitive to the environment is a key element of this work, therefore an in-depth analysis of the differentt properties of the probes used will be presented. In particular, analysis of emission and excitation spectra of Laurdan and Di-4 and their relation to phase behavior of lipid bilayer will be described.

Differential scanning calorimetry

Differential scanning calorimetry (DSC) is a calorimetric technique that allows to quantitatively study phase transitions upon changes in temperature. This method has been used extensively to investigate the lipid bilayer gel to liquid transition and phase behavior in dry and hydrated membranes [START_REF] Lewis | Methods in Membrane Lipids[END_REF]. It has been succesfully used to probe the phase diagrams for binary and ternary mixtures with and without cholesterol, as well as the effects of micromolecules on the phase behavior of model lipid membranes [52,[START_REF] Prenner | [END_REF][75][76][77][78][79][80].

Chapter 2. Materials and Methods

Measurement chamber

Reference chamber

Heating system FIGURE 2.1: A schematic representation of a typical DSC apparatus. The measurement chamber contains the sample while the reference chamber is filled with solvent of reference.

The feedback system mantains both cells at the same temperature while heating or cooling the chambers at the same rate.

The apparatus consists of an adiabatic chassis with two separate chambers: a measurement and a reference cell. Sample of lipid bilayer in solution is placed in the measurement cell while reference cell contains the same mass of pure solvent. The apparatus heats both cells at the same rate and records the energy provided to the system to keep both chambers at the same temperature. Because the phase transition of lipid bilayer requires adsorption or release of energy the resulting signal is a peak centered at T m . For first order phase transitions such as the bilayer transition from gel to liquidcrystalline, the transition temperature, T m , is where the heat capacity, C p , reaches 2.2. SANS its maximum value. The value of the calorimetric enthalpy (∆H cal ) for the phase transition is determined by integrating the area under the peak,

∆H cal = C p dT (2.1)
and the entropy of the transition is extracted by using the simple relation:

∆S = ∆H cal T m (2.2)
Comparison of ∆H cal , ∆S and T m shows the effect of a structural modification (e.g. chain length) on the thermodynamics of the phase transition.

Theoretically for pure lipid systems the transition should be completely symmetrical, however experimentally the thermograms of biological systems and lipid bilayer are asymmetrical. T m is therefore no longer the midpoint of the transition and T 1/2 , the width of the distribution, becomes an important parameter.

Analysis of the shape of the peak provides additional information about the melting process. The T 1/2 parameters, defined as the width at half height of the peak, is a valuable tool to estimate purity, protein-lipid interactions, as well as lipid-lipid interactions, and provides information about the cooperativity of the phase transition [81], as explained in Chapter 1.

The cooperativity of a pure lipid transition is in fact related to the shape and sharpness of the peak and is described by a cooperative unit (CU), the number of lipids involved in the transition [81]. The cooperative unit can be calculated by the ratio of ∆H vH /∆H cal , where ∆H cal is the enthalpy of the transition and ∆H vH is the van't

Hoff enthalpy [82].

∆H vH = 4RT 2 m T 1/2 (2.3)
with R is the gas constant.

SANS

Although many techniques allow characterization of biological structures and interactions, scattering techniques, such as X-ray and neutron scattering, have allowed to probe objects at the nanometric level with great precision. Among the different methods using neutron scattering, small-angle neutron scattering (SANS) is one of During a SANS experiment a beam of neutrons is directed at a sample, which can be an aqueous solution, a solid, a powder, or a crystal (Fig. 2.3). The neutrons are elastically scattered by nuclear interaction with the nuclei or interaction with magnetic momentum of unpaired electrons. The detector measures the scattering intensity I(q) at varying angles as a function of the scattering variable q, defined a

q = 4π λ sinφ 2 ≈ 2πφ λ (2.4)
where φ is scattering angle, λ the wavelength of neutron radiation. Taking account the detector distance, D, from sample, the scattered radiation striking the detector at distance, S, from its center defines the low limit of scattering angle. When D > S, the scattering wave vector equals

q = 4π λ sinφ 2 ≈ 4π λ tan(φ/2) = 2π λ S D (2.5)
The scattering of neutrons upon impinging on the sample is collected by a detector, and afterwards is corrected by detector efficiency, which is determined in separate measurement. The scattering cross section, dΣ/dΩ, is related to the count rate I(q)

2.2. SANS measured by the detector

I(q) = I 0 AdT ( dΣ(q) dΩ ) sample ∆Ω t (2.6)
where ∆Ω is a solid angle, A the area of the sample, d the sample thickness, the detector efficiency, t the counting time, and T the transmission efficiency of filled cell.

Scattering lengh and scattering lengh density

Scattering of a neutron is due to interaction between neutron and the atomic nucleus, therefore isotopes of the same atom scatter neutrons in different way. The intensities of scattered neutrons is proportional to the scattering lengh, b, which describes the scattering potential of a single atom. It can be shown that the intensity by the detector is related to b by the simple equation:

I s I 0 = |b| 2 • A D 2 = |b| 2 ∆Ω I s dΩI 0 = |b| 2 (2.7)
Which gives the relative amount of scattered neutrons by sample towards the detector. To obtain the scattering into all directions, this quantity must be integrated over all solid angles:

Ω |b| 2 dΩ = 4π|b| 2 = σ dσ dΩ = |b| 2 (2.8)
where: σscattering cross section, dσ/dΩ differential scattering cross section.

Usually it is more convienient to describe the scattering potency of the sample as built up by atoms with an associated scattering length in a finite volume, called

scattering length density (SLD) ρ. ρ = n i b i V (2.9)
where V is the molecular volume.

Chapter 2. Materials and Methods

The scattering intensity of sample in solution is a combination of scattering from particles and solvent. Assuming a homogenous scattering signal from the solvent, the excess scattering length density, ∆ρ, of the particles compared to solvent gives:

∆ρ = ρ molecule -ρ solvent (2.10)
Therefore intensity and shape of scattering curves is determind only by the scattering density deviations from the solvent. If the sample and solvent have the same SLD values, ∆ρ vanishes with no signal recorded by the detector. Thus, ∆ρ is usually referred to sample contrast.

In this thesis we used equation 2.9 to calculate the SLD of hydrogenated lipid head groups and fatty acids together with their deuterated analogs. Since we worked at two different temperatures, i.e. above and below T m of DPPC the changes in SLD due to volume variations at different temperature were taken into account as well.

The calculated SLD values of lipids and polymer used are collected in Table 2.1.

Molecule

Part of the molecule Chemical formula Temperature Scattering length Volume Scattering length density

[ • C] [10 -5 Å] [Å 3 ] [10 -6 Å -2 ] h-DPPC Lipid C

Contrast variation technique

As mentioned in the last paragraph the scattering intensity depends solely on the contrast between the sample and the solvent. This allows, by selective deuteration

SANS

Data analysis

A complete analysis of scattering curves generally requires the choice of a model with a form factor for the object and a size distribution of the sample. This analysis can be fairly difficult and very sensitive to the choice of parameters and scattering length densities. However, it is possible to analyse the scattering data and extract significant information on the structure of the sample by studying the behaviour of the scattering profiles in different regions (Fig. 2.4) [START_REF]Neutron Scattering -Applications in Biology, Chemistry, and Materials Science[END_REF]. In particular, for lipid bilayer the model free analys allows to estimate the average size of the object and the thickness of the bilayer.

I(q) (cm -1 ) q (Å -1 )
Liposomes size and polydispersity

Bilayer inner structure

Shape of liposomes

Guinier regime Porod regime Intermediate regime FIGURE 2.4: Example of a SANS scattering curve, highlighting the q to their corresponding bilayer structural information.

Guinier analysis

In the limit of q close to 0, for n orienteded particles, the scattered intensity can be approximated by, as first proposed by Guinier in 1939,

I(q) ∝ exp(-q 2 R 2 g /3) (2.11)
where R g is the radius of gyration, which represents the effective size of the scattering "particle", whether it is a polymer chain, part of a protein, a micelle, or a domain in a multiphase system. This approximation provides a simple way to estimate R g of the measured system, by plotting ln[I(q)] vs q 2 in order to obtain the slope R 2 G /3. However, it has to taken into account that expression is valid only for q • R g ≤ 1.

Kratky-Porod analysis

Although the Kratky-Porod plot is a powerful tool in probing the membrane thickness, it has to be stressed that this method is an average of the scattering density profile from the bilayer. Moreover the penetration of solvent molecules within the bilayer interface must be small enough to avoid changes in the SLD distribution.

In the intermediate q regime the signal corresponds to smaller lenght scales, where the bilayer appears as a sheet with given thickness d. By fitting linearly ln[I(q) • q 2 ] vs q 2 and extracting the slope R, the corresponding value for the thickness can be written:

d = R √ 2 (2.12)

Fluorescence, microscopy and environment-sensitive fluorescent probes

Fluorescence is the property of a molecule or a material to absorb energy from incoming light, and emit it in form of light at a different wavelength. It occurs when an orbital electron of a molecule, atom, or nanostructure, relaxes to its ground state by emitting a photon from an excited singlet state.

As illustrated in the Jablonski diagram (Figure 2.5), a fluorophore absorbs light at λ exc wavelength rising from the ground state to the first electronic state S 2 . Subsequently it returns to the ground state emitting a photon at a higher wavelength λ em .

Emitted fluorescent light has a longer wavelength and lower energy than the absorbed light. This phenomenon, known as Stokes shift, is due to the internal energy loss between the time a photon is absorbed and when it is emitted (Fig. 2.5 B).

Microscopy

Microscopy has become over the years an indispensable tool in biological research, due its many applications and variants that allow to investigate different properties In contrast, a confocal microscope uses point illumination and a pinhole in an optically conjugated plane in front of the detector to eliminate out-of-focus signal -the name "confocal" stems from this configuration. Successive slices make up a 'z-stack' which can either be processed by softwares to create a 3D image, or it is merged into a 2D stack (predominately the maximum pixel intensity is taken, other common methods include using the standard deviation or summing the pixels).

Confocal microscopy provides better contrast with respect to a conventional epifluorescence microscope. Moreover, since the fluorescence intensity recorded comes only from the focal plane, it is possible to perform quantitative analysis on the fluorescent probe.

In the analysis of lipid bilayer phase behavior, confocal imaging provides indeed better contrast for visualization of domains, at the cost of direct imaging of phase coexistence. The ability to perform 3D reconstruction allows for better quantification of domains area coverage (Fig. 2.9).

Laurdan and other environment-sensitive probes

Laurdan (6-Dodecanoyl-2-Dimethylaminonaphthalene) is an organic compound that has been used as a fluorescent dye, particularly in fluorescence microscopy and steady state fluorescence, due to its environment sensitive properties. In particular, Laurdan is very sensitive to membrane phase transition and alterations of membrane fluidity. It was firstly synthesized by Gregorio Weber [START_REF] Weber | [END_REF] in 1979 as a probe to study specifically dipolar relaxation on cell and lipid membranes, as it is more sensitive to variation of these characteristics.

Because of its polar sensitive properties Laurdan is used extensively for probing bilayer phase transitions in the presence of membrane altering compound, such as cholesterol [85], proteins [86,[START_REF] Antollini | Methods in Membrane Lipids[END_REF], peptides [START_REF] Lúcio | [END_REF]89], and polymers [90]. These stud- A blue emission is typically associated with a non-polar solvent or environment [START_REF] Parasassi | [END_REF],

ies
whereas a red shifted emission is characteristic of polar solvents [94], with maximum emission wavelength of 440 nm and 490 nm, respectively. Due to the lauric tail, Laurdan is easily solubilized within lipid bilayers, with the naphthalene moiety located towards the headgroup -water interface (Fig. 2.11).

When the lipids are in a gel-phase, the emission maximum of Laurdan is centered at 440 nm and when the lipids are in a liquid-crystalline phase the emission maximum is centered at 490 nm. This spectral shift is the result of the dipolar relaxation of Laurdan in the lipidic environment. The origin of this dipolar relaxation has been attributed to a few water molecules present in the bilayer at the level of the glycerol backbone, where the Laurdan naphthalene moiety resides [94].

The shift from blue to red emission can be therefore used to probe phase transition in single and multicomponent systems, as the emission spectra reflect packing structure of the bilayer and local hydration. Conversely, analysis of the excitation spectra and emission anisotropy are as important as the emission, since they provide additional information on the bilayer phase and fluidity.

Emission spectra

The shift of the emission maximum of Laurdan can be quantified using the general polarization (GP), defined as [START_REF] Valeur | Santos in Molecular Fluorescence[END_REF]94]:

GP = I 440 -I 490 I 440 + I 490 (2.13)
where I 440 and I 490 are the emission intensities at 440 and 490 nm respectively, albeit other possible wavelength can be chosen [85]. Theoretically, GP can assume any values between 1 and -1, however experimentally typical values for gel phase are ∼ 0.5 -0.6 and ∼-0.3 --0.2 for liquid phase [START_REF] Parasassi | [END_REF][95][96][97]. Plotting the GP values over the range of temperatures investigated (Fig 2 .12) allows information on the phase behavior of the lipid bilayer, as in the case of the broadening of the GP transition in DPPC vesicles induced by the presence of cholesterol.

Gratton and Parasassi [98] firstly showed the significance in GP analysis of choice of the excitation wavelength. For a single liquid crystalline phase, excitation in the red band of absorption yields lower GP values compared to spectra obtained from λ exc in the blue band (Fig. 2.13). Conversely, in case of S o -L α coexisting phases, excitation at ∼400 nm results in a higher GP compared to excitation at ∼350 nm.

In case of coexisting phase these differences can be explained by the preferential photoselection of one of the two phases under different excitation wavelengths. For a single liquid crystalline phase, Parasassi explained this phenomenon by considering the selection of energetically unfavorable configurations upon excitation in the blue band, which will result in a higher amount of blue emission of Laurdan [98]. FIGURE 2.13: GP plots for DOPC:DPPC 70:30 obtained by exciting the system at 350 nm (black squares) and 400 nm (orange circles). At low temperature the system display S o -L α coexistence and the GP is higher for the 400 nm excitation. At high temperatures the system transitions into a homogenous liquid crystalline phase, and the excitation at 400 nm yields a more negative GP.

Another method used to analyse Laurdan emission spectra is a spectral decomposition, used firstly by Lucio [START_REF] Lúcio | [END_REF] and subsequently used by Bacalum [99]. The principle behind this method is to represent the two emission bands of the dye using a Gaussian model for each, with center of each curve fixed at the maximum of emission for each phase (Fig. 2.14).

Chapter 2. Materials and Methods

Using this approach allows to quantitatively measure the portion of gel and liquid contribution in the overall spectra, and characterize the effects of any molecule on the bilayer in terms of the individual components. Using this method Lucio e al.

[88] showed that even in pure gel phase exists a fraction of 490 nm emission, that explains the experimental values lower than their theoretically counter part. This presence of liquid-phase signal in a pure gel phase membrane has been explained as being due to the presence of the relaxed excited states that are present in the bilayer at any phase. were developed to be sensitive to voltage changes. Di-4, however, was found to be very sensitive to lipid bilayer phase and cholesterol content, therefore has been used recently to study lipid packing in model membranes and cells [100][101][102][103].

Di-4-ANEPPDHQ

It has a λ exc in the blue region, which allows for direct imaging using a conventional single-photon confocal microscope. Moreover, it has a high fluorescence quantum efficiency and large stokes shift when bound to the membrane, but very little fluorescence when dissolved in water [104].

Much like Laurdan, Di-4-ANEPPDHQ is influenced by several environmental factors such as acyl chain saturation, charge of the head groups, presence of cholesterol, hydration of the membrane and presence of proteins.

Although both Laurdan and Di-4 are sensitive to membrane packing, their sensitivity has been found to be very different depending on the conditions. Di-4 is less sensitive to temperature phase changes than Laurdan, but it is more receptive to cholesterol content [105]. These differences in the environmental sensitivity have

Concluding remarks

The phase behavior of the lipid bilayer arises from different properties of the membrane and involves both thermodynamics of the interaction between the molecules, as well as structural changes occuring in the bilayer. In particular, in the case of multicomponent systems the use of complementary techniques is crucial in obtaining a comprehensive picture of the interaction, as changes may occur at different length scales and affect different properties of the membrane.

The techniques presented in this chapter provide an optimal platform to study the phase behavior in model membranes and they have been extensively employed, as they allow to probe different length scales, both from a thermodynamics and structural point of view.

3

Polystyrene in a single component lipid bilayer

The increasing amount of plastic present in ocean waters has become a major issue in recent years, with increasing concerns regarding the potential hazardous effects it may have on living systems. Annual production of plastic has reached almost 300 million tons per year, of which 5 to 13 million tons are estimated to reach the ocean waters by different means [106]. Despite initially being present in large size, external environmental factors like temperature, oxidizing radiation and bacterial degradation can reduce the plastic to micrometric and nanometric fragments [107]. Objects of this scale can easily enter the food chain via digestion and there is increasing evidence of plastic micro objects found in the marine life forms [108][109][110].

Presence of nanoobjects have not yet being reported, however the lack of evidence can be associated with difficulties in detecting such objects rather than absence of them. Besides plastic degradation, nanometer-size polymer particles are also produced industrially for specific research and technological applications, such as imaging, sensing, and preparation of nanocomposites [111]. The presence of nanosized plastics in research laboratories, industry, and the environment raises questions on their potential toxicity.

Studies have indeed shown that plastic nanoparticles can accumulate in the tissues of living organisms and disrupt their metabolism [112,113], and that size plays an important role in determining the accumulation of nanoparticles within the organism [113]. Moreover, the surface of plastic particles can adsorb and transport persistent organic pollutants, such as the hydrophobic polycyclic aromatic hydrocarbons, transforming the plastic particles into vehicles of toxic substances into the organism. However, a physicochemical characterization of the interaction between plastic nanoobjects and living organisms is still lacking, especially regarding the mechanisms of potential oxicity.t

The cell membrane is the first barrier encountered by any foreign object entering an Chapter 3. Polystyrene in a single component lipid bilayer organism, and therefore is the primary candidate of investigation in assessing possible toxicity of plastic nano fragments. In particular, the membrane phase behavior plays a crucial role in maintaining cell functionality, and even minute changes in membrane phase properties can result in a potential threat for the organism.

Several coarse-grained molecular simulation studies suggest that polystyrene (PS) may alter the phase behavior and properties of lipid membranes. Rossi et al. [114] described that the presence of PS oligomer and polymers changes the beding rigidity of POPC membranes and moreover affects the phase coexistence when cholesterol is present in the bilayer. In a continuation of the study, Bochicchio et al. [115] showed that PS stabilized phase coexistance in model bilayers. These results indeed point out to a potential hazard since protein functionality is partially controlled by the membrane organization.

However, experimental study on the effects of polystyrene in cell membrane and model membranes are still lacking. Jung et al. [116] reported that incorporation of styrene monomers into DODAB liposomes fluidifies the membrane and reduces the transition temperature of the membrane. Radlinska et al. [117] showed that charged polystyrene sulfonated chains of about 2500 monomers (equivalent to a radius of gyration of 10 nm) can penetrate within surfactant bilayers and alter their structural and elastic properties.

In this work we perform a systematic study on the effects of polystyrene oligomers (M n = 500 Da) on the phase transition of unsaturared lipids. We investigated the changes in transition using small neutron scattering, differential scanning calorimetry, and Laurdan fluorescence spectra to extract information on the structure and the thermodynamics. Moreover, we directly visualized the changes on the membrane at different scale lengths using Cryo-TEM and epifluorescence microscopy.

Results and discussion

In the following paragraph we will outline the main feature of the interaction between polystyrene and single component lipid bilayer composed of DMPC, DPPC and DSPC. For clarity, we will describe in detail experimental results for only one lipid and summarize the role of chain length in the interplay. Complete experimental results for all lipids investigated can be found in Appendix A.

3.1. Results and discussion 35

Estimation of the polymer lateral distibution

To provide for a benchmark for the area coverage of the polystyrene dispersed in the lipid bilayer, we considered first the purely two-dimensional confinement of a polymer in a fluid lipid bilayer.

Let us define σ P S the number of PS oligomers per unit surface. Given the area per lipid A l and the molar fraction f P S , one has

σ P S = 2 A l • f P S 1 -f P S (3.1)
We now consider the total area occupied by the chain monomers S con = N • s 0 , estimated by considering the projected area s 0 (Fig. If the polymer is dissolved in a good solvent in two dimensions, the polymer will assume a coil conformation [START_REF] Flory | Principles of Polymer Chemistry[END_REF]. The confined polymer area is calculated by considering a two dimensional self avoiding walk (SAW), which gives a total radius R coil [START_REF] Flory | Principles of Polymer Chemistry[END_REF][START_REF] Teraoka | Polymer solutions: An introduction to physical properties[END_REF]:

R coil = N 3/4 • b (3.2)
where b is the the polymer segment which was found to be b = 3.1 Å. In this case we obtained R coil P S = 11 Å, which yields a projected area of the polymer of S coil P S ∼ 380 Å 2 . P S . This will however increase the energy of the system providing for a propensity for PS to escape from two dimensional confinement.

By considering the values of area coverage obtained, we observe that already at f P S = 0.1 the area coverage exceeds the overlapping density, and the coils interact, as shown in Table 3.1.

Note also that samples with f P S = 0.2 and f P S = 0.3 are close to or above the complete monomer coverage σ P S •S con = 1, further supporting the existence of strong energetic reasons for escape from two-dimensional confinement.

SANS

We investigated the changes of the bilayer structural parameters upon incorpora- bilayer thickness observed for gel to liquid transition [121]. For the h-DPPC/PS system the inflection is suppressed, albeit less strongly compared to the gel phase.

tion
Morevoer, the decrease of intensity is comparable to h-DPPC liposomes, suggesting similar thicknesses between the two membranes. 3.2). To obtain the values for membrane thickness, the data were fitted in the 0.007< q < 0.12 interval, since at lower values data shows nonlinear behaviour (Fig The membrane thickness obtained for h-DPPC decreases with increase in temper-

Contrast matching

The information obtained using only D 2 O as a solvent provide scattering signal as an average of lipid/polystyrene systems, therefore it does not allow to distinguish the scattering due to the lipid bilayer from the scattering of the polymer. In order to estimate the distribution of polystyrene within the membrane, as well as the changes in the lipid bilayer structure, we acquired the scattering signal using a matched contrast for either the bilayer or the polymer (Fig. For DPPC liposomes at 20 • C, we still observe a non-negligible scattering signal (Fig. To further estimate the distribution of the polymer within the membrane we fitted the scattering curves using the Sasview software (www.sasview.org).

For samples at 20 In the case of 50 • C, due to the lack of a plateau at low q, we fitted the curves using a polydisperse lamellar model (equations A. The resulting fitting shows that the distribution of polystyrene within the bilayer is different between S o and L α phase. Specifically we observed that in the gel phase the polymer aggregates with a typical size smaller than the liposome, with a thickness smaller than the hydrophobic region thickness of the acyl chains. In the fluid phase the polymer distribution becomes uniform, suggesting the absence of aggregates or segregation. We also measured an increasing thickness of the PS for d62-DPPC:PS liposomes in the fluid phase suggesting an intercalation of the polymer in the acyl chains.

Cryo-TEM

Cryo-TEM allows for visualization of liposomes in their native state, with a reso- We measured an average thickness of 5.4 ± 0.5 nm for DOPC membranes and 5.2 ± 0.5 nm for DOPC:PS systems. The two values are comparable and therefore incorporation of PS does not produce significant changes in the membrane thickness for the fluid DOPC system.

lution
To verify that the loss of contrast is not due to de-focus of the sample or artifacts, we mixed the DOPC and DOPC:PS liposomes dispersion at 1:1 volume ratio. As shown in figure 3.8, the field of view shares liposome with contrasted bilayer and vesicles with blurred contrast. Since both vesicles are acquired at the same focus, the loss of contrast cannot be explained by any artifact and therefore must be due to the presence of the polymer within the bilayer. The changes in contrast are evident also in the intensity profiles of the two systems (Fig. 3.9). In pure DOPC liposome (Fig. We observe that the contrast between the two external bands and the middle one is highly reduced.

The differences in contrast can be associated with the presence of polystyrene due to the higher electron density of the benzene ring compared to the hydrocarbon chains, which would result in less intensity in the midplane region of the bilayer. Indeed, Jung et al. [123] showed that styrene monomers polymerized with a lipid membrane are observed as dark areas in Cryo-TEM acquisitions. Moreover, we observe that the loss of contrast is uniform over the whole vesicle membrane suggesting a homogeneous distribution of the polymer, in good agreement with our SANS scattering data for DPPC in the fluid phase.

Laurdan emission spectra

The typical emission spectra of Laurdan in DPPC bilayers with different percentages of polystyrene at different temperatures are shown in Fig. 3.10.

In pure DPPC we observe a shift of the emission maximum from 440 nm to 490 nm when temperature changes from 20

Intensity (A.U.) 20° C 35° C 42° C 50° C 60° C 20° C 35° C 42° C 50° C 60° C Intensity (A.U.) Wavelength (nm) 20° C 35° C 42° C 50° C 60° C Wavelength (nm) 20° C 35° C 42° C 50° C 60° C A B C D

General polarization

To further quantify the spectral changes of Laurdan for bilayers upon interaction with polystyrene, we calculated the general polarization of the emission curves (Fig.

Spectral decomposition

The spectral decomposition performed following the procedure described by Lucio [START_REF] Lúcio | [END_REF] in Chapter 2 is illustrated in Figure 3.12. The relative coverage of the 440 nm contribution was plotted for each temperature to further elucidate the effects Chapter 3. Polystyrene in a single component lipid bilayer of polystyrene in the lipid bilayer. For pure DPPC we observe a relative coverage of ∼0.8 at temperatures below T m , consistent with values reported [START_REF] Lúcio | [END_REF]. At higher temperatures the contribution sharply decreases and remains stable at ∼0.15 in the liquid phase. The persistence of a 440 nm contribution for temperatures in the liquid phase has been attributed before to the presence of relaxed excited states.

For increasing molar fraction of polymer we observe a higher 440 nm contribution in the spectra, up to ∼0.33 for f P S = 0.

.12: Area fraction of 440 nm contribution in DPPC liposomes over temperature at 0% (black squares), 10% (orange circles), 20% (blue triangles) and 30% (green inverted triangles) polystyrene molar fraction.

Chain length effect

The effects induced by polystyrene on the lipids are summarized in Figure 3. This suggests that the distribution of the polymer within the bilayer is uniform. The creation of two distinct phases in the membrane due to polystyrene would produce a strongly non linear effect on the Laurdan emission spectra as the ratio between the Laurdan sensitivity to chain packing arises from the different number of solvent molecules at the level of the glycerol backbone. The increase in GP therefore suggests that in the liquid phase the bilayer has lower number of molecules due to the presence of polystyrene, hinting a higher packing order of the lipid molecules.

However, in the case of a two dimensional confinement of the polymer in the membrane midplane, packing of the lipid would not be significantly affected as the contact between hydrophobic polymer and water molecules is minimal. Conversely, a tighter packing would arise from the presence of oligomers in the acyl chains closer to the glycerol backbone. Indeed, Pham et al. [124] reported that incorporation of hydrophobic monoterpenes increases the order parameter of the acyl chains, specifically the carbon groups closer to the interface. Similar results have been also found for benzyl alcohol.

In the case of polystyrene, the lack of a hydroxyl group prevents the polymer from being exposed to the water-acyl chain interface, however the observed shift in GP, indicative of a higher order of the chain closer to the headgroup region, suggest that at least portion of the oligomers is present in the acyl chain rather than only in the midplane. This picture is consistent with neutron scattering results reported by Richter et al. [125] in case of styrene monomers interacting with DMPC vesicle, where the distribution of the monomers was found to be a coexistence of molecules highly segregated in the midplane and molecules aligned with the hydrocarbon tails. In particular we observe a linear decrease of ∆H and increase of T 1/2 with respect to polymer content (Table A.1 and A.2), whereas T m does not vary significantly. Our results differ significantly from the trend reported by Jung for DODAB vesicle contaning ∼ 60% molar fraction of styrene monomers [116]. For styrene it was indeed reported that transition temperature would strongly decrease, whereas the enthalpic contribution would be only slightly diminished. However, the trend we observe for polystyrene oligomers is in agreement with the variation of T m observed for DPPC bilayers containing hydrophobic molecules [126,127].

DSC

Polystyrene fraction The variation of the thermodynamic parameters is observed for all lipid investigated. Changes of T m are independent of acyl chain length, as shown in Figure 3.15 We attempted to quantify the differences between different chain lengths using a simple descriptive model [128], for which the enthalpy ∆H for an unsaturated lipid gel-to-liquid transition can be described as:

∆H T m T 1/2 [% mol] kJ• mol -1 • C • C 0 38.
∆H = A(n -10) (3.3)

Results and discussion

51

where A is the enthalpic contribution of acyl chain pair and n is the acyl chain length.

This model, despite minimal, describes well the energy of transition in terms of chain length. We fitted the enthalpies for pure lipid system from our experimental DSC thermographs and obtained a value for A of 6.2 ± 0.4 kJ• mol -1 , in agreement with values found by Matsuki et al. [128].

We assume here that the incorporation of polystyrene changes the individual contribution A of a single acyl pair by a factor k. We can then describe the enthalpy of a lipid bilayer containing polystyrene with:

∆H = A(1 -k • f P S )(n -10) (3.4)
where f P S is the molar fraction of polymer within the membrane. The obtained values for the interaction parameter k shows an apporixmately linear dependence on the bilayer thickness (Fig 3 .16) of each lipid in the fluid phase [24], indicating that changes in enthalpic contribution correlate well with perturbations propagating through the whole membrane. Other quantities such as area per lipid and chain length correlate poorly with k. Although differences on the variation on the chain length cannot yet be fully explained, the overall trend of depression of the melting temperature and broadening of the transition are consistent with some of the trends reported for hydrophobe/lipid bilayer interactions. Wolka et al. [129] and Rolland et al. [126] reported that incorporation of penetration enhancers reduce the transition temperature of DPPC to 40 • C at 10% molar fraction of molecule, as well as a significant increase in the width of the transition peak. Similar results have been found for membrane containing flavonoids, with increasing hydrophobicity of the molecule producing a stronger effect [130,131]. Borsacchio et al. also reported similar behavior for incorporation of pheromones in DOPC bilayers [132].

The majority of strong effects reported show a significant drop in T m and/or ∆H at relatively small fraction of hydrophobes. Our calorimetric results instead show only strong effects on the enthalpy of the transition, but not for temperature, which is the primary effect to be expected for a bilayer containing hydrophobic contaminants.

However, the strong effects reported appear for single molecules of small molecular weight (< 300 Da). Indeed, Abboud et al. [133] reported that incorporation of hydrophobes of higher molar mass (∼ 400 Da) results in a smaller depression in the T m of DPPC liposomes. The size of the molecule has also been reported to play a role in the magnitude of the changes induced by hydrophobic molecules [134,135].

Our SANS experiments show that polystyrene in the gel phase partially aggregates into larger structures partially confined in the bilayer midplane. This would greatly inhibit the penetration level of the oligomers in the S o phase thus explaining the small decrease in transition temperature observed. We can therefore partially ascribe the differences in magnitude between our experimental data and the trends reported for other hydrophobes to the higher molecular weight and chain structure of the oligomer we utilized compared to smaller single molecules for which strong effects have been observed.

GUV

Giant unilamellar vesicles (GUVs) of DMPC and DMPC:PS (Fig. 3.17 we observe for both systems the appearance of phase separation, characterized by dark domains with no detectable fluorescence due to the partinioning of the fluorescent probe [65,138]. This is consistent with observation previously reported for membranes close to transition temperature [139]. However, in our observations the phase coexistence persists at higher temperature, albeit with lower area coverage of S o domains. Above 35 • C we do not observe any domains and the membrane appears homogenously in the fluid phase. Although the wide range of temperature displaying S o -L α coexistence, which has not been reported before, incorporation of polystyrene does not produce any significant changes in the membrane morphology or phase behavior at any probed temperature. Analysis of the ratio of phase separation for DMPC and DMPC:PS GUVs indeed shows that the phase coexistence region is not affected by the presence of polymer (Fig. 3.18). A possible explanation for the observed effects is that the typical time of rearrangement of the polymer distribution is much faster than for the lipid domains. Under this assumption, the polymer, upon variation of temperatures, would Chapter 3. Polystyrene in a single component lipid bilayer re-distribute across the membrane without affecting the rearrangement of the coexisting domains, thus not producing any appreciable effect on the macroscopic scale.

Conclusions

Our results indicate that low molecular weight polystyrene incorporated within the hydrophobic region of a lipid bilayer has a different distribution between the two bilayer phases. For the S o phase polystyrene is segregated in the membrane midplane, as shown by SANS scattering curves and Laurdan emission spectra. This confinement is due to the poor solubility of the polymer in the tightly packed acyl chain region. As the bilayer melts to the L α phase, the polymer distribution becomes more uniform, as probed by Cryo-TEM imaging, and intercalates between the acyl chains towards the water interface. The strong variation in GP in the fluid phase, as well the linear dependence of the Laurdan emission and transition enthalpy with polymer content changes strongly supports this scenario.

Incorporation of the polymer was also found to alter the gel-to-liquid main transition and the lipid packing of the membrane in the fluid phase, as indicated by Laurdan emission spectra. These changes can be ascribed to a general hydrophobe/membrane interplay where the the polymer preferentially partitions into the liquid phase, thus depressing the melting temperature of the lipid bilayer. The differences in packing between polymer-free membrane and bilayer containing polystyrene can be accounted for the changes of enthalpic contribution in the gel-liquid transition.

Our results are in good agreement with previously reported data regarding preferential partinioning of polystyrene and polymer distribution in the fluid lipid bilayer, and with the thermodynamic trends observed for other hydrophobic compounds.

The alterations of the membrane phase behavior do suggest a possible disruption of cell membranes, calling for studies on more complex and biologically relevant lipid systems. A step in this direction is attempted in the next chapter.

Materials and methods

Materials

Chloroform solution of DOPC (

Liposomal preparation

2.5 mg of lipid in chloroform were transferred to a glass vial, and organic solvent was evaporated using an argon stream until completely dried followed by 8h of vacuum pumping. For fluorescence measurements, the lipids were stained with 1% mol Laurdan in chloroform prior to evaporation. For Cryo-TEM experiments the multilamellar vesicles suspension was also extruded 21 times through a 100 nm diameter pore polycarbonate filter with an Avanti Mini-Extruder. Vesicles prepared in this way generally showed a narrow size distribution as confirmed with Dynamic Light Scattering (DLS).

Liposomes for SANS measurements were instead extruded using an Avanti Mini-Extruder. The sample was first extruded 21 times through 200 nm, subsequently 21 times through 100 nm and finally 21 times through 50 nm diameter pore polycarbonate filters.

Giant unilamellar vesicles preparation

GUVs were prepared by electroformation following the protocol introduced by Angelova [START_REF] Angelova | Faraday Discuss[END_REF]. Simply, 5 µl of 2 mg/mL solution of DMPC and polystyrene at the desired molar ration in chloroform. stained with 1% mol of diI, were spread on each cathode of a custom made electroformation stage. The stage was kept under vacuum for at least 1 hour to ensure complete evaporation of solvent and subsequently the lipid film was hydrated using the necessary solution (water or sucrose at different concentrations) at 55 • C.

We applied a sinusoidal electric field of 1 V peak-peak intensity at 10 kHz for 1 hour while keeping the sample heated above the transition temperature. The resulting GUV suspension was kept at 20 • C water bath to ensure complete stabilization of the sample. Vesicles were used on the same day of preparation.

Fluorimeter

emission spectra was performed with a Jobin Horiba FluoroMax equipped with a Peltier unit to control temperature. Excitation wavelength was set at 350 nm with a bandpass of 1 nm and emission was also recorded with slit of 1 nm. The solution was equilibrated at given temperature for 10 min before each acquisition. For each sample (n=3) we performed two cycles of heating and cooling.

General polarization (GP) was calculated using the standard expression provided by Parasassi [140]:

GP = I 440 -I 490 I 440 + I 490 (3.5)
where I 440 and I 490 are the intensity recorded at 440 nm and 490 nm respectively.

Differential Scanning Calorimetry

The calorimetry measurements were performed with high sensitivity differential scanning calorimeter (µDSC Setaram). The measuring cell was filled with the sonicated sample (MLVs at different concentrations of sucrose), while the reference cell was filled with the same sucrose solution. The temperature of the cells was changed with a constant rate (heating rate: 0.5 K•min -1 , cooling rate: 0.3 K•min -1 ). The system was equilibrated ∼ 20 min before each heating or cooling ramp. The analysis of DSC data was performed using OriginPro 9.0 (Northampton, Ma, USA). For each sample (n=2) we performed three cycles of heating and cooling

Optical Microscopy

Imaging of GUVs labelled with DiI was performed using a confocal laser scanning microscope Nikon Eclipse TE2000-E equipped with a Nikon camera. The objective was a Nikon 60x water immersion, NA 1.2 (Nikon). GUVs samples prepared at different sugar concentrations were initially swelled by diluting the external medium with ∼5% volume of pure water. The samples were kept at 5 • C for at least 2 hours after preparation to ensure complete transition to gel (also called S o ) phase. Prior to experimental observation, GUVs were kept at 20 • C for at least 1 hour to stabilize the temperature. 100 µL of a GUV solution were placed in a custom-made heating stage through which we could control the temperature of the sample. For each temperature, we stabilized the sample for at least 1 hour before acquiring images.

Cryo-transmission electron microscopy (Cryo-TEM)

A laboratory-built humidity-controlled vitrification system was used to prepare the samples for Cryo-TEM. Humidity was kept close to 80% for all experiments and the temperature was set at 22 • C. 5 µL of the sample was placed onto a grid covered by the lacey carbon film (Ted Pella), which was rendered hydrophilic via glow discharge (Elmo, Cordouan Technologies). Excess sample was removed by blotting with filter paper and the sample grid was vitrified by rapid plunging into liquid ethane (-180 • C). The grids were kept in liquid nitrogen before being transferred into a Gatan 626 Cryo-holder. Cryo-TEM imaging was performed on an FEI Tecnai G2 TEM (200 kV) under low dose conditions with an Eagle slow scan CCD camera.

Small Angle Neutron Scattering (SANS)

The samples were mounted in a 2 mm thick quartz cuvettes (Hellma). The small angle Neutron Scattering data were collected on the beam line D11 at the Institut Laue-Langevin (ILL) in Grenoble, France. 4

Changes in multi-component lipid bilayers upon incorporation of polystyrene

The phase behaviour of cell membranes is modulated by the lipid composition and cholesterol content of its lipid bilayer [65,141]. It has been reported that the functionality of many membrane proteins highly depends on the lipid-protein interactions, and factors such as hydrophobic mismatch, membrane fluidity, lipid head group and cholesterol content can affect the protein conformation and activity [START_REF] Simons | [END_REF]142]. Moreover, proteins have been shown to preferentially partition into a specific phase of the membrane. This interplay is crucial for cell viability, pointing to potential hazardous effects of plastic nanoparticles incorporating in the cell membrane, a concern that has become over the years an important focus of research. As briefly mentioned in Chapter 3, the effects of polystyrene on the phase behaviour of lipid membranes have been investigated, using coarse grained molecular dynamics simulations. Rossi et al. [114] showed that incorporation of 10 % in mass of polystyrene polymers (chain length = 100) disrupts the phase behaviour of DLiPC:DPPC:Chol lipid bilayers. In particular they demonstrated that the polymer preferentially partitions in the liquid crystalline phase and uniformly distributes within it. Moreover, upon interaction with PS, the L d -L o coexistence was stabilized upon increase in temperature, whereas in a polymer free bilayer the temperature change would induce a transition to a homogenous liquid disordered phase. The stabilizing effect of polystyrene was further confirmed by Bochicchio et al. [115] via coarse grained simulations.

Despite several simulation studies performed on the subject, experimental studies on the polystyrene interaction with membranes displaying liquid-liquid coexistence are lacking. Moreover, to date no studies, theoretical or experimental, are present on the interaction with membrane possessing liquid-solid phase coexistence.

In this work we expand our investigation of the interaction of polystyrene with lipid The increase of GP both at low and high temperature in the presence of polystyrene suggests that the polymer strongly partitions in the L d phase, as was indicated by the results of Chapter 3. The decrease of miscibility temperature cannot be solely explained by the contribution of PS to the GP, as a constant increase of GP would produce a higher miscibility temperature. therefore variation of T misc are due to polystyrene and its ability to reduce the T m of DPPC.

Excitation wavelength dependence

Further information on the phase coexistence of the bilayer can be obtained by probing Laurdan emission spectra at different λ exc . For all composition we observe that the excitation at 400 nm results in a higher GP values compared to 350 nm for temperatures below melting. This has been previously reported to be a clear indication of phase coexistence between two states with different packing order [98]. Conversely, in the fluid phase, exciting in the red band would produce more negative GP values, which we observe for all systems above T misc .

Incorporation of polystyrene therefore does not disrupt the phase coexistence, however the variation of GP in presence of polymer is smaller compared to DOPC:DPPC system. This variation suggests a lower amount of gel phase coexisting in the membrane, possibly as a result of the drop in T misc observed. For vesicle composed only of DOPC:DPPC we observed a total area coverage of 0.17 A reduction of gel phase domains in GUVs has been reported before by Ostroumova et al. [145] for DOPC:DMPC GUVs interacting with flavonoids, and the effect was found to be stronger for more hydrophobic molecules. The reduction of S o area coverage, indicative of a depression of the melting point of the gel phase, has been commonly observed for small hydrophobic molecules incorporated within the lipid bilayer, and it has been ascribed to a preferential partitioning of the hydrophobe towards the L α phase [146].

DOPC:DPPC phase diagram

We compared the melting temperature obtained from the Laurdan emission spectra for DOPC:DPPC liposomes with previously reported phase diagram for the binary mixture. Our result for polymer-free membranes are in good agreemnt with values of the liquidus line obtained by Chen [143] using GUVs.

f g (T ) = X a (T ) -X X b (T ) -X a (T ) (4.1)
where X is the DPPC molar fraction of the sample, and X a (T ) and X b (T ) are the DPPC molar fraction corresponding to a temperature T in the liquidus and solidus lines, respectively. Values of area fraction at different molar fractions of DPPC are summarized in Table 4.1, and are consistent with values previosuly reported [143]. We compared our experimental results with a thermodynamics model for binary phase diagrams developed by Wolff et al. [144]. Simply, for a binary lipid mixture in a mean-field Ising model the gel-to-liquid transition can be written as:

G l (T,φ 1 , φ 2 , m) = -m[h 1 (T )φ 1 + h 2 (T )φ 2 ] -2Jm 2 + ( 1 + m 2 )ln( 1 + m 2 ) + ( 1 -m 2 )ln( 1 -m 2 ) (4.2)
with

h 1 (T ) = ∆H 1 2RT 2 1 (T -T 1 ) h 2 (T ) = ∆H 2 2RT 2 2 (T -T 2 ) (4.3)
where φ 1 and φ 2 are the area fractions of the two lipid species, J is the energy associated with the interaction between two neighboring lipids, m is a scalar order We calculated the number of domains in GUVs 1 hour and 36 hours after preparation to evaluate the coarsening and coalescence of domains, which are related to the mobility of the lipids in their respective phase. Domain coarsening is expected to be relatively fast, in the order of minutes, as probed by many studies on supported bilayers and GUVs. However, beyond the diffusion limit, L o domains can still coalesce, albeit at a smaller rate. In the case of DOPC:DPPC:Chol, we observe that the number of L o domains steadily decreases over a large period of time, favoring a complete phase separated membrane. Due to the time scale, this effect is associated with coalescence rather than growth or coarsening.

Incorporation of polystyrene significantly changes the domains behavior, as observed in Figure 4.9 B. Already after 1 hour we observe a significant increase in the average number of of domains, with prevalently smaller domains (Fig. 4.9 B). After 36 hours of stabilization the coalescence is greatly inhibited, with an average number of domains larger than the average obtained for DOPC:DPPC:Chol ( The presence of a larger number of domains might indicate that PS prevents large scale domains from merging together. Therefore polystyrene appears to inhibit the coalescence of L o domains. These results are in good agreement with simulations performed by Rossi [114] and Bochicchio [115], which reported that polystyrene can stabilize the phase coexistence of liquid/liquid membranes. In the case of systems containing both cholesterol and polystyrene, the characteristic emission for the L o phase is unaffected, centered at 589 ± 5 nm (Fig. 4.10 A). However the L d phase shows a shift in emission maximum to smaller wavelength, at 619 ± 2 nm (Fig. 4.10 B). This variation indicates that the polymer preferentially partitions into the liquid phase, as suggested by our results in single and binary systems, as well by Rossi et al. using molecular dynamics simulation [114]. In particular, such a shift in the emission maxima has been reported by Szegin et al. [147] for DOPC membrane containing cholesterol and is associated with a higher order of the bilayer. The emission shift we observe in presence of polystyrene might be therefore associated with a higher ordered liquid phase, as observed in the case of single lipid membranes in Chapter 3. In particular we can observe that at least part of the polymer chains are aligned with the lipid acyl chains. Di-4 dipole moment resides, when incoporated in a lipid bilayer, at the level of the acyl chains, and therefore is more sensitive to presence of molecules in the hydrophobic regions compared as to Laurdan. Indeed it was shown that compared to Laurdan, Di-4 is highly insensitive to temperature changes [105],

but more susceptible to cholesterol content. Therefore variations in the emission spectra indicate presence of molecules in the acyl chain region, consistently with the results we obtained in single component membranes (Chapter 3).

Conclusions

We observed that polystyrene significantly alters the phase behavior of multicomponent lipid bilayers with a different effect depending on the presence of cholesterol.

In the case of binary system the presence of polymer shifts the S o and L α miscibility towards lower temperatures, due to the similar order between the two phases induced by polystyrene. This behavior is similar to the reported phase behavior alteration of hydrophobic molecules incorporated in the hydrophobic region of the lipid bilayer which, by preferentially partitioning in the fluid phase, broadens the main phase transition and reduces the melting temperature. Our results are in good agreement with inhibition of membrane phase coexistence previously reported. However in our case, insertion of PS does not change the transition temperature of single component lipid membranes.

Moreover we observed that polystyrene partitions preferentially in the liquid disordered phase with less cholesterol, consistently with molecular simulations and experimental studies on hydrophobic molecules incorporated in membrane containing cholesterol. The presence of the polymer in membranes displaying liquid ordered domains stabilizes the lateral phase segregation and prevents the coalescence of domains.

Our results provide good agreement with previously reported studies and indicate that even a small amount of polystyrene might be potentially dangerous for biological membranes, specifically due to its antagonist behavior with cholesterol.

Liposomal preparation

2.5 mg of lipid in chloroform were transferred to a glass vial, and organic solvent was evaporated using an argon stream until completely dried followed by 8h of vacuum pumping. For fluorescence measurements, the lipids were stained with 1% mol Laurdan in chloroform prior to evaporation. The lipid film was then hydrated with aqueous solution (buffer or sucrose solution) at 70 • C to reach desired concentration and gently vortexed. Resulting MLV suspensions were sonicated for 15 min to disperse larger aggregates. Liposomal solutions remained stable over a period of days.

Giant unilamellar vesicles preparation

GUVs composed of DOPC:DPPC and DOPC:DPPC:Chol, both in absence of presence of polystyrene, were prepared by electroformation following the protocol introduced by Angelova [START_REF] Angelova | Faraday Discuss[END_REF]. Simply, 5 µl of 2 mg/mL solution of DOPC:DPPC or DOPC:DPPC:PS at the desired molar ratio, stained with 1% mol of diI, in chloroform were spread on each cathode of a custom made electroformation stage. The stage was kept under vacuum for at least 1 hour to ensure complete evaporation of solvent and subsequently the lipid film was hydrated using the necessary solution (sucrose at different concentrations) at 55 • C.

We applied a sinusoidal electric field of 1 V peak-peak intensity at 10 kHz for 1 hour while keeping the sample heated above the transition temperature. The resulting GUV suspension was kept at 20 • C water bath to ensure complete stabilization of the sample. Vesicles were used on the same day of preparation.

For GUVs stained with Di-ANEPPDHQ, a solution of the fluorescent probe in water was added to the GUV suspension to reach a 1% molar ratio with respect to lipids and incubated above melting temperature for 30 min in absence of illumination.

Chapter 4. Changes in multi-component lipid bilayers upon incorporation of polystyrene Fluorimeter 3 mL of liposomal suspension stained with Laurdan of total concentration 3 mg/mL was placed in a quartz silica cuvette with 1 mm path length. Acquisition of Laurdan emission spectra was performed with a Jobin Horiba FluoroMax equipped with a Peltier unit to control temperature. Excitation wavelength was set at 350 or 400 nm nm with a bandpass of 1 nm and emission was also recorded with slit of 1 nm. The solution was equilibrated at given temperature for 10 min before each acquisition.

For each sample (n=2) we performed two cycles of heating and cooling.

General polarization (GP) was calculated using the standard expression provided by Parasassi [140]:

GP = I 440 -I 490 I 440 + I 490 (4.4)
where I 440 and I 490 are the intensity recorded at 440 nm and 490 nm respectively.

Optical Microscopy

Imaging of GUVs labelled with DiI was performed using a confocal laser scanning microscope Nikon Eclipse TE2000-E equipped with a Nikon camera. The objective was a Nikon 60x water immersion, NA 1.2 (Nikon) using an excitation line of 543 nm with a mercury lamp. For GUVs labelled with Di-ANEPPDHQ the conventional three-band filter was substituted with a 20/80 R/T filter to perform spectral imaging, using a 488 excitation line Ar laser.

GUVs samples were initially swelled by diluting the external medium with a slighly ipoosmotic glucose solution (<10 mosm/Kg) to also facilitate the fall of the vesicles on the glass surface.

DOPC:DPPC samples were kept at ambient temperature • C for at least 2 hours after preparation to ensure complete stabilization of domains. 100 µL of a GUV solution were placed on a borosilicate glass slide and sealed with a silicon spacer and an additional glass slide to prevent any leakage or solvent evaporation.

For each lipid composition of binary mixtures we acquired images from two (n = 2) separate samples of 40 vesicles each, and for mixtures containing cholesterol two separate samples (n = 2) of 70 vesicles each.

DPPC bilayers in solution of high sucrose content

Disaccharides and sugars in general play a key role in preserving the structure and the functionality of biological membranes during period of environmental stress [148]. Many reports have shown that membranous structures are particularly stabilized by small sugars [149,150].

Besides their significant role in cellular regulation, carbohydrates have also a broad range of applications in biophysics and industrial research, particularly in the field of biopreservation and cryopreservation. Their interactions with membranes have therefore been the focus of many studies.

Some sugars such as sucrose and trehalose are very efficient cryprotectors [149,151,152]. They have shown to readily reduce the liquid-gel transition temperature T m in highly dehydrated lipid bilayers, and to increase survivability of membrane undergoing freeze/thawing processes. While this mechanism was initially associated with the ability of disaccharides to insert between adjacent lipid head groups during dehydration and hydrogen bond to them, an alternative model has been proposed that explains the observed effects in terms of the sugars' changes on the hydration repulsion [START_REF] Lenné | The journal of physical chemistry[END_REF].

Moreover, it has been proven that sugars play a role in the properties of hydrated bilayers. Dobereiner et al. [154] obtained a strong influence of glucose on the spontaneous curvature of liposomes. Genova et al. [155] showed by fluctuation analysis that high concentration of sucrose reduces the bending modulus of SOPC giant vesicles up to 25%, whereas Vitkova [156] found 60% reduction using micropipette aspiration. Nagle [157] showed as well by X-ray scattering that the bending rigidity of DOPC bilayers is reduced, although it should be noted that recently the opposed effect has been reported [158].

Chapter 5. DPPC bilayers in solution of high sucrose content

Characteristic chain melting temperatures T m of phospholipid dispersions was found to increase as the activity of water decreases in the presence of increased solutes [159,160]. Strauss et al. [160] found that addition of more than 10% sucrose to hydrated multilamellar vesicles of DPPC elevated the melting temperature; they suggested a hydrogen bonded sucrose network as the cause. Crowe and Crowe [161] found that several mono-and disaccharides raise T m and broaden the main transition of DPPC (MLV). However, addition of sugars to unilamellar vesicles created multiple thermodynamic populations. High concentrations of trehalose, sucrose, and fructose created a low-temperature shoulder on the DPPC endotherm, indicating a second population with a lower T m than that of pure hydrated DPPC.

Despite the many effects of sugars on dry, semi-dry and hydrated bilayers, relatively few studies have been conducted in order the understand the main effect of disaccharide on the lipid bilayer phase transition, and the mechanisms of interaction are yet to be understood. While there is some agreement that high concentration of sugar increases the transition temperature of the bilayer melting, the effect on the enthalpic contribution are quite discording, reporting in certain cases no effect on the enthalpy of the transition, and in other studies a significant decrease of the energy.

In this work we expose well hydrated bilayers of DPPC to high concentrations of sucrose and investigate the effects on the membrane phase behavior for increasing concentrations of sucrose using a combination of differential scanning calorimetry and Laurdan emission spectra to obtain structural and thermodynamical information. We also utilize giant unilamellar vesicles to visualize changes in phase behavior and kinetics of transition. Based on our experimental observation we propose a thermodynamical model for the lipid bilayer -sugar interaction to explain our results and the ones reported by previous studies. As temperature was increased above the transition temperature of DPPC, we observe a red emission shift with maximum at 490 nm, with a decrease of total intensity and a broader emission spectra (Fig 5 .1 B). Such a decrease of intensity has been reported before [97]. Increasing concentration of sucrose results in persistence of a 440 nm shoulder in the emission spectra. This additional contribution is found to be dependent on the sucrose concentration and remains at any temperature above T m . Laurdan emission centered at 490 nm is attributed to a more relaxed Laurdan excited state, which would be favoured by a more hydrated environment [95,162].

Results and discussion

Laurdan emission spectra

Chapter 5. DPPC bilayers in solution of high sucrose content

Conversely, contribution at 440 nm reflects a stiffer and/or less hydrated system surrounding the Laurdan naphthalene moiety.

To further quantify the spectral changes, we calculated the GP from each emission spectra using 440 nm and 490 nm as maximum emission for each phase. In the liquid phase, above T m , the GP curve shows a significant increase of GP values 5.1. Results and discussion 79 with higher sucrose concentration, with GP of 0.09 ± 0.01 and -0.22 ± 0.02 for T m and 60 • , respectively, at 1.5 M sucrose. Interestingly, the maximum variation can be observed for T m , and at higher temperatures the difference with respect to GP values of DPPC in pure water gradually decreases (Fig. 5.2 inset). The variation owed to the presence of sucrose appears to follow a linear dependence with sugar concentration in bulk for any analysed temperature Laurdan is insoluble in water, therefore any information from the emission spectra and GP arises entirely from the probe in the membrane. In lipid bilayers the fluorescent moiety of Laurdan is located at the level of the glycerol backbone and the emission shift upon changes in temperature is due to a dipolar relaxation process [162]. For MLVs formed in water, the red shift and decrease in GP generally associated with phase transition are caused by a change in lipid packing, as the more disordered membrane allows deeper penetration of polar solvent molecule in the interfacial region.

Variations of emission spectra and GP as the one showed in this study have been reported before in the case of high ionic strength or in the presence of cations in the solvent for DMPG liposomes [START_REF] Lúcio | [END_REF]163]. In these studies the effect was due to changes in lipid packing upon electrostatic attraction caused by ionic charges. Despite sucrose being highly polar, our results clearly show that when it is present in high concentration, the Laurdan emission spectra is similar to that of an apolar environment [97]. Conversely, increased GP values in presence of sugar are linked to a lower mobility of water or a lesser number of water molecules around the Laurdan naphthalene moiety.

Two possible mechanisms can be invoked to explain such an effect: i) A tighter lipid packing of the membrane, as observed for example in liquid ordered (L o ) phases in presence of cholesterol, ii) A depletion of water molecules at the lipid headgroup region which reduces the emission shift of Laurdan.

Simulations and previous experimental studies showed that the adsorption of sugar to the membrane leads to a lower number of water molecules around the lipid headgroup [152]. These results are consistent with our GP data that show a progressive reduction of water molecules surrounding the Laurdan napthalene moiety resulting in a higher GP values.

Differential scanning calorimetry

To better understand the changes in the bilayer structure due to the presence of sucrose, we performed differential scanning calorimetry on DPPC multilamellar vesicles (MLSVs) hydrated with water, 0.39 M, 0. The DSC profiles display a striking asymmetry under all conditions, with a sharp variation of intensity at higher temperatures and a smoother curve at low temperatures. This feature has been reported before and it is usually associated with instrumental imprecision, therefore is not characteristic of the transition itself [164].

The intensity of the peaks decreases with increasing amount of sugar, and the centre of the peaks shifts to slightly higher temperature when MLVs are in high sucrose concentration. Overall the curves remain sharp, indicating that the transition is still highly cooperative. Broadening of peaks is usually associated with disruption of the lipid packing and cooperativity [165], as it has been reported by Mannock [166] for cholesterol. In our experimental observations, such large broadening is not present, suggesting that the packing and the phase of the lipid bilayer remains the same at all sucrose content.

To quantitatively measure the variation on the transition, we extracted the enthalpy (∆H), transition temperature (T m ) and half intensity temperature interval (T 1/2 ) of the peaks. The results are summarized in Table 5.1. For system formed in pure water we obtained an enthalpic contribution of 38.5 kJ

• mol -1 and T m of 41.8 • C, well in agreement with previously reported values. In presence of sucrose we observe a significant drop of enthalpy, with a final value 23.9 kJ • mol -1 at 1.5 M sucrose. Such an effect of sucrose on the DPPC enthalpy has been observed before by Chowdry and Chen [167,168]. Our values are in good agreement with a variation of 10.9 kJ • mol -1 for 1 M sucrose observed by Chowdry.

Chen, however, reported a drop of 11.6 kJ • mol -1 at already 0.2 M sucrose, which we do not observe. Surprisingly, our results show a nonlinear trend for enthalpy change with respect to sucrose concentration (Fig 5 .3 B). The enthalpy reaches a steady state at 1.5 M, suggesting a saturation of the sucrose effect on the membrane transition.

This saturation could be linked to a maximum adsorption of sucrose at the surface, as it was observed with ESEEM measurements by Konov [169], which observed a Langmuir adsorption of sucrose for DPPC bilayers.

The transition temperature for DPPC with sucrose is slightly increased, with final value of 42.7 • C for 1.5 M of sucrose. These changes in T m , albeit small, are in good agreement with an increase of 0.6 Many studies report variations of T m of lipid bilayer upon interaction with macromolecules located either at the surface or in the hydrophobic region of the membrane.

An increase of the transition temperature is generally linked to a tighter packing of the lipids, which can arise from many reasons. In this study, we argue that the observed shift of T m to higher temperature is due to a dehydration of the lipid bilayer. Sugar molecules, by accumulating on the membrane surface, substitute water molecules from the outer most hydration layer and increase the transition temperature. Our observation of Laurdan emission spectra indicates a decrease of the number of water molecules at membrane interface.

Gabrielle-Delmont [172] reported for DPPC vesicles an enthalpy drop and T m comparable to the value obtained in this study in the case of 20% water content. However, under our experimental conditions water content has been calculated to be ∼60% for 1.5 M sucrose. Moreover the DSC curve does not diplay a high temperature contribution reported for lipid bilayers under low hydration conditions. Therefore simple dehydration or substitution of water molecules by sucrose is not enough to explain our experimental data.

Finally, we observe a slight increase of T 1/2 , consistently with values reported by Chen [168].

Giant unilamellar vesicles

We observed giant unilamellar vesicles of DMPC to probe the micrometric phase Our observation for pure DMPC GUVs reveals a large temperature interval of phase The disappearance of phase coexistence for temperatures above T m can be interpreted as a lowering of the energy involved with gel-liquid contact. The size of gel domains in the liquid phase is mainly driven by the minimization of the energy involved with hydrophobic mismatch (such as a S o /L d coexistence) [173]. Several studies have proposed that sucrose can alter the head-head distance in lipid bilayers under low hydration conditions [149,174]. Sucrose can therefore act as a reliever of hydrophobic mismatch allowing thus for smaller domains existence, by covering the portion of chains exposed by the mismatch. Therefore, it is possible that the presence of sugar can enhance the dissolution of domains or reduce the time scale of meta-stable states.

Thermodynamics model

Based on our experimental observations we attempt to derive a thermodynamics description of the interaction between sucrose and the DPPC lipid bilayer. In discussing the interaction between dry bilayers and sugars, a frequent model is the water replacement hypothesis (WRH), which postulates direct hydrogen bonding between sugars and phospholipids [149]. By creating H-bonds with lipid headgroups, sugars keep lipids separated in the liquid phase upon dehydration. In some case the hypothesis considers the sugars to be able to penetrate in the interfacial region of the membrane, and therefore keeping the lipids apart. Although our data do support the removal of water molecules from the membrane, the WRH model cannot explain both increase in T m and decrease of enthalpy.

Another proposed model is the hydration forces explanation (HFE) [175,176], which assumes no direct interaction between sugars and lipids. Sugars' non-specific volumetric and osmotic effects are the cause of changes in the bilayer phase transition.

This model has been able to quantitively predict changes in the transition temperature [START_REF] Lenné | The journal of physical chemistry[END_REF]177], however the variations of enthalpy reported here and in previous studies have not been accounted for.

A full thermodynamics model that would fully explain the observed phenomena must include: i) an increase of transition temperature, ii) a significant drop in enthalpy and iii) a decrease of the cooperativity of the transition.

We propose a model in which the adsorption of sucrose at the membrane surface locally dehydrates the lipid bilayer, resulting in the formation of clusters with an intrinsic different transition temperature. As phase transition occurs only a portion of lipid participates, resulting in a lower enthalpic contribution and a higher effective melting temperature, coupled with broader peak.

To physically describe this model, we use a formalism previously used by Heimbourg [178] and others [179,180] to succesfully describe the gel-to-liquid transition.

This description is akin to an Ising spin model, where however the spin represents the phase of the lipid, and a full derivation of the model can be found in Appendix C. Following this formalism, the Gibbs free energy for a bilayer can written as

∆G = n l (∆h -T ∆S) -Jn gl (5.1)
where n l is the number of lipids in the liquid phase, ∆h is the invidual lipid enthalpic contribution to transition, ∆S is the enthropic contribution, J is the cooperativity energy and n gl the number of gel-liquid lipid pairs in contact with each other. We assume that sucrose, by adsorbing to the membrane, can change the number of water molecules surrounding a lipid and that this process is not necessarily homogenous. Effectively this will affect the Gibbs free energy and T m of a lipid so that we have a new ∆G for lipid interacting with sucrose:

∆G = n l (∆h -T ∆S) -J n gl (5.2)
and the total energy of the system can be then described by

∆G tot =n l (∆h -T ∆S) + n l (∆h -T ∆S) - -J n gl -Jn gl -J n gl (5.3)

Results and discussion

87

where Jn gl represents the free lipid-free lipid contact, J n gl the free lipid-bound lipid contact and J n gl the bound-bound contact. The number of lipids in the gel or liquid state will depend thus on the site coverage of sucrose σ and the probability p for a lipid to be in a specific state. We have then

n l = N • (1 -σ) • p l n g = N • (1 -σ) • (1 -p l ) (5.4)
for free lipids and

n l = N • (1 -σ) • p l n g = N • (1 -σ) • (1 -p l ) (5.5)
for bound lipids. To estimate the cooperativity term we use a mean field approach

n gl = (1 -σ) 2 • ηN 2 • 2p l (1 -p l ) n gl = 2σ(1 -σ) • ηN 2 • [p l (1 -p l ) + p l (1 -p l )] n gl = σ 2 • ηN 2 • 2p l (1 -p l ) (5.6)
Based on this we can write the total enthalpy as ∆H = ∆hp l + ∆h p l + J tot (5.7)

where J tot is the sum of all enthalpic cooperativity terms. We should point out that in this study we choose to consider the cooperativity term as an enthalpic term. This choice is arbitrary as the formalism has a degree of freedom in choosing the ratio of enthalpic and enthropic contribution for J tot . We performed numerical simulation using our model with the following parameter: T m = 314.95K, T m = 319.95K, J = 1.94; J = J = 0.97,

N A ∆h = N A ∆h = 56 kJ • mol -1 .
By comparing the values of ∆H obtained from DSC and theoretical predictions, we obtained the dependence of the area coverage on the sucrose concentration (Fig.

Conclusions

In this study we have shown that sucrose can alter the gel-liquid phase transition of DPPC lipid bilayers, increasing its T m and lowering the enthalpy of the transition.

These results confirm and expand the observations reported in previous studies concerning the effects of sugars in well hydrated lipid bilayers, with possible implications in the field of byopreservation. We grouped the observed effects from different techniques as a result of a local dehydration of lipids due to high concentration of sucrose near the membrane, with a formation of small lipid clusters that would melt at a different temperature than T m . This interaction is successfully described by a simple thermodynamic model that takes into account the two populations that undergo phase transition at different temperatures, and provides excellent agreement with our experimental data.

Materials and methods

Materials

Chloroform solution of DPPC (

Liposomal preparation

2.5 mg of DPPC in chloroform were transferred to a glass vial, and organic solvent was evaporated using an argon stream until completely dried followed by 8h of vacuum pumping. For fluorescence measurements, the lipids were stained with 1% mol Laurdan in chloroform prior to evaporation. The lipid film was then hydrated with aqueous solution (buffer or sucrose solution) at 70

Giant unilamellar vesicles preparation

GUVs were prepared by electroformation following the protocol introduced by Angelova [START_REF] Angelova | Faraday Discuss[END_REF]. Simply, 5 µl of 2 mg/mL solution of DMPC stained with 1% mol of diI in chloroform were spread on each cathode of a custom made electroformation stage. The stage was kept under vacuum for at least 1 hour to ensure complete evaporation of solvent and subsequently the lipid film was hydrated using the necessary solution (water or sucrose at different concentrations) at 55 • C.

We applied a sinusoidal electric field of 1 V peak-peak intensity at 10 kHz for 1 hour while keeping the sample heated above the transition temperature. The resulting GUV suspension was kept at 20 • C water bath to ensure complete stabilization of the sample. Vesicles were used on the same day of preparation.

Fluorimeter

3 mL of liposomal suspension stained with Laurdan of total concentration 3 mg/mL was placed in a quartz silica cuvette with 1 mm path length. Acquisition of Laurdan emission spectra was performed with a Jobin Horiba FluoroMax equipped with a Peltier unit to control temperature. Excitation wavelength was set at 350 nm with a bandpass of 1 nm and emission was also recorded with slit of 1 nm. The solution was equilibrated at given temperature for 10 min before each acquisition. For each sample (n=3) we performed two cycles of heating and cooling.

General polarization (GP) was calculated using the standard expression provided by Parasassi [140]:

GP = I 440 -I 490 I 440 + I 490 (5.8)
where I 440 and I 490 are the intensity recorded at 440 nm and 490 nm respectively.

Differential Scanning Calorimetry

The calorimetry measurements were performed with high sensitivity differential scanning calorimeter (µDSC Setaram). The measuring cell was filled with the sonicated sample (MLVs at different concentrations of sucrose), while the reference cell was filled with the same sucrose solution. The temperature of the cells was changed with a constant rate (heating rate: 0.5 K•min -1 , cooling rate: 0.3 K•min -1 ). The system was equilibrated ∼ 20 min before each heating or cooling ramp. The analysis of DSC data was performed using OriginPro 9.0 (Northampton, Ma, USA). For each sucrose concentration, three consecutives heating/cooling cycles were performed in two separate samples.

Optical Microscopy

Imaging of GUVs labelled with DiI was performed using a confocal laser scanning microscope Nikon Eclipse TE2000-E equipped with a Nikon camera. The objective was a Nikon 60x water immersion, NA 1.2 (Nikon). GUVs samples prepared at different sugar concentrations were initially swelled by diluting the external medium with ∼5% volume of pure water. The samples were kept at 5 • C for at least 2 hours after preparation to ensure complete transition to gel (also called S o ) phase. Prior to experimental observation, GUVs were kept at 20 • C for at least 1 hour to stabilize the temperature. 100 µL of a GUV solution were placed in a custom-made heating stage through which we could control the temperature of the sample. For each temperature, we stabilized the sample for at least 1 hour before acquiring images.

6

Interaction of cholesterol grafted polymer with lipid membranes

Traditional cancer therapeutics are designed to interact with proteins and nucleic acids to treat the tumoral cells. However, in recent years, development of scientific tools concerning lipids and lipid metabolism opened new aspects of tumor cells understanding, indicating possible differences in lipid composition and membrane function of tumor cells compared to healthy cells [181]. The development of lipidomics therefore has postulated that modulation of, or interaction with, lipids could change lipid composition, membrane properties or alter the properties in tumoral cells to interfere specifically with cancer cell membranes [182]. Indeed, proteins like integrins, adherins and receptors, involved in tumor progression and invasion, are located in lipid rafts [183,184] and their abundance in tumor cells are linked with a higher invasive potential and a decreased fluidity of membranes [185].

The functionality of proteins interacting with the membrane is often modulated by the host bilayer [186,187]. The regulation induced by membrane lipids may occur through chemical interaction between proteins and individual lipid molecules, and to non-specific interaction due to the collective physical properties of the lipid bilayer [188,189]. In particular, membrane parameters such as thickness, fluidity, charge, and curvature have been found to regulate the folding, activity and stability of membrane proteins [142].

Cholesterol, a lipid present in all eukaryotic cells, has been found to significantly affect the activity of membrane proteins and ion channels, due to its relevant properties in affecting both the hydrophobic mismatch and fluidity in lipid model membranes [190].

Recently, a newly developed cholesterol grafted polymer has been synthetized as an effort to develop a delivery agent that could alter the membrane fluidity and cholesterol content in a triggerable manner (Chen, data not published). The grafting of cholesterol on the PLP backbone therefore inhibits the pH sensitive feature of the polymer, which remains in a globular or less extended conformation even at neutral pH. This is caused by the low grafting percentage (15%) of the lipid to the poly(L-lysine isophthalamide) backbone. The pH-mediated conformational change observed in amino acid grafted polymers is regulated by the balance between the hydrophobic moieties and the charged carboxyl groups, with the hydrophobic backbone with carboxyl groups undergoing random-coil to globular conformational transitions [191].

GUVs formed in PPCHOL solution

We compared the phase of GUVs for DOPC:DPPC 6:4 formed in PBS (Fig. Vesicles formed in polymer-free buffer display clear phase coexistence between S o and L α domains. Gel domains appear as dark spot due to the preferential partinioning of the fluorescent probe in the fluid phase [147]. Analysis of the emission spectra shows a single emission maximum located at 648.3 ± 8.3 nm, a value consistent with Di-4 spectra reported for pure DOPC membranes [147].

Formation of vesicles in 500 µg/mL of polymer results in significant changes in the membrane phase behaviour. The bilayer displays homogenous fluorescence intensity with no visible domains (Fig. 6.3 B). The lack of domains indicates an increase Chapter 6. Interaction of cholesterol grafted polymer with lipid membranes

We argue that the presence of the polymer and of cholesterol in the membrane reduce the hydrophobic mismatch between DOPC and DPPC, thus increasing the miscibility between the two lipids.

Conclusions

Our results show that the presence of the cholesterol-grafted polymer significantly disrupts the phase behavior of DOPC:DPPC membranes, inducing a mixing between the two lipids either by intercalation of cholesterol or polymer chains. The emission spectra obtained show that although mixing occurs at optical scale, two separate populations exists, close to the L o and L d observed for ternary lipid membranes containing cholesterol.

Albeit preliminary, our data suggest that grafting of membrane altering molecules on the backbone of polymers is a viable approach in developing therapeutic strategies for varying the properties of cell membranes.

Materials and methods

Materials

Chloroform solution of DOPC (

Giant unilamellar vesicles preparation

GUVs composed of DOPC:DPPC were prepared by gel assisted formation following the protocol introduced by Weinberger [194]. Simply, 5 µl of 2 mg/mL solution of DOPC:DPPC at the desired molar ratio in chloroform were spread a PVA coated borosilicate glass. The formation glass was kept under vacuum for at least 1 hour to ensure complete evaporation of solvent and subsequently the lipid film was hydrated using the necessary solution (PBS or PPCHOL in PBS) at 55 • C. Solution of Di-ANEPPDHW in PBS was added to the GUV suspension to reach a 1% molar ratio with respect to lipids and incubated above melting temperature for 30 min in absence of illumination.

Optical Microscopy

Imaging of GUVs was performed using a confocal laser scanning microscope Nikon Eclipse TE2000-E equipped with a Nikon camera. The objective was a Nikon 60x water immersion, NA 1.2 (Nikon) using an excitation line of 543 nm with a mercury lamp. The conventional three-band filter was substituted with a 20/80 R/T filter to perform spectral imaging, using a 488 excitation line Ar laser. DOPC:DPPC samples were kept at ambient temperature • C for at least 2 hours after preparation to ensure complete stabilization of domains. 100 µL of a GUV solution were placed on a borosilicate glass slide and sealed with a silicon spacer and an additional glass slide to prevent any leakage or solvent evaporation. For each lipid:PS composition we acquired images of two (n = 2) separate samples of 40 vesicles each.

Chapter 7. Conclusions and future outlook emission spectra showed an increase of the bilayer order with increasing molar ratio of polymer. The information on the bilayer was complemented with SANS measurements and Cryo-TEM imaging, which demonstrated that polystyrene is more segregated in the S o phase, and upon phase transition distributes homogenously in the lipid bilayer. The uniform distribution of the polymer within the membrane produces a tighter packing of the lipids due to the intercalation of the polymer in the acyl chains.

We further studied the potential hazard of small polystyrene chains on the cell membrane by investigating the effects of polystyrene on multicomponent lipid membranes. We demonstrated that for binary lipid systems, polystyrene induced a depression of the liquidus line between the S o /L α phase coexistence region and the fluid phase, as shown by Laurdan fluorescence spectroscopy and analysis on GUVs.

The shift in temperature of the phase diagram induced by the polymer was successfully described using a thermodynamics model, which showed that the reduction of enthalpic contribution can be ascribed as the main driving force. Moreover, it was demonstrated that polystyrene induces different alterations in mem- DSC. We successfully described the interaction using a thermodynamic model and performed numerical simulations that are in good agreement with our experimental data. Our results provide a novel interpretation on the sucrose/lipid interplay and is relevant in understanding interactions with other hydrophilic molecules.

Last, together with our ITN SNAL collaborators at the University of Cambridge, we started the characterization of the interaction between model lipid membranes and a cholesterol grafted polymer as a potential membrane altering agent. The molecule synthetized by our collaborators (named PPCHOL) has a cholesterol grafted to its pH-sensitive backbone, and due to this feature, it is believed to change the fluidity of the membrane upon adsorption at physiological pH, therefore it is a potential carrier for novel lipidomic therapeutic strategies. Our preliminary results show that the grafting of cholesterol inhibits the pH sensitivity of the polymer and decreases its solubility, as measured by DLS. In physiological conditions, however, formation of DOPC:DPPC giant unilamellar vesicles containing the polymer show a drastic increase of the miscibility between the two components. Moreover, the DI-ANEPPDHQ emission spectra, probed locally via confocal spectral imaging, show a unique signal which has never been reported before, that we interpreted as the coexistence of small clusters of L o and L d phases. This exciting result does prove that the polymer may act as a membrane altering system, and calls for further investigation of the interaction.

In conclusion, this Ph.D. thesis presents a comprehensive study of the effects of hydrophobic/hydrophilic molecules on the phase behaviour of lipid membrane.

We demonstrated that the combination of environment sensitive fluorescent probes, calorimetry and confocal imaging is a powerful set of tools to characterize variations in phase behavior, thus providing a clearer picture of the interactions taking place.

A.2 SANS disk model

This model calculates the form factor for a monodisperse right circular cylinder with uniform scattering length density. The form factor is normalized by the particle volume such that:

P (q) = scale V cyl π/2 0 f 2 (q, α) sin αdα (A.1)
where V cyl = πr 2 L is the volume of the cylinder, q is the scattering vector, α is defined as the angle between the cylinder axis and the scattering vector and the function f is defined as:

f (q, α) = 2∆πV cyl j 0 (qH cos α) J 1 (qr sin α) q 2 (qr sin α) (A.2)

with ∆ρ the scattering length difference between the sample and the solvent, j 0 (x) = sin(x)/x and j 1 is the first order Bessel function. The integral over alpha averages the form factor over all possible orientations of the cylinder with respect to q. Therefore θ can be written as:

θ = π/2 -(α 1 + α 2 ) (B.6)
given by: H(T ) = N (p l (N c , T )h l + p g (N c , T )h g ) = N (p l (N c , T )∆h + h g ).

(C.4)

where liquid-gel mismatch energy contributions are neglected and h l and h g are taken independent of temperature at the vicinity of the transition. Then

C.2 Phenomenology of the lipid main transition

A simple insightful treatment of the lipid main transition was introduced by Doniach, and improved by several authors [178][179][180][195][196][START_REF] Ivanova | Physical review. E Statistical nonlinear and soft matter physics[END_REF]. It is based on a scalar order parameter showing two preferred values, one corresponding to the gel state, and the other to the fluid state. This statistical model can be implemented in practice by assigning binary variables (Ising spins) to the fixed vertices of a two dimensional lattice. Reference [178] presents in detail the historical development of the model.

In this approach, lipid molecules spontaneously adopt either a gel or a fluid conformation, depending on external thermodynamic conditions (temperature, but also isotropic pressure and membrane tension). In the Ising language, this is achieved by applying a uniform temperature (pressure, tension) dependent magnetic field that vanishes precisely at the coexistence temperature T m , where fluid and gel are observed with equal probability. Additional nearest neighbour couplings (Js parameters) are introduced to enforce cooperativity. Above a critical J c value, the system shows phase coexistence and metastability, with hysteretic thermal behavior upon heating and cooling cycles. Below the critical value, the system state evolves FIGURE C.2: Enthalpy variation with temperature as σ increases from 0 to 0.5. We integrate the area under the specific heat curve from T m -5 to T m + 5.

FIGURE C.3:

Resulting enthalpy change vs 1 -σ (pure water at the right of the graph). We note that for the selected values, the ∆H curves seems initially to decrease linearly with 1 -σ. Mattia MORANDI Disruption of model membranes' phase behavior upon interaction with hydrophilic/hydrophobic molecules Résumé Ce travail concerne l'altération du comportement de phase de membranes lipidiques lors de leur interaction avec des molécules hydrophiles ou hydrophobes. L'utilisation de sondes moléculaires de fluorescence sensibles à leur micro-environnement constitue un aspect majeur de ce travail. Les techniques de spectroscopie de fluorescence et de microscopie confocale ont été mises à profit pour l'étude du comportement de ces sondes, donnant accès au degré de compacité et d'ordre dans les membranes.

Nos résultats montrent que le polystyrène, un plastique rencontré de façon commune dans les régions polluées des océans, présente la capacité de modifier le comportement de phase des membranes lipidiques, entrant notamment en compétition avec le cholestérol.

Nous avons montré que la présence élevée de sucres, tel que l'on peut le rencontrer dans certaines situations relevant de la bio-préservation, a pour effet de rompre la qualité de compaction des lipides, et nous avons proposé un nouveau modèle thermodynamique pour interpréter nos résultats.

Enfin, les effets sur la membrane de l'incorporation d'un polymère amphiphile comportant un cholestérol greffé ont été étudiés, dans le cadre de l'élaboration de nouvelles stratégies thérapeutiques à base de lipides.

Membranes modèles de lipides ; Transition de phase ; Coexistence de phase ; Polystyrène ; Cholesterol ; Saccharose ; Microscopie confocal ; Laurdan

Résumé en anglais

This work focuses on the alterations of lipid membrane phase behavior upon interaction with hydrophobic and hydophilic molecules. One major aspect of this thesis is the employement of environment sensitive probes to obtain information on the lipid bilayer packing by means of confocal spectral imaging and fluorescence spectroscopy.

Our results show that polystyrene, a commonly found plastic in ocean wastes, has the ability to disrupt the lipid bilayer phase behavior and has a competitive interaction with cholesterol.

The presence of high concentration of sugars, relevant in the field of biopreservation, has been found to alter the lipid bilayer packing and a new thermodynamics model has been proposed to complement the experimental results.

Finally, the effects of an amphiphilic cholesterol-grafted polymer on model membrane was investigated, providing insight into potential new lipid therapeutic strategies.

First

  and foremost, I would like to express my gratitude to the MCube team and all the colleagues of the Institut Charles Sadron. My three years at the institute have been constantly filled with research, learning and human relationships, and I have spent a wonderful time doing what I love.

ix xiv 3 . 1 111 xix

 31111 Prjected areas of polymer in 2-D confinement . . . . . . . . . . . . . . . 3.2 Schematics of the polymer distribution within the lipid bilayer . . . . . 3.3 SANS scattering curves for h-DPPC and h-dPPC:PS liposomes in D 2 O 3.4 Schematics of matching contrast conditions . . . . . . . . . . . . . . . . 3.5 SANS scattering curves for d62-DPPC and d62-dPPC:PS liposomes in lipid matched solvent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.6 SANS fitting of d62-DPPC:PS in lipid matched solvent . . . . . . . . . 3.7 Cryo-TEM images of DOPC and DOPC:PS 40:60 liposomes . . . . . . . 3.8 Cryo-TEM images of mixed DOPC/DOPC:PS 40:60 liposomes . . . . . 3.9 Bilayer intensity profiles of mixed DOPC/DOPC:PS 40:60 liposomes . 3.10 Laurdan emission spectra for DPPC at different polystyrene molar fractions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.11 Laurdan GP plot for DPPC at different polystyrene molar fractions . . 3.12 Laurdan high energy band fraction for DPPC liposomes containing polystyrene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.13 Dependence of GP variation over polystyrene molar fraction . . . . . . 3.14 DSC thermographs of DPPC bilayers at different polystyrene molar fractions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.15 Normalized ∆H and T m for DMPC, DPPC and DSPC . . . . . . . . . . 3.16 Dependence of the interaction parameter k on the bilayer thicknes . . .3.17 DMPC and DMPC:PS GUVs at different temperatures . . . . . . . . . . 3.18 Statistics of DMPC GUVs phase separation at different polystyrene molar fractions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1 Laurdan emission spectra for DOPC:DPPC and DOPC:DPPC:PS . . . . 4.2 Laurdan GP plot for DOPC:DPPC containing polystyrene . . . . . . . . 4.3 Laurdan GP plot for DOPC:DPPC at different λ exc . . . . . . . . . . . . 4.4 GUVs of DOPC:DPPC at different DPPC fraction with and without polystyrene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.5 Comparison between experimental data points and DOPC:DPPC phase diagram . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.6 Comparison between theoretical DOPC:DPPC phase diagram and experimental results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.7 Location of analyzed sample in the DOPC:DPPC:Chol phase diagram . 4.8 Distribution of the number of domains in GUVs at 1h and 36h stabilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.9 GUVs of DOPC:DPPC:Chol and DOPC:DPPC:Chol:PS after 1 h and 36 h of stabilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.10 Emission maxima of Di-4 in ternary GUVs containing polystyrene . . . 5.1 Comparison of Laurdan emission spectra of DPPC at 20 • C and 60 • C . 5.2 Laurdan GP plot for DPPC at different sucrose concentrations . . . . . 5.3 DSC thermographs of DPPC bilayers at different sucrose concentrations xv 5.4 DMPC GUVs formed in water and sucrose at different temperatures . 5.5 Statistics of DMPC GUVs phase separation at different sucrose concentrations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.6 Schematic representation of thermodynamics model . . . . . . . . . . . 5.7 Comparison between experimental data and thermodynamics model . 6.1 Chemical structure of PPCHOL . . . . . . . . . . . . . . . . . . . . . . . 6.2 Average diameter of PPCHOL over pH . . . . . . . . . . . . . . . . . . 6.3 GUVs of DOPC:DPPC in PBS and DOPC:DPPC in PPCHOL solution . 6.4 Variation of Di-4 emission spectra in presence of PPCHOL . . . . . . . 6.5 Fitting of Di-4 emission spectra in presence of PPCHOL . . . . . . . . . A.1 Guinier and Kratky-Porod plots for h-DPPC and h-DPPC:PS in D 2 O solvent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A.2 Guinier plot for d62-DPPC:PS in lipid matched solvent . . . . . . . . . A.3 Laurdan emission spectra for DMPC at different polystyrene molar fractions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A.4 Laurdan emission spectra for DSPC at different polystyrene molar fractions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A.5 Laurdan GP plot for DMPC at different polystyrene molar fractions . . A.6 Laurdan GP plot for DSPC at different polystyrene molar fractions . . A.7 Laurdan high energy band fraction for DMPC liposomes containing polystyrene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A.8 Laurdan high energy band fraction for DSPC liposomes containing polystyrene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A.9 GP values at different polystyrene content in gel and fluid phase . . . . A.10 DSC thermographs of DMPC bilayers at different molar fractions of polystyrene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A.11 DSC thermographs of DSPC bilayers at different molar fractions of polystyrene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B.1 Arc length approximation for area coverage estimation . . . . . . . . . B.2 Solid angle approximation for area coverage estimation . . . . . . . . . C.1 Laurdan emission spectra for DPPC at different sucrose concentrations C.2 Enthalpy variation with temperature at increasing σ . . . . . . . . . . . C.3 Resulting enthalpy change vs 1 -σ . . . . . . . . . . . . . . . . . . . . . C.4 Variation of the apparent T m . . . . . . . . . . . . . . . . . . . . . . . . . xvii List of Tables 2.1 Calculated scattering length densities . . . . . . . . . . . . . . . . . . . 18 3.1 Calculated polystyrene area coverage for extended and coiled conformations. Dotted line represent crossing point to overlapping chains. . 36 3.2 Bilayer structural parameters for h-DPPC and h-DPPC:PS in D 2 O . . . 38 3.3 Calculated ∆H, T m and T 1/2 for DPPC with increasing molar fractions of polystyrene. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49 4.1 Summary of S o area coverage for DOPC:DPPC GUVs at different composition. Each value represents average and standard deviation of two separate samples of 40 vesicles each. . . . . . . . . . . . . . . . . . 66 4.2 Summary of average number of domains for ternary GUVs composed of DOPC:DPPC:Chol and DOPC:DPPC:Chol:PS. Each value represents average and standard deviation of two separate samples of 70 vesicles each. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69 4.3 Emission maxima of Di-4 in ternary lipid membrane and ternary lipid membranes containing cholesterol. Each value represents average and standard deviation of two separate samples of 70 vesicles each. . . 71 5.1 Summary of DSC results for DPPC liposomes in sucrose solution . . . 82 A.1 Calculated ∆H, T m and T 1/2 for DMPC with increasing molar fractions of polystyrene. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111 A.2 Calculated ∆H, T m and T 1/2 for DSPC with increasing molar fractions of polystyrene. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .List of AbbreviationsDOPC = 1,2-dioleoyl-sn-glycero-3-phosphocholine DMPC = 1,2-Dimyristoyl-sn-glycero-3-phosphocholine DPPC = 1,2-dipalmitoyl-sn-glycero-3-phosphocholine DSPC = 1,2-Distearoyl-sn-glycero-3-phosphocholine d62-DPPC = deuterated 1,2-dipalmitoyl-sn-glycero-3-phosphocholine Chol = Cholesterol PS = polystyrene Laurdan = 6-Dodecanoyl-2-Dimethylaminonaphthalene DiI = 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine SANS = Small angle neutron scattering DSC = Differential scanning calorimetry GP = general polarization Cryo-TEM = Cryo-transmission electron microscopy LSCM = Laser scanning confocal microscopy PLP = Poly (L-lysine isophthalamide) PPCHOL = Cholesterol grafted PLP xxi

Finally, Chapter 7

 7 summarises the results obtained and provides future directions to pursue based on the experimental results. Mattia Morandi, November 2017 xxiii Resumé de these en français Introduction La complexité de la membrane cellulaire a motivé le développement de systèmes modèles minimaux pour étudier le rôle des lipides dans les mécanismes cellulaires.

FIGURE 1 : 30 FIGURE 2 :FIGURE 3 :FIGURE 4 :FIGURE 5 :FIGURE 6 :FIGURE 7 :FIGURE 8 :FIGURE 9 :

 13023456789 FIGURE 1: (A) Cryo-TEM image d'un échantillon mixte de DOPC / DOPC: PS 40:60, montrant la coexistence de vésicule avec bicouche bien contrastée et vésicules avec flou contraste. (B) Détail du liposome affichant un contraste inférieur. (C) Détail de liposome affichant un contraste élevé. Barre d'échelle 50 nm.

Chapter 1 .

 1 Introductionand other molecules, it regulates the transport of nutrients and metabolic products between cells and extracellular environments. Other processes crucial to cellular activities, such as recognition, immunological response are regulated or performed by the plasma membrane.The plasma membrane is composed by three main elements: lipids, proteins and sugars (Fig 1.1). The relative rates of these components vary between species and there is also variability in composition in eukaryotic cell. Another important property of the plasma membranes of human and animal cells is that they exhibit transverse lipid compositional asymmetry, i.e.the lipid composition of the outer and inner monolayers or leaflets of the lipid bilayer are different. The structure of the plasma membrane has been studied extensively, and in 2017 the first scattering image of the live cell was reported[6].

FIGURE 1 . 1 :

 11 FIGURE 1.1: A schematic represeantion of the plasma membrane of a cell, displaying the lipid bilayer, saccharides and membrane proteins .

FIGURE 1 . 4 :

 14 FIGURE 1.4: (A) Structure of hydrated lipid bilayer in gel phase. The acyl chains are completely extended and packed in a rectangular lattice. The lipids can be either straight (L β ) or tilted (L β ).(B) A fluid phase bilayer, with chains posessing trans-and gauche-conformations and lipids disorderly distributed.

FIGURE 1 . 5 :

 15 FIGURE 1.5: (A) Typical phase diagram of a binary lipid system composed of a lowmelting and high-melting lipid. (B) Typical micrometric behavior for GUV composed of DOPC:DPPC 75:25 at 15 • C showing pure S o phase. (C) 20 • C with L α -S o coexistence and (D) and at 35 • C with bilayer in L α state. Scale bar 20 µm.

1. 2 .FIGURE 1 . 6 :

 216 FIGURE 1.6: Different type of S o domains that can be observed, depending on membrane tension and heating rate. (A) irregular shape (B) hexagonal domains and (C) striped domains. Scale bar 10 µm.

FIGURE 1 . 8 :

 18 FIGURE 1.8: (A) Typical phase diagram of ternary lipid mixtures composed of low-melting lipid, high-melting lipid and cholesterol. (B) Characteristic micrometric domains of ternary phase separation (extracted from ref. [39]) displaying L d -L o . (C) L d -L o -S o three phase coexistence and (D) L d -S o coexistence. Scale bar 20 µm.

  FIGURE 1.9: (A) Example of a four component mixture phase diagram (DOPC:POPC:DSPC:Chol) highlighting normal coexistence regions and modulated phase separation region. (B) Image of GUV in the modulated phase region exhibiting disitinctive domains. Both images are from ref. [72]. Scale bar 10 µm.

FIGURE 2 . 2 :

 22 FIGURE 2.2: Example of a typical thermograph of liposomal suspension of DPPC in water. The mid point of the curve is defined as the transition temperature T m , whereas the width at half maximum is T 1/2

Chapter 2 .

 2 Materials and Methodsthe most implemented and has had a major impact in polymer science, biology and material science.Particularly, in the field of membrane physics, neutron scattering data were used to investigate structural parameters of the lipid bilayer like thickness, hydration, area cross section, bending rigidity and domain size upon phase coexistence. The range of applicability and uses of scattering techniques were successfully employed to obtain the first structure of live bacterial membrane[6].

FIGURE 2 . 3 :

 23 FIGURE 2.3: Schematics of a SANS experimental apparatus. The incident beam from the source impinges on the sample, resulting in a scattered beam and a transmitted beam that are collected by the detector.

  of sample, to highlight parts of interests by matching the SLD of certain components by choosing the proper H 2 O/D 2 O ratio. Contrast variation technique can improve the information extracted from sample, however special care should be taken. The incoherent scattering increases by adding extra H 2 O and for weakly scattering samples, the resulting lower signal to noise ratio may mask structural signal.

FIGURE 2 . 5 :

 25 FIGURE 2.5: )A) A typical Jablonski diagram illustrating the main principle of fluorescence. Light at λ exc is absorbed by the molecule, raising the electrons from the ground state S 0 to the excited state S 2 . The electronic state relaxes back to ground state by emitting light at lower wavelength λ em . (B) Schematics of Stokes shift, the difference between λ em and λ exc .

6 .FIGURE 2 . 6 :

 626 FIGURE 2.6: A schematic diagram of a conventional fluorescence microscope. The light source illuminates the sample at a specific λ exc and the resulting fluorescence is filtered by the mirror and recorded by the camera.

2. 3 .FIGURE 2 . 7 :FIGURE 2 . 8 :

 32728 FIGURE 2.7: Typical images obtained by staining the lipid membrane with a fluorescent probe in case of vesicle displaying phase separation. (A) The membrane appears completely uniform in bright field, but when visualized using fluorescence (B) the different phases are visible due to the preferential partitioning of the dye towards one of the two phases. Scale bar 10 µm

2. 3 .FIGURE 2 . 9 :

 329 FIGURE 2.9: Comparison between conventional fluorescence microscopy and confocal imaging in visualizing phase separation of GUVs. (A) In epifluorescence the domains are clearly visible, however the contrast is low. (B) Confocal microscopy yields improved contrast, but visualization is dependent of the focal plane. (C) The possibility to reconstruct in 3D the vesicle allows for better quantification of domain area coverage. Scale bar 10 µm.

FIGURE 2 .FIGURE 2 .

 22 FIGURE 2.10: (A) Chemical structure of Laurdan, with dipole moment highlighted as a red arrow. (B) Jablosnki diagram for a fluorescent probe possessing dipole moment. The reorientation of the dipoles of solvent molecules changes the emission wavelength.

FIGURE 2 .

 2 FIGURE 2.12: (A) Typical emission spectra of Laurdan for DPPC bilayer at different temperatures. The information of each spectra can be summarized in a single value using the GP definition, resulting in (B) a GP plot over temperature, which gives information on the lipid packing.

2. 3 .

 3 Fluorescence, microscopy and environment-

FIGURE 2 . 14 :

 214 FIGURE 2.14: An example of Gaussian decomposition of Laurdan emission spectra for DPPC bilayer at 45 • C. The spectra are plotted in energy rather than wavelength to obtain a better fitting. Black dotted line represents the experimental Laurdan emission spectra and blue line is the resulting fitting. Red and green line represent repsectively the 440 nm and 490 nm contribution obtained via fitting.

FIGURE 2 . 15 :

 215 FIGURE 2.15: Chemical structure of Di-4-ANEPPDHQ.

FIGURE 2 .

 2 FIGURE 2.16: (A) Example of spectral imaging of DOPC:DPPC:Chol giant vesicles stained with Di-4. It is possible to simultaneously visualize L d (orange square) and L o (blue square) phases and to (B) extract the local emission spectra.

FIGURE 3 . 1 :

 31 FIGURE 3.1: (A) Projected area S 0 of a single styrene monomer. (B) Projected surface of a polymer chain given as the sum of s 0 over the number of chains. (C) Projected surface of a polymer coil allowing to compute a projected coil area and thus (D) crossover surface density at which coils start to interact.

Chapter 3 .

 3 FIGURE 3.2: (A) Schematic of the polymer distribution within the lipid bilayer strictly confined in the membrane midplane below the overlapping surface density. (B) Polymer with coiled conformation confined in the membrane midplane above the overlapping density.

  of polystyrene and change in temperature by acquiring the scattering profile of h-DPPC liposomes with 30% molar fraction of polystyrene at 25 • C and 50 • C. To maximize the contrast with the hydrocarbon region and minimize incoherent scattering, we used 100% D 2 O as a solvent.Figure 3.3 shows scattering profiles for 25 • C (Fig. 3.3 A) and 50 • C (Fig. 3.3 B) for pure DPPC and DPPC:PS liposomes. At low temperature (Fig. 3.3 A), in the gel phase, the curve for unloaded h-DPPC displays an inflection point at 0.01 Å -1 , followed by a linear decrease of scattering intensity in the intermediate region. The kink observed is consistent with liposomes formed via extrusion method, and the position is associated with formation using a pore size of 50 nm [120], consistent with our preparation method. Incorporation of polymer results in suppression of the inflection point, indicating a higher polidispersity of the sample induced by inclusion of polystyrene. Moreover, scattering intensity decreases non linearly and more rapidly compared to a lipidonly system. At 50 • C (Fig. 3.3 B) in pure h-DPPC we observe the same kink at 0.01Å -1 , showing that crossing from the gel to the fluid phase of DPPC does not significantly affect the polydispersity of the size distribution of the liposomes. The intensity decrease linearly, albeit with smaller slope compared to 25 • C, consistent with a reduction of

FIGURE 3 . 3 : 38 Chapter 3 .

 33383 FIGURE 3.3: (A) Raw SANS data for h-DPPC (black) and h-DPPC:PS 70:30 (orange) in D 2 O at 25 • C. (B) Raw SANS data for h-DPPC (black) and h-DPPC:PS 70:30 (orange) in D 2 O at 50 • C.

  . A.1 B).Pure h-DPPC vescicles show a decrease of the radius of gyration of about 33.5 Å upon increase in temperature. The changes in R g in liposomes with 30% molar fraction PS are more pronounced with a variation of ∼ 57.2 Å.

  ature. A smaller thickness of the fluid phase compared to the gel phase has been reported by Nagle & Tristam-Nagle [121], from SAXS and volumetry experiments, who observed 45.7 Å at 20 • C and 35.1 Å at 50 • C. We observe a strong decrease in membrane thickness at low temperature when polystyrene is present. However, in the fluid phase the membrane thickness show comparable values to pure h-DPPC liposomes. Interestingly we observe that already in the gel phase the presence of polymer decreases the d H to values comparable to h-DPPC in the fluid phase.

3 . 4 )FIGURE 3 . 4 :FIGURE 3 . 5 :

 343435 FIGURE 3.4: (A) Bilayer scattering profile for h-DPPC:PS systems in D 2 O solvent. (B) Bilayer scattering profile for h-DPPC:PS liposome in polymer matching solvent. (C) Bilayer scattering profile for d62-DPPC:PS liposome in lipid matching solvent.

3. 5 A

 5 ). This arises from the choice of the SLD of the solvent chosen to match the SLD of DPPC in the fluid phase. For systems containing polystyrene we observe a strong increase of scattering intensity. The plateau located at ∼0.007 Å -1 indicates a typical size of the scattering sample smaller than the liposome radius. This size could be correlated to aggregates of the polymer in the bilayer which are laterally segregated.Increase in temperature shows complete loss of signal in the case of polymer-free liposomes, due to the better matching between the SLD of DPPC and the solvent (Fig.3.5 B). Incorporation of polymer shows once again an increase of scattering intensity comparable to the one measured in the gel phase. Moreover, the scattering Chapter 3. Polystyrene in a single component lipid bilayer curve does not display any inflection point in the low q range. This indicates that the typical size of the polymer distribution increases with temperature, suggesting the loss of lateral segregation. Guinier analysis of the scattering curves indeed shows that at 20 • C the typical size of the polymer is 153.78 ± 12.35 Å -1 , much smaller than the liposome radius of 50 nm. At 50 • C the lack of a plateau at low q region, due to the different q range probed in the experiments with respect to D 2 O solvent, does not allow to perform any estimation on R g (Fig. A.2).

54 ± 3 FIGURE 3 . 6 :

 54336 FIGURE 3.6: (A) Disk model fitting (grey) for d62-DPPC:PS 70:30 (blue) in H 2 O/D 2 O 92:8 at 20 • C. (B) Lamellar model fitting (grey) for d62-DPPC:PS 70:30 (gren) in H 2 O/D 2 O 92:8 at 50 • C.

FIGURE 3 . 7 :

 37 FIGURE 3.7: (A) DOPC liposome acquired via Cryo-TEM. It is possible to see the bilayer as a three stripe region. (B) Cryo-TEM picture of DOPC:PS 40:60 liposomes. The bilayer appears as a uniform gray band. Scale bar 50 nm.

Figure 3 .

 3 Figure 3.7 shows typical DOPC liposomes and DOPC:PS liposomes prepared withf P S = 0.6 acquired via electron microscopy. For pure DOPC the bilayer is clearly visible as a three stripes region, due to the different electron density between headgroups (black stripes, high density) and the acyl chain region (white stripe, low density). For DOPC:PS liposomes we observe a significant change in the contrast, as the bilayer appears as a rather uniform grey region due to the higher intensity in the acyl chain region (Fig.3.7 B).

FIGURE 3 . 8 :

 38 FIGURE 3.8: (A) Cryo-TEM image of a mixed sample of DOPC/DOPC:PS 40:60, showing coexistence of vesicle with well contrasted bilayer and vesicles with blurred contrast. (B) Detail of liposome displaying lower contrast. (C) Detail of liposome displaying high contrast Scale bar 50 nm.

3. 9 AFIGURE 3 . 9 :

 939 FIGURE 3.9: (A) Intensity profile of the bilayer of DOPC liposomes. We observe a sharp contrast between the two external bands and the middle one. (B) Intensity profile of the bilayer of DOPC:PS liposomes with f P S = 0.6. We observe that the contrast between the two external bands and the middle one is highly reduced.

FIGURE 3 . 10 :

 310 FIGURE 3.10: Comparison of emission curves of Laurdan for pure DPPC (A), DPPC:PS 90:10 (B), DPPC:PS 80:20 (C) and DPPC:PS 70:30 (D) at 20 • C (black line), 35 • C (orange line), 42 • C(blue line), 50 • C(green line) and 60 • C (red line).

3. 11 30 FIGURE 3 . 11 :

 1130311 FIGURE 3.11: Variation of general polarization (GP) over temperature for multilamellar liposomes of DPPC formed in water at 0 (black squares), 10% (orange circles), 20% (blue triangles) and 30% (green inverted triangles) molar fraction of polystyrene.

3 .

 3 For DMPC (Fig. A.7) and DSPC (Fig. A.8),we observe similar values of high energy coverage for the gel phase, while in the fluid phase the blue band contribution strongly differs among the lipids. However, the increase of the fraction with incoporation of polystyrene shows similar trends for all three lipids. The blue band contribution remains constant upon increase of temperature following a quasi-linear dependence on the polymer fraction.

FIGURE 3 .

 3 FIGURE 3.13: (A) GP variation at different molar fraction of polystyrene incorporated for DMPC (black squares), DPPC (orange circles) and DSPC (blue triangles). (B) High energy coverage for T m + 15 at increasing molar fraction of polystyrene for DMPC (black squares), DPPC (orange circles) and DSPC (blue triangles).

Chapter 3 .

 3 Polystyrene in a single component lipid bilayer two phases changes. Moreover, our results in DOPC liposomes observed in Cryo-TEM show an uniform change in the bilayer contrast (Fig. 3.8 B), rather than a laterally segregated effect. The difference in variation of GP, minimal in the gel phase and maximal for the liquid crystalline phase (Fig. A.9), indicate a different partitioning and behavior of the polymer between the S o and L α . These results are in good agreement with our neutron scattering curves, which shows an increase of the typical size of the polymer with increasing temperature.

FIGURE 3 . 14 :

 314 FIGURE 3.14: DSC thermographs of DPPC liposomes containing increasing amount of polystyrene. Each curve represents the second thermographic signal from the full run experiment.

Chapter 3 .FIGURE 3 .

 33 FIGURE 3.15: (A) ∆H(f P S )/∆H(f P S = 0) calculated from DSC thermographs for DMPC (black squares), DPPC (orange circles) and DSPC (blue triangles). (B) T m (f P S )/T m (f P S = 0) calculated from DSC thermographs for DMPC (black squares), DPPC (orange circles) and DSPC (blue triangles). Each data point is the average of 2 samples over 3 heating/cooling cycles.

FIGURE 3 . 16 :

 316 FIGURE 3.16: Variation of interaction parameter k over bilayer thickness.

  ), with f P S = 0.3, at low temperature (20 • C) display a homogenous phase characterized by facets and corrugations, a morphology already attributed to giant vesicles in the S o phase [136, 137]. At 25 • C, close to transition temperature of DMPC (T DM P C m ∼ 24.5 • C)

FIGURE 3 . 17 :

 317 FIGURE 3.17: Epifluorescence microscopy images of GUVs composed of DMPC and DMPC:PS, with f P S = 0.3, at different temperatures.

30 FIGURE 3 . 18 :

 30318 FIGURE 3.18: Summary of 30% molar fraction of polystyrene on the phase behavior of DMPC GUVs. Each data is the average and standard deviation from analysis of 50 GUVs from 2 separate samples.

Chapter 4 .

 4 Changes in multi-component lipid bilayers upon incorporation of polystyrene model membranes probed in Chapter 3, employing the same experimental techniques. We incorporate small molar fractions (< 20%) of short chained PS within membranes composed of DOPC:DPPC and DOPC:DPPC:Chol of different molar ratios, and extrapolate changes in their phase behaviour using Laurdan and Di-4 spectroscopy and confocal microscopy.

Fig. 4 . 5 FIGURE 4 . 1 :

 4541 Fig. 4.1 shows typical Laurdan emission spectra for DOPC:DPPC liposomes at different DPPC molar fraction containing no polymer or 10 mol % of polystyrene. We observe that for liposomes composed of 0.4 molar fraction of DPPC (Fig. 4.1 A) the spectrum at low temperature is broad with emission maximum at 440 nm. The broad peak can be associated with phase coexistence due to the presence of both liquid and gel phase in the membrane. With increasing temperature we observe a continuous red shift of the spectra, reaching a stable broad peak centered at 490 nm at 40 • C, associated with fluid phase. Incorporation of polymer yields a sharper peak for low temperature (Fig. 4.1 B). Increasing temperature still displays the red shift, however we observe a shoulder centered at 440 nm which persists even in the fluid phase.

FIGURE 4 . 2 :

 42 FIGURE 4.2: (A) Variation of General Polarization over temperature for multilamellar liposomes of DOPC:DPPC 60:40 (black squares) and DOPC:DPPC:PS 54:36:10 (orange circles). (B) Variation of general polarization over temperature for multilamellar liposomes of DOPC:DPPC 40:60 (blue triangles) and DOPC:DPPC:PS 36:54:10 (green inverted triangles).

  Fig 4.3 shows the GP values for 4.1. Results and discussion 63 DOPC:DPPC at different DPPC content with and without polystyrene obtained using different excitation wavelength. DOPC:DPPC:PS500 54:36:10 exc 350 nm DOPC:DPPC:PS500 54:36:10 exc 400 nm -

FIGURE 4 . 3 :

 43 FIGURE 4.3: (A) Variation of general polarization over temperature for multilamellar liposomes of DOPC:DPPC 60:40 excited at 350 nm (black squares) and 400 nm (orange circles). (B) Variation of general polarization over temperature for multilamellar liposomes of DOPC:DPPC:PS 54:36:10 excited at 350 nm (black squares) and 400 nm (orange circles).(C) Variation of general polarization over temperature for multilamellar liposomes of DOPC:DPPC 40:60 excited at 350 nm (blue triangles) and 400 nm (green inverted triangles). (D) Variation of general polarization over temperature for multilamellar liposomes of DOPC:DPPC:PS 36:54:10 excited at 350 nm (blue triangles) and 400 nm (green inverted triangles).

Chapter 4 .

 4 Changes in multi-component lipid bilayers upon incorporation of polystyrene Domains coverage in GUVS Giant unilamellar vesicles formed of DOPC:DPPC display characteristic gel domains of irregular or hexagonal morphology (Fig. 4.4 A B), consistent with previous reported studies. For each vesicle we calculated the area fraction of S o domains in the pure lipid case and upon incorporation of polystyrene, using the arc lenght approximation described in Appendix B.

± 0 .FIGURE 4 . 4 :

 044 FIGURE 4.4: (A)(B) Typical DOPC:DPPC 60:40 GUVs stained with DiI. (C)(D) Typical DOPC:DPPC:PS 54:36:10 GUVs stained with DiI. (E)(F) Typical DOPC:DPPC 40:60 GUVs stained with DiI. (G)(H) Typical DOPC:DPPC:PS 36:54:10 GUVs stained with DiI. The bright area represents presence of fluorophore, which partitions in the L α phase. Dark spots are S o domains. Scale bar 5µm.

FIGURE 4 . 5 :

 45 FIGURE 4.5: (A) Comparison between liquidus line points obtained via analysis of Laurdan emission spectra for DOPC:DPPC (orange) and DOPC:DPPC:PS (blue) and liquidus line reported by Chen [143]. (B) Obtained liquidus line points for DOPC:DPPC:PS obtained by area coverage of S o domains in GUVs (orange) and liquidus line reported by Chen [143].

Chapter 4 .

 4 Changes in multi-component lipid bilayers upon incorporation of polystyrene To compare our observations on the gel phase area coverage in GUVs, we employed the lever rule using the phase diagram proposed by Chen. The liquidus line up to T = 20 • C was extrapolated from the experimental data of Chen in the range [0.2 -0.7] of X DP P C . Assuming a constant solidus line X b = 0.95 for the considered DPPC molar fraction interval, it is possible to estimate the area fraction of gel phase using the relation:

TABLE 4 . 1 :

 41 Summary of S o area coverage for DOPC:DPPC GUVs at different composition. Each value represents average and standard deviation of two separate samples of 40 vesicles each. The values of solid area fraction obtained with GUVs allow to estimate the tie line for 20 • C in presence of 10% polysterene. By considering a constant X b = 0.95, we obtained values for X P S a of 0.35 ± 0.05 for 20 • C and 0.38 ± 0.06 for 23 • C.

4. 1 .= 41 . 8 •= - 21 . 3 •

 1418213 Results and discussion 67 parameter restricted to interval [-1, 1], R is the gas constant, ∆H 1 and ∆H 2 are the lipid characteristic gel-to-liquid enthalpies and T 1 and T 2 their respective transition temperatures. For DOPC:DPPC bilayers we calculated the phase diagram using values of ∆H DP P C = 38.5 kJ•mol -1 and T DP P C m C for DPPC, obtained from our DSC experiments, and ∆H DOP C = 7.7 kJ•mol -1 and T DOP C m C obtained from literature. For the cooperativity of the interaction between neighboring lipids we chose a value of J = 0.31. The theoretical model is in good agreement with our experimental data from Laurdan emission spectra and with the DOPC:DPPC phase diagram reported by Chen [143] (Fig. 4.6). For systems containing polystyrene, we kept all model parameters constant, except for the different enthalpy and transition temperature obtained from calorimetry experiments, namely ∆H P S = 30.7 kJ•mol -1 and T P S m = 41.5 • C, to calculate the new boundary line. The cooperativity was kept at the same value based on the measurement of T 1/2 from DSC experiments. The boundary line in presence of polystyrene indeed shows good agreement with our experimental observation. This indicates that the changes induced by the polymer in DPPC bilayers is the main driving force behind depression of the liquidus line towards lower temperature.

FIGURE 4 . 6 : 68 Chapter 4 .FIGURE 4 . 7 :

 4668447 FIGURE 4.6: Comparison between experimental data point for the liquidus line obtained by Chen (orange, ref.[143]), experimental data point for DOPC:DPPC (blue triangles) and DOPC:DPPC:PS (black squares) obtained from Laurdan emission spectra, and theoretical predictions of liquid lines for DOPC:DPPC (red) and DOPC:DPPC + 10%PS (green).

FIGURE 4 . 8 :

 48 FIGURE 4.8: (A) Statistical distribution of number of domains in GUVs composed of DOPC:DPPC:Chol 45:30:25 (black) and DOPC:DPPC:Chol:PS 36:24:20:20 (orange) after 1 hour of stabilization. (B) Statistical distribution of number of domains in GUVs composed of DOPC:DPPC:Chol 45:30:25 (blue) and DOPC:DPPC:Chol:PS 36:24:20:20 (green) after 36 hour of stabilization.

FIGURE 4 . 9 :

 49 FIGURE 4.9: (A) Typical GUVs composed of DOPC:DPPC:Chol 45:30:25 after 1 hour and 36 hour of stabilization. (B) GUVs composed of DOPC:DPPC:Chol:PS 36:24:20:20 after 1 hour and 36 hour of stabilization. Green represents the liquid disordered phase while blue represent the liquid ordered domain. White arrow indicate L o domains. Scale bar 20 µm.

FIGURE 4 .

 4 FIGURE 4.10: (A) Typical emission spectra of Di-4 for the liquid ordered phase for GUVs composed of DOPC:DPPC:Chol 45:30:25 (blue) and DOPC:DPPC:Chol:PS 36:24:20:20 (green). (B) Typical emission spectra of Di-4 for the liquid disordered phase for GUVs composed of DOPC:DPPC:Chol 45:30:25 (blue) and DOPC:DPPC:Chol:PS 36:24:20:20 (green). Lines represent gaussian fitting of the distribution.

3 TABLE 4 . 3 :

 343 Emission maximum L o phase Emission maximum L d phase Emission maxima of Di-4 in ternary lipid membrane and ternary lipid membranes containing cholesterol. Each value represents average and standard deviation of two separate samples of 70 vesicles each.

Chapter 4 .

 4 Changes in multi-component lipid bilayers upon incorporation of polystyrene

Figure 5 .

 5 Figure 5.1 illustrates emission spectra of Laurdan for DPPC MLVs at different concentration of sucrose at 20 • C (Fig. 5.1 A) and 60 • C (Fig. 5.1 B). During the phospholipid main transition the spectra show a continuous shift to longer wavelengths (Fig. C.1).

FIGURE 5 . 1 :

 51 FIGURE 5.1: (A) Laurdan emission spectra for DPPC at different concentration of sucrose at 20 • C. (B) Laurdan emission spectra for DPPC at different concentration of sucrose at 60 • C .

Fig. 5 . 6 FIGURE 5 . 2 :

 5652 FIGURE 5.2: Variation of General Polarization over temperature for multilamellar liposomes of DPPC formed in water and at different sucrose concentration. It is possible to observe a rise in GP values proportional to sucrose concentration. Inset: Dependance of GP on sucrose concentration for 20 • C (blue), 42 • C (green) and 60 • C (red)

  7 M and 1.5 M sucrose (Fig 5.3). The energy associated with the transition from gel to liquid crystalline phase gives information on the local packing of the membrane upon melting. Fig 5.3 A shows DSC curves of the liposomal systems in pure water and at each sucrose concentration.

FIGURE 5 . 3 :

 53 FIGURE 5.3: (A) DSC calorimetric signal (B) Variation of T m over sucrose concentration (C) Decrease of enthalpy at different sucrose concentration. In both B) and C) straight line represent initial value in pure water.

FIGURE 5 . 4 : 4 C

 544 FIGURE 5.4: Summary of sucrose effects on GUVs formed with DMPC. (A) Typical GUV phase behavior for DMPC in water, (B) in 0.2 M and (C) in 0.39 M sucrose in the temperature interval [20 • C -40 • C]. Scale bar 10 µm.

FIGURE 5 . 5 :

 55 FIGURE 5.5: Statistics of DMPC GUVs phase separation at different sucrose concentrations. Each bar represents average and standard deviation of two different samples of 60 vesicles each.

FIGURE 5 . 6 :

 56 FIGURE 5.6: Schematic representation of interaction of sucrose with the lipid bilayer. The sugar bounds to a lipid, creating two populations with different transition themodynamics. Free lipids (grey) melt as expected while bound lipids (green) are dehydrated and therefore undergo a transition at different temperature.

. 7 AFIGURE 5 . 7 :

 757 FIGURE 5.7: (A) Dependence of sucrose coverage σ on the sucrose concentration (black squares) and linear fitting (red line). (B) Experimental values of ∆H for the DPPC gel-toliquid transition at different concentration of sucrose (orange circles), alongside with the theoretical predictions from the thermodynamics model (black dotted line).(B) Experimental values of T m for the DPPC gel-to-liquid transition at different concentration of sucrose (green), alongside with the theoretical predictions from the thermodynamics model (blue dotted line).

5. 2 . Conclusions 89 The

 289 experimental values for enthalpic contribution (Fig. 5.7 B) and transition temperature (Fig. 5.7 C) are in good agreement with the theoretical predictions obtained via numerical simulations. The thermodynamics model therefore proves valid for low sucrose coverages.

  .3 A) and DOPC:DPPC 6:4 formed in 0.5 mg/mL polymer solution in PBS (Fig. 6.3 B) by analyzing the emission spectra of Di-4 incorporated in the lipid bilayer.

  branes containing cholesterol. Direct visualization of Di-4 emission spectra in GUVs composed of DOPC:DPPC:Chol showed a preferential partitioning of the polymer in the L α phase, with a stabilizing effect on the L d /L o coexistence. Our results indeed show that polystyrene disrupts the phase coexistence of multicomponent membrane, thus reinforcing the suggestion that nanometric polymers are hazardous for membrane viability and provides indications for future studies on the effects of plastic nanomolecules on lipid membranes. The second project of this work is devoted to the study of changes induced by high concentration of sucrose on the phase transition of DPPC model membranes, motivated by the crucial role sugars play in the biopreservation and cryopreservation field. Our experimental observations suggest that sucrose can induce a dehydration of the bilayer, as recorded by Laurdan emission spectra. The removal of water molecules, coupled with the formation of lipid-sucrose clusters, reduces the overall enthalpy of the transition and increases the transition temperature, as measured by

FIGURE A. 2 :FIGURE A. 3 :FIGURE A. 4 :

 234 FIGURE A.2: (A) Guinier plot for d62-DPPC:PS at 20 • C (blue triangle) and 50 • C (green inverted triangles) in H O/D 2 O 92:8 solvent.

30 FIGURE A. 5 :

 305 FIGURE A.5: Variation of General Polarization over temperature for multilamellar liposomes of DMPC formed in water at 0 (black squares), 10% (orange circles), 20% (blue triangles) and 30% (green inverted triangles) molar fraction of polystyrene.

FIGURE A. 6 :

 6 FIGURE A.6: Variation of General Polarization over temperature for multilamellar liposomes of DSPC formed in water at 0 (black squares), 10% (orange circles), 20% (blue triangles) and 30% (green inverted triangles) molar fraction of polystyrene.

FIGURE A. 7 :

 7 FIGURE A.7: Area fraction of 440 nm contribution in DMPC liposomes over temperature at 0% (black squares), 10% (orange circles), 20% (blue triangles) and 30% (green inverted triangles) polystyrene molar fraction.

FIGURE A. 8 :FIGURE A. 9 :

 89 FIGURE A.8: Area fraction of 440 nm contribution in DSPC liposomes over temperature at 0% (black squares), 10% (orange circles), 20% (blue triangles) and 30% (green inverted triangles) polystyrene molar fraction.

  FIGURE B.2: (A) Image treatment for solid angle approximation. (B) Schematics of the solid angle estimation.

2 m(N A ∆h) 2 = 4 C

 24 e βNc∆g ) 2 , = nN c (N A ∆h) 2 4RT 2 1 ch(βN c ∆g/2) 2 , (C.5)with N A the Avogadro number, N A ∆h the molar enthalpy change at the transition, n the number of moles of lipids.The peak maximum is at ∆g = 0, C p,max = dH/dT| T =Tm = nRN c (N A ∆h) 2 /(4R 2 T 2 ),from which N c can be expressed in terms of molar quantitiesN c = C p,maxn4RT

FIGURE C. 4 :

 4 FIGURE C.4: Variation of the apparent melting temperature (inflexion point of ∆H(T ))

  40 H 80 NO 8 P

				20	27.6	1144	0.24
	h-DPPC	Lipid	C 40 H 80 NO 8	50	27.6	1232	0.22
	d-DPPC	Lipid	C 40 H 18 NO 8 PD 62	20	673	1152	5.84
	d-DPPC	Lipid	C 40 H 18 NO 8 PD 62	50	673	1242	5.42
	h-DPPC	two tails (h)	C 30 H 62	20	-32.5	825	-0.39
	h-DPPC	two tails (h)	C 30 H 62	50	-32.5	913	-0.36
	d-DPPC	two tails (d)	C 30 D 62	20	613	833	7.36
	d-DPPC	two tails (d)	C 30 D 62	50	613	923	6.64
	DPPC	head group (h)	C 10 H 18 NO 8 P		60.1	319	1.88
	Water	all	H 2 O	25	-1.7	30	-0.56
	Heavy water all	D 2 O	25	19.2	30	6.44
	Polystyrene	all	(C 8 H 8 ) n				1.41

TABLE 2 .

 2

1: Neutron scattering lengths, molecular volumes, and corresponding scattering length densities of lipids used in this work.

TABLE 3 .

 3 1: Calculated polystyrene area coverage for extended and coiled conformations. Dotted line represent crossing point to overlapping chains.

TABLE 3 .

 3

2: Bilayer structural parameters for h-DPPC and h-DPPC:PS in D 2 O obtained from Guinier and Kratky-Porod analysis

  • C to 60 • C (Fig.3.10 A). At temperatures below T m the emission spectra is sharp and remains relatively stable and centered at 440 nm. At T m the spectra display an equal contribution from both emission bands, as expected for a gel-to-liquid melting. For temperatures above transition, Laurdan spectra show a continuos red shift, which indicates a larger number of solvent molecules interacting with the naphtalene moiety of Laurdan.

			Chapter 3. Polystyrene in a single component lipid bilayer
					9.0x10 6
	2x10 6
					6.0x10 6
	1x10 6
					3.0x10 6
	0				0.0
	400	450	500	550	600	400	450	500	550	600
	2x10 6				1.0x10 6
	1x10 6				5.0x10 5
	0				0.0
	400	450	500	550	600	400	450	500	550	600

  59 H 97 ClN 2 O 4 , M w = 933.8793) was provided by ThermoFisher Scientific (Waltham, MA, USA).Sucrose (C 12 H 22 O 11 M w = 342.3) and Laurdan (6-Dodecanoyl-N,N-dimethyl-2-naphthylamine) were purchased from Sigma-Aldrich (Saint-Quentin, France). Atactic polystyrene ((C 8 H 8 ) n ) M n = 500) was purchased from Polymer Source Inc. All chemicals had high purity and were used without further purification. The osmolarities of the sucrose solutions were measured with a cryoscopy osmometer Osmomat 030 (Gonotec; Berlin, Germany)

	1,2-dioleoyl-sn-glycero-3-phosphocholine, C 44 H 84 NO 8 P, M w 786.11), DSPC (1,2-Distearoyl-sn-glycero-3-phosphocholine, 55 C 3.3. Materials and methods Tetramethylindocarbocyanine Perchlorate C

44 H 88 NO 8 P, M w 790.15), DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine, C 40 H 80 NO 8 P, M w = 734.039) and DMPC (1,2-dimyristoyl-sn-glycero-3phosphocholine, C 36 H 72 NO 8 P M w = 677.933) were purchased from Avanti Polar Lipid (Birmingham, AL). DiI Stain (1,1'-Dioctadecyl-3,3,3',3'-

  The lipid film was then hydrated with aqueous solution (H 2 O, D 2 O or a H 2 O/D 2 O mixture) at 70 • C to reach desired concentration and gently vortexed. Resulting MLV suspensions were sonicated for 15 min to disperse larger aggregates. Liposomal solutions remained stable over a period of days.

  The h-DPPC and h-DPPC/h-PS samples were prepared in D 2 O, while d 62 DPPC (both deuterated chains) and d 62 -DPPC:PS samples in H 2 O/D 2 O to match the scattering length density of the lipids or of the polymer. The neutron wavelength resolution was 2 • ∆λ/λ = 0.1. All scattering data were corrected for background, and incoherent scattering from 1 mm thick D 2 O or H 2 O solutions were used to correct for the deviation in uniformity of the detector response. The final data were converted to absolute scale. All experiments were first performed at low temperature (25 • C) and then at 50 • C.

Table 4

 4

	.2.

TABLE 4 .

 4 2: Summary of average number of domains for ternary GUVs composed of DOPC:DPPC:Chol and DOPC:DPPC:Chol:PS. Each value represents average and standard deviation of two separate samples of 70 vesicles each.

  59 H 97 ClN 2 O 4 , M w = 933.8793) and Di-ANEPPDHQ were provided by ThermoFisher Scientific (Waltham, MA, USA). Sucrose (C 12 H 22 O 11 M w = 342.3) and Laurdan (6-Dodecanoyl-N,N-dimethyl-2naphthylamine) were purchased from Sigma-Aldrich (Saint-Quentin, France). Atactic polystyrene ((C 8 H 8 ) n ) M n = 500) was purchased from Polymer Source Inc. All chemicals had high purity and were used without further purification. The osmolarities of the sucrose and glucose solutions were measured with a cryoscopy osmometer Osmomat 030 (Gonotec; Berlin, Germany)

	4.3. Materials and methods	73
	4.3 Materials and methods
	Materials
	Chloroform solution of DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine,
	C 44 H 84 NO 8 P, M w 786.11), DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine,
	C 40 H 80 NO 8 P, M w = 734.039) and cholesterol ((3S,8S,9S,10R,13R,14S,17R)-10,13-
	dimethyl-17-[(2R)-6-methylheptan-2-yl]-2,3,4,7,8,9,11,12,14,15,16,17-dodecahydro-
	1H-cyclopenta[a]phenanthren-3-ol, C 27 H 46 O, M w 386.65) in powder were purchased
	from Avanti Polar Lipid (Birmingham, AL). DiI Stain (1,1'-Dioctadecyl-3,3,3',3'-
	Tetramethylindocarbocyanine Perchlorate C

  • C at 1 M reported by Chowdhry, measured via calorimetry[167]. Stumpel[170] also observed a shift of ∼ 1 • C in T m of DMPC MLVs in presence of 40% sucrose. Similar effects have been reported on DMPC vesicles[171]. Other studies instead reported little or no changes in T m , contrary to our results.

	Sucrose concentration	T m	T 1/2	∆H
	[M]	[ • C]	[ • C]	[kJ • mol -1 ]
	0	41.8 ± 0.2 0.40 ± 0.01	38.5 ± 0.7
	0.39	41.9 ± 0.2 0.44 ± 0.01	33.6 ± 1.9
	0.7	42.1 ± 0.1 0.48 ± 0.01	27.9 ± 1.6
	1.5	42.7 ± 0.2 0.55 ± 0.02	23.9 ± 0.7

TABLE 5 .

 5 1: Summary of DSC results for DPPC liposomes in sucrose solution

  5.1. Results and discussion85coexistence, which has not been reported before in giant vesicles experiments. Although phase coexistence is expected to appear around T m[139], the persistence of gel domains at higher temperatures may indicate metastability produced by the low rate of heating employed in the experimental setup.

  59 H 97 ClN 2 O 4 , M w = 933.8793) was provided by ThermoFisher Scientific (Waltham, MA, USA). Sucrose (C 12 H 22 O 11 M w = 342.3) and Laurdan (6-Dodecanoyl-N,N-dimethyl-2-naphthylamine) were purchased from Sigma-Aldrich (Saint-Quentin, France). All chemicals had high purity and were used without further purification. The osmolarities of the sucrose solutions were measured with a cryoscopy osmometer Osmomat 030 (Gonotec; Berlin, Germany)

	1,2-dipalmitoyl-sn-glycero-3-phosphocholine,
	C 40 H 80 NO 8 P, M w = 734.039) and DMPC (1,2-dimyristoyl-sn-glycero-3-
	phosphocholine, M w = 677.933) were purchased from Avanti Polar Lipid (Birm-
	ingham, AL). DiI Stain (1,1'-Dioctadecyl-3,3,3',3'-Tetramethylindocarbocyanine Per-
	chlorate C

  1,2-dioleoyl-sn-glycero-3-phosphocholine, C 44 H 84 NO 8 P, M w 786.11) and DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine, C 40 H 80 NO 8 P, M w = 734.039) were purchased from Avanti Polar Lipid (Birmingham,AL). PBS and Di-ANEPPDHQ were provided by ThermoFisher Scientific (Waltham, MA, USA). All chemicals had high purity and were used without further purification. PPCHOL was synthesized and provided by Alex Chen, University of Cambridge, as a part of a joint collaboration within the ITN SNAL consortium. The osmolarities of the PBS solutions were measured with a cryoscopy osmometer Osmomat 030 (Gonotec; Berlin, Germany)

TABLE A .

 A 1: Calculated ∆H, T m and T 1/2 for DMPC with increasing molar fractions of polystyrene. ± 0.7 54.9 ± 0.3 0.23 ± 0.01 10 36.1 ± 0.8 54.4 ± 0.3 0.35 ± 0.01 20 24.1 ± 0.1 54.4 ± 0.7 0.47 ± 0.01 30 18.4 ± 0.5 54.1 ± 0.4 0.80 ± 0.01

	Polystyrene fraction	∆H	T m	T 1/2
	[% mol]	kJ/mol	• C	• C
	0	43.1

TABLE A .

 A 2: Calculated ∆H, T m and T 1/2 for DSPC with increasing molar fractions of polystyrene.

Dans ce travail, nous avons étudié avec succès les effets de molécules simples hydrophiles et hydrophobes sur le comportement en phase des membranes modèles lipidiques. En utilisant une combinaison de fluorescence en régime permanent, de calorimétrie différentielle (DSC) et de microscopie à fluorescence, nous avons extrait des informations structurelles de la membrane, ainsi que sur la thermodynamique de la transition de phase, en interaction avec différentes molécules. L'utilisation de sondes fluorescentes sensibles à l'environnement pour extraire des informations pertinentes aux échelles de longueur nanométrique et micrométrique est également un aspect clé de ce travail. De plus, en utilisant l'état de l'art Cryo-TEM et la diffusion des neutrons, nous avons obtenu une image complète de l'interaction au niveau nanométrique.En conclusion, cette these de doctorat présente une étude complète des effets des molécules hydrophobes / hydrophiles sur le comportement en phase de la membrane lipidique.Nous avons demontre que la combinaison de sondes fluorescentes sensibles a l'environnement, de la calorimetrie et de l'imagerie confocale forme un puissant ensemble d'outils pour caracteriser les variations de comportement de phase, fournissant ainsi une image plus claire des interactions qui s'établissent dans ces systèmes complexes.

mL of liposomal suspension stained with Laurdan of total concentration 3 mg/mL was placed in a quartz silica cuvette with 1 mm path length. Acquisition of Laurdan

Acknowledgements

To further investigate the effects of the polymer, we analyzed the local emission spectra of Di-4 in the L d and L o phases. For systems containing only cholesterol we observe two distinctive emission maxima, centered at 587 ± 4 nm and 629 ± 2 nm, associated respectively with DPPC:Chol and DOPC phase. The values we obtained are in good agreement with previously reported Di-4 spectra for DPPC:Chol and DOPC systems [147]. This cholesterol-grafted PLP, coined PPCHOL, belongs to the family of PLP derivatives first developed by Chen [191]. They are CPP-mimicking, lysine based polymers derived from hydrophobic modification of poly(L-lysine isophthalamide) with amino acids, and display different conformation under varying pH conditions. They have been succesfully reported to enable efficient cytoplasmic delivery of smallmolecule model drugs and bioactive macromolecules in vitro and in vivo [192,193].

In this work we investigate the effects of the cholesterol-grafted polymer on the phase behavior of giant unilamellare vesicles displaying gel-liquid phase coexistence using confocal spectra imaging of DI-4-ANEPPDHQ emission spectra.

Results and discussion

Polymer conformation at different pH conditions

To characterize the changes of the polymer conformation at varying pH, we performed DLS measurements of 250 µg/mL PPCHOL (Fig. 6.1) solution in PBS at progressively acidic pH. We observe an average diameter of 35.2 ± 2.9 nm at pH 7.3, with increasing acidification resulting in decrease of polymer size up to 8.73 ± 2.9 nm at pH 5.9. The size values of the diameters at neutral pH are significantly lower than the values of ∼ 100 nm for phenylalanine-grafted polymers (Wang, data not published). This difference suggests that already at neutral pH the polymer is already in a globular or condensed conformation, with a higher hydrophobicity than the amino acids grafted counterparts. The reduction of diamater upon acidification however indicates that the polymer possesses a partial pH-sensitivity.

Chapter 6. Interaction of cholesterol grafted polymer with lipid membranes of miscibility between the two lipids induced by the presence of the polymer. Moreover, Di-4 emission spectra are significantly different and display two separate contributions.

The presence of the two peaks suggest the existence of two distinct populations at sub-resolution level. Moreover, the higher wavelength maximum for vesicle formed in polymer solution are shifted with respect to polymer-free GUVs to lower wavelengths (Fig. 6.4). To confirm whether the resulting emission spectra is due to two separate populations or a single uniform signal, we performed a fitting on the emission spectra using a single contribution and a two contribution Gaussian model. Fitting with a single curve (Fig. 6.5 A) shows good agreement (χ 2 = 0.016) with experimental data, yielding an emission maximum of 625.6 ± 2.7 nm, close to values reported for DOPC:DPPC:Chol bilayers [147]. Using a two-component model also provides very good fitting (χ 2 = 0.005) with respective emission maxima located at 576.

Conclusions and future outlook

The plasma membrane is the primary barrier between the cell interior and the external environment; it regulates its functionality and it is a part of the cell signalling processes. Lipids are the primary membrane components, responsible for the structural integrity and mechanical properties. Active processes in the cell regulate lipid composition in the membrane, so to maintain membrane fluidity. Lipids coexist and display different structures depending on the type of lipid and external factors like temperature, an important property known as phase behaviour. Biological systems are inherently complex, therefore any small variation on the membrane phase behaviour can potentially perturb cell functionality.

In this work we succesfully investigated the effects of simple hydrophilic and hydrophobic molecules on the phase behaviour of lipid model membranes. Using a combination of steady-state fluorescence, differential scanning calorimetry (DSC), and fluorescence microscopy, we extracted structural information of the membrane, as well as on the thermodynamics of phase transition, upon interaction with different molecules. The use of environment-sensitive fluorescent probes to extract relevant information at the nanometer and micrometer length scales is also a key aspect of this work. Moreover, by using state-of-the art Cryo-TEM and neutron scattering we obtained a comprehensive picture of the interaction at the nanoscale level.

The main project of this thesis focuses on changes in the lipid bilayer phase behavior upon incorporation of polystyrene oligomers, motivated by an increasing concern regarding the presence of plastic waste in oceans, and their consequent degradation into possibly hazardous nanoparticles.

We Polystyrene in a single component lipid bilayer

A.3 SANS lamellar model

This model calculates the form factor from a lyotropic lamellar phase, assuming lamellae of uniform scattering length density that are randomly distributed in solution. No inter-lamellar structure factor is calculated. The scattering intensity I(q) is:

where q is the scattering vector, δ is the bilayer thickness and P (q) is the form factor defines as:

with ∆ρ the scattering length difference between the sample and the solvent.

B

Changes in multi-component phase diagrams upon incorporation of polystyrene

B.1 Estimation of gel domains area coverage in GUV

To estimate the area fraction of S o domains in liquid phase, we analyzed epifluorescence images of giant unilamellar vesicles. the area of solid domain was approximated to a circle of equivalent area. To correct for the sphericity of the vesicles, we tested two different approximations. For both approximations we obtained similar values of area coverage.

In case of vesicles presenting multiple domains or very elongated domains we subdivided the area covered by the solid phase into smaller approximated circles (Fig. ).

B.1.1 Approximation A: determination of arc length

The area of the domain can be obtained by the measured diameter l (Fig B

where α is the angle in radiants spanned by the length l. The angle α can be calculated by considering that

where h = R 2 -(l/2) 2 is the distance from the center of the projected l to the center of the circle. The fraction f of the solid area with respect to the area of the vesicle is defines as

B.1.2 Approximation B: determination of solid angle

An alternative approach to measure the area spanned by a domain is to use spher- Molecular quantities are defined as

Finally, we introduce the differences ∆g = g l -g g , ∆h = h l -h g , ∆s = s l -s g between the high and low temperature phases. We denote by T the absolute temperature, and β = 1/(k B T ) the inverse temperature factor. The probability of occurrence of a phase is proportional to exp(-βG). As the Gibbs freeenergy scales with the number of lipid, phases cannot coexist except but in a narrow temperature interval centered around T m .

A standard thermodynamic relation states that

On the other hand, at the coexistence temperature ∆G(T m ) = 0. One can expand to first order in T -T m the difference in Gibbs free-energy

The probability of occurrence of the gel and liquid phases reads

The transition takes place on a temperature interval ∆T given by βN ∆g =

To provide a phenomenological description of the melting transition, one introduces a cooperativity number N c as the effective number of lipids that share the same internal state. The system is then treated as an assembly of N/N c "bundles" changing state independently, with N/N c 1. The equilibrium enthalpy at temperature T is DPPC bilayers in solution of high sucrose content smoothly and reversibly with temperature. The latter case is therefore suitable to describe most experimental situations with a finite width, regular and reversible thermal capacitycurve determined e.g. in differential scanning calorimetry (DSC) experiments.

There is some freedom left in deciding whether the binary state is assigned to a whole lipid or just a lipid chain, or which lattice is most representative (the hexagonal lattice seeming the more appropriate), with all models in the end able to describe the observed behaviour [179]. Several Monte-Carlo studies were shown to successfully account for various situations of interest [START_REF] Ivanova | Physical review. E Statistical nonlinear and soft matter physics[END_REF].

To explain the main features of sucrose induced changes in the DPPC melting transition, we implement one such model and solve it by means of a mean-field approximation. Each binary state takes a value 0 (gel) or 1 (fluid) and describe a single lipid molecule internal state. The average internal value is therefore a real number p comprised between 0 and 1, which is readily interpreted as the probability to find a lipid molecule in the fluid conformation. For a given microscopic configuration, one introduces a configurational energy

as the difference between the actual system state, and a reference state where all lipids would be in the gel state. N l and N gl are respectively the number of lipids in fluid state, and the number of lattice bonds linking lipids in a different state (state mismatch). J is the mismatch gel-fluid state penalty parameter. The quantity ∆H determines the probability of the microscopic state, proportional to exp(-β∆H).

Note that it is unusual to deal with temperature dependent "Hamiltonians" in statistical physics. The approach used here means that a coarse-graining step is performed by averaging over the inner conformations of each lipid molecule, while partitionning them into two broad classes (gel and fluid state). Eq. (C.7) is something of an intermediate quantity between the true (molecular) microscopic energy and the macroscopic Gibbs free-energy.

β∆H can be expanded around the coexistence temperature:

The Gibbs free-energy associated to the configurational energy above reads in the large N limit:

where appears the entropy S associated to the many internal gel-fluid microscopic

or equivalently

Self-consistent equations are trivially satisfied by

at vanishing coupling J = 0, and must be numerically solved in the general case.

There is freedom in deciding if the interaction term -Jzp(1 -p) is of enthalpic or entropic origin. Assuming that J is enthalpic and does not depend on temperature T , one derives the mean-field enthalpy difference

that can be compared with the experimental DSC thermograms once the solution of eq. (C.12) is obtained. Moreover, one observes that, irrespective of the choice done regarding the interaction term, the difference ∆H(p = 1) -∆H(p = 0) reaches the expected limit value N ∆h, corresponding to the total latent heat upon melting completely the system from the gel to the fluid state.

Introducing the dimensionless coupling J = N A J RTm z, one finds that the critical value separating reversible and hysteretic temperature behaviour is Jc = 2 in the meanfield approximation. A quite sharp specific heat peak can therefore be obtained with DPPC bilayers in solution of high sucrose content Jc 1.94.

In the practical situation of a DPPC bilayer, when assigning binary variables to lipid molecules (not chains), treating lipids as basic degrees of freedom coupled with Jc = 1.94, and taking N A ∆h equal to the experimental value 38 kJ • mol -1 (9.1 kcal •mol -1 ) leads to a non negligible amount of the minor component into the major component around the location of the phase transition. This means that the area under the peaked curve d∆H/dT on a 10 • C temperature interval centered around T m = 273.15 + 41.8 = 314.95K gives a value 28 kJ•mol -1 , smaller than the experimental one. This is inherent to the "Ising" like treatment of the internal degrees of freedom, and is also true for Monte-Carlo "exact" sampling of the configurations.

To get around this shortcoming, one can decide on a phenomenological ground to assign a larger value to the constant N A ∆h. We found that at mean field level, with Jc = 1.94, the correct ∆H(T m + 5) -∆H(T m -5) = 38 kJ • mol -1 value is recovered for N A ∆h = 56 kJ • mol -1 (13.4 kcal•mol -1 ). There is then 16% of fluid lipid at

C.3 Influence of the sucrose on the gel transition

Increasing concentrations of sucrose in solution lead to a noticeable drop in latent heat (area under the specific heat curve) with only a tiny increase in the appearent melting temperature (of the order of 1K).

In first order phase transitions, the coexistence temperature T m coincides with the ratio ∆h/∆s. If the sucrose was only acting on changing the enthalpy jump ∆h , then keeping the melting temperature constant by 1 part in 300 would require a quasi-perfect matching of the entropy variation ∆s , with T m = ∆h/∆s ∆h /∆s . On the other hand, it is well known that lipid melting temperature is extremely sensitive to molecular details. Perdeuteration of the DPPC alkyl chains, for instance, lowers the transition temperature by 4 • C. Shifting one C16 fatty acid chain link with glycerol from sn-2 to sn-3 position has the same consequence. Going from cis to trans double bond insaturations raise the melting transition of DOPC by 60 K.

If one thinks of the action of sucrose as simply dehydrating the lipid headgroups, then a strong elevation of the melting transition temperature would be expected. Yet, the observed change goes in this direction, but in much weaker proportions.

In addition, hydrophilic sucrose molecules are not really expected to interact with the bulk of hydrophobic alkyl chains region, which is where the largest part of the contribution to the enthalpy change ∆h is expected to arise from. C.3. Influence of the sucrose on the gel transition 123 Drops in latent heat at the transition can be alternatively explained by the presence of domains. If one assumes that in the presence of sucrose, lipids get separated into sucrose-depleted and sucrose enriched domains, and that only sucrose depleted domains melt as usual, with other domains remaining in the gel phase, then the result would also be a neat decrease in experimental latent heat. However, here is no clear reason for such domains to form, and this mechanism lacks experimental support.

We propose here an alternative mechanism where sucrose adsorbs indistinctly in the gel and fluid phases. Lipids that are in close contact with sucrose molecules are assumed to melt at a slightly higher temperature T m , and more importantly, to behave in a less cooperative way than in pure lipid water solutions. This could be justified for instance by saying that gel-fluid mismatch configurations are eased by surrounding sucrose molecules.

Adapting the previous model, the configurational energy becomes

with N l the number of free lipids in fluid state, N l the number of lipid in fluid state in contact with sucrose, N gl the number of unlike gel-fluid free lipid pairs, N gl the number of unlike gel-fluid lipid pairs, both in contact with sucrose and N gl the number of unlike gel-fluid pairs with one lipid free and one lipid in contact with sucrose, J, J , J being the corresponding mismatch penalties.

We assume now that the probability for a lipid to be in contact with sucrose is σ, that p is the average probability of finding free lipids in fluid state, and p the average probabiliy of finding lipids in contact with sucrose in fluid state. The average configurational energy can be expressed in the mean-field limit.

DPPC bilayers in solution of high sucrose content

where for simplicity we assume ∆h ∆h , T m T m at the first order of the temperature expansion. The mean field configurational entropy then becomes:

(C.17)

The Gibbs free-energy β∆G(p, p , σ, T ) = β∆H -S/k B must now be minimised with respect to p and p . We do not perform a minimisation over σ because we assume σ imposed by the sucrose molarity ([Sucrose]) of the hydrating solution.

The self-consistent equations become

With the numerical solution for p(T ), p (T ) determined, the temperature dependent enthalpy is readily obtained from eq. (C.16).

In practice, equations (C.18) are solved for each temperature T using the Newton-Raphson iteration scheme, starting initially from the exact solution at J = J = J = 0, and iteratively converged for increasing values of the coupling constant. Below critical coupling Jc = 2, the method is fast and accurate.

An interesting behavior is obtained for the following choice of parameters:

• T m = 273.15 + 41.8K, T m = 273.15 + 41.8 + 5.0K,

• J = 1.94; J = J = 0.97,

• N A ∆h = N A ∆h = 56 kJ • mol -1 .

The three graphs below explains how the decreased cooperativity mechanism works: By comparison, the exact value of the critical point on a 2d hexagonal lattice Ising model is βJ Ising = 0.2746 . . . (see [START_REF] Fisher | [END_REF], page 671). In our notations, this corresponds to J = 0.5432. Back to the original parameter J, one finds (with z = 6, and 1 cal=4.18 J):

• mean-field:

• exact:

The mean-field approximation underestimates the magnitude of the coupling constant that is needed to correlate the spins to a given degree.

We can compare now the values used in this study to those of Jerala et al. [179]. In (415 J•mol -1 ). We therefore globally operate closer to the critical point.