During this PhD journey I have met and interacted with several wonderful people weather it was for work, exchange

I would like to thank all the members of the committee defence of my thesis, Dr. Damien Quemener, Dr. Stéphane Pezennec, Pr. Murielle Rabiller-Baudry and Pr. Christelle Guigui, first for accepting to evaluate my thesis work, but also for their insightful comments and for making the discussion that pleasant.

Within the framework of this PhD, I had the chance to travel to Taiwan as an invited research student for 6 weeks in the Research and Development Center for Membrane Technology (Chung Yuan Christian University, ChungLi, Taiwan). Thus, I thank Pr. Chang Yung, Pr.

Antoine Venault and all the students of the lab for welcoming me and helping me to carry out my experiments in the best possible conditions. Special thanks to Corrine Routaboul of the Toulouse Institute of Chemistry (ICT) for having trained me on the ATR-FTIR microscopes, and for facilitating my access to the instruments. I would like to acknowledge also the entire LGC staff, professors, engineers, technicians, administrative officers and especially the students, among whom I have made friends, for the three pleasant years I have shared with them, and for all the good convivial moments. I thank in particular my very good friend Dihia, for being there through the ups and downs and for her support, as well as Nabiil and Fatma for also being good friends to me and for all the things we shared together.

Words are not enough and will never be enough to thank the people who are dearest to me, my parents, to whom I owe my existence, my happiness and all the success belongs to them in the first place. I would like to express my deep sense of thanks to them for their encouragements, their unconditional trust, endless love that raised me up again when I got weary and for believing in me and being behind me whatever the circumstances are. With my parents always come my brothers Habib and Abdelhamid and my lovely sister Besma who also fill me with their unconditional love and support, I would like to thank them simply for being the best family I could have, and I also thank all the other members of the family for their support.

Finally, I would like to dedicate this thesis to my family and especially to my dear uncle Abdou and my dear aunt Zahra, two people who meant a lot to me and who suddenly passed away together because of the COVID pandemic, without giving me the chance to see them one last time, I hope they rest in peace in heaven.

The selective separation of blood proteins and cells is of a major importance to address some target diseases and for several medical applications such as wound healing. The challenge is to provide the most efficient and cost-effective technology that can ensure that. Among many other technologies, membranes processes have been revealed as promising techniques.

However, their nonspecific interactions with proteins make them prone to biofouling, which in turn limits their application in biomedical fields. Therefore, the aim of this work is to design tailored membrane for blood proteins screening via surface modification. This latter is performed by coating onto the membrane surface some functional groups such as polystyrenepoly acrylic acid (PS-PAA) and polystyrene-poly ethylene glycol (PS-PEG) copolymers, either to promote a specific interaction with target proteins or to reduce their non-specific interaction.

First, the potential interactions that could take place between PAA, PEG brushes and some abundant proteins in plasma such as human serum albumin (HSA) and ɣ-globulin (IgG) have been investigated in solution using Small Angle X-ray Scattering (SAXS) combined to chromatography. Thus, PAA has been found to form complexes with HSA in some specific physicochemical conditions of pH and ionic strength. This HSA-PAA binding has then been revealed to be reversible depending on the pH and the ionic strength of the medium and its stoichiometry was shown to be related to the PAA size. Whereas IgG did not exhibit the same behaviour as HSA towards PAA, it was found to be partially aggregated in our conditions. PEG did not display any interaction with both proteins. After that, Fourier Transform Infrared spectroscopy (FTIR) mapping showed that surface modification of the membranes by copolymers coating was successful, although the distribution of copolymer was heterogeneous over the membrane surface. The static adsorption of HSA onto membranes coated with PS-PAA was found to be also correlated to the pH and ionic strength as it was in solution, while that of IgG was not. Then, the adsorption of HSA onto a membrane coated with PS-PEG has been shown to be mitigated. Eventually, filtration experiments disclosed the same trend of HSA adsorption in dynamic mode even though the membrane permeability was reduced by the increase of the size of the copolymers brushes deposited onto the surface.

Overall, this study enabled us to understand the behaviour of some abundant blood proteins in contact of some functional groups either in solution or at the interface of a modified membrane.

Résumé II

Résumé

La séparation sélective des protéines et des cellules sanguines est d'une importance capitale pour traiter certaines maladies cibles et pour plusieurs applications médicales telles que la cicatrisation des plaies. Le défi consiste à fournir la technologie la plus efficace et la plus rentable qui puisse garantir cette séparation. Parmi de nombreuses autres technologies de séparation, les technologies membranaires se sont révélées être prometteuses. Cependant, leurs interactions non spécifiques avec les protéines les rendent vulnérables au bio-colmatage, ce qui limite leur application dans le domaine biomédical. Par conséquent, l'objectif de ce travail est de concevoir une technologie membranaire adaptée pour le dépistage des protéines sanguines en utilisant la modification de surface. Cette dernière est réalisée en déposant certains groupements fonctionnels sur la surface de la membrane, tels que des copolymères polystyrènepoly acrylique acide (PS-PAA) et polystyrène-polyéthylène glycol (PS-PEG), soit pour favoriser une interaction spécifique avec des protéines cibles ou pour réduire leur interaction non spécifique. Dans un premier temps, les interactions potentielles qui pourraient avoir lieu entre les brosses de PAA, PEG et certaines protéines abondantes dans le plasma telles que la sérum-albumine humaine (HSA) et la ɣ-globuline (IgG) ont été étudiées en solution en utilisant la Diffusion des rayons-X aux petits angles (Acronyme anglais : SAXS) combinée à la chromatographie. Ainsi, nous avons constaté que le PAA forme des complexes avec l'HSA dans certaines conditions physico-chimiques spécifiques de pH et de force ionique. Cette liaison HSA-PAA s'est ensuite révélée réversible en fonction du pH et de la force ionique du milieu, et il a été démontré que sa stoechiométrie était liée à la taille du PAA. En revanche, l'IgG n'a pas présenté le même comportement que l'HSA vis-à-vis du PAA, mais elle s'est avérée être partiellement agrégée. Quant au PEG, il n'a montré aucune interaction avec les deux protéines.

Ensuite, la cartographie par spectroscopie infrarouge à transformée de Fourier (FTIR) a confirmé le succès de la modification de surface par dépôt de copolymères sur la surface de la membrane, bien que la distribution du copolymère y soit hétérogène. L'adsorption statique de l'HSA sur les membranes revêtues de PS-PAA s'est avérée également corrélée au pH et à la force ionique comme elle l'était en solution, alors que celle de l'IgG ne l'était pas. Ensuite, il a été démontré que l'adsorption de l'HSA sur la membrane recouverte de PS-PEG était réduite.

Enfin, les expériences de filtration ont révélé la même tendance à l'adsorption de l'HSA en mode dynamique, même si la perméabilité de la membrane était réduite par l'augmentation de la taille des brosses de copolymères déposées sur la surface. Résumé III Globalement, cette étude nous a permis de comprendre le comportement de certaines protéines sanguines abondantes au contact de certains groupes fonctionnels que ce soit en solution ou à l'interface d'une membrane modifiée. 2. 12. Experimental SAXS scattering intensity I(q) of HSA 75 µM (blue line) compared to the SAXS intensity calculated with CRYSOL from HSA crystal structure 6EZQ (red curve). The inset represents the Guinier plot (Ln I(q) vs q 2 in red line) with the residual (green curve) that allow to determine the radius gyration using Primus. The HSA 3D structure shown in cartoon representation using PyMOL software. . 3. 30. FTIR maps for the adsorption of HSA at different ionic strength on PVDF membranes coated with PS30-b-PAA5 and PS100-b-PAA107 using ID process, 2 hours of immersion and a copolymer concentration of 5 mg•mL -1 ; [HSA]= 1 mg•mL -1 . .................... 145 Figure 3. 31. The evaluated mean intensities of the peaks area at 700 cm -1 and 1660 cm -1 showing respectively the coating on different samples and the adsorption of HSA onto these same samples at different ionic strength; [HSA]= 1 mg•mL -1 . .............................................. 146 Figure 3. 32. FTIR maps for the adsorption of IgG at different pH (5, 7 and 8) on PVDF membranes coated with PS30-b-PAA5 and PS100-b-PAA107 using ID process, 2 hours of immersion and a copolymer concentration of 5 mg•mL -1 ; [IgG]= 1 mg•mL -1 . ..................... 147 Figure 3. 33. The evaluated mean intensities of the peaks area at 700 cm -1 and 1660 cm -1 showing respectively the uniformity of coating on different samples and the adsorption of IgG onto these samples at different pH;

List of figures

Membrane technology for medical applications

A membrane is a thin layer of semipermeable material that could be used for separation processes in liquid or gas phase as a driven force is applied across the membrane (pressure, electrical potential, etc.). It is an interphase between two adjacent phases acting as a selective barrier, regulating the transport of substances between two compartments. In this manuscript, membranes for liquid phase separations operated under pressure are mainly reported. In fact, membrane separation technology could be used to purify some solutes contained in a mixture due to the difference of their properties such as their size or of a specific interaction that they exhibit towards the membrane. The main advantages of membrane technology as compared with other unit operations in (bio)chemical engineering are related to the transport selectivity that could possess the membrane. Separations with membranes do not require additives, and they can be performed isothermally without change of state, hence at low temperatures and at low energy consumption compared to other thermal separation processes [1]. Certain properties of membrane separation technology, such as durability, porosity, permeability, stability, and selectivity, make them an indispensable element in various industrial applications [2]. In addition, upscaling and downscaling of membrane processes as well as their integration into other separation or reaction processes are easy. where improvement of biocompatibility remains a challenge [5]. Blood oxygenators and filters for infusion solutions represent also a large group with respect to membrane consumption for medical devices. Among other medical applications, membranes are used for liver support systems, artificial pancreas, therapeutic apheresis and plasma donation [6]. Membranes are also used in drug delivery systems and diagnostic devices [7]. Those applications for membranes are by far not all, there is a continuous development of devices for specific separation applications. Considering only extracorporeal devices containing membranes for blood purification several new therapy devices are emerging.

Membrane technologies have now been industrially

Membrane materials

Chapter

I

Thereby, artificial membranes are used in medicine to treat blood for a broad variety of therapeutic purposes. In most treatments, blood is continuously withdrawn from the patient's blood circulation and brought in extracorporeal circulation into direct contact with the membranes assembled in the device. Membranes are used as permselective barriers to permit transport of selected solutes to/from the blood, while hindering the loss of essential blood constituents, and to regulate the rate at which solutes are transferred across the membrane so as to maintain the patient's homeostasis [8].

For such processes rather complex, a number of factors contribute to make the development of adequate membranes and devices, among which are the physical-chemical properties of the species to be transported or rejected, the interactions between membranes and body fluids that continuously modify both membrane and blood properties, and the need for materials with suitable properties.

Membrane materials

Membrane materials lie at the center of membrane technology by determining membrane separation performance, cost, operational stability, and process design. That's because the material a membrane is made of determines its physical and chemical properties, for example, its physical strength and workability; its resistance to temperature and chemicals, which determines the sterilization methods that can be used without altering its structure and separation properties; and its biocompatibility in the case of biomedical applications.

The membrane process conditions must be engineered very carefully, but the performance limits are clearly determined by the membrane itself. Depending on the application the membrane is used for, this last could be formed of different materials.

Extensive research efforts have been dedicated to investigating novel membrane materials leading to the emergence of three major membrane material categories: organic (polymeric), inorganic (ceramic) materials, and biological materials [9]. Polymeric membranes dominate the present separation market due to their relatively low cost and excellent mechanical strength.

Organic membranes are either cellulose based or composed of modified polymers. The main polymers constituting the organic membranes include Polyamide (PA), Polysulfone (PSf), Polyethersulfone (PES), Polyvinylidene fluoride (PVDF), and Polypropylene (PP).

Membrane fouling and surface modification

Chapter

I

Those materials are less resistant to high temperature and aggressive chemicals compared to inorganic or metallic membranes. However, they are still widely used due to easy preparation, reasonable expense (low cost), high efficiency, lower energy requirement and flexibility in membrane configuration [1]. For that reason, polymeric membranes are attractive materials for various applications, such as filtration, microelectronic devices, biomaterials, and so on. For instance, polymeric materials have been widely applied in the biomedical field such as in dental/bone materials, implants, wound dressing, prosthetic materials, drug carriers, and tissue engineering scaffolds. This is owing to their desired mechanical and physical properties, manufacturability into various shapes, sterilizing capability, and ease of modification with multiple functionalities [10].

The performance of polymeric materials in many applications relies largely upon the combination of bulk (e.g. mechanical) properties and the properties of their surfaces [11].

However, those materials very often do not possess both the surface properties needed for these applications and good mechanical properties at the same time, which is critical for their successful application.

Due to this dilemma, surface modification of membrane without changing the bulk properties has been a topical aim in research for many years, mostly because surface modification provides a potentially easier route to obtain new surface properties than other techniques (e.g. polymer blending) [11]. Another advantage of surface modification is that it permits to enhance or prevent specific interactions (affinity) during separation processes. The field is still receiving extensive attention as new applications of polymeric materials emerge rapidly, especially in the fields of biotechnology, bioengineering, and nanotechnology [12].

Membrane fouling and surface modification

Fouling is the deposition of retained particles, colloids, macromolecules, salts, etc., at the membrane surface or inside the pores which either temporarily or permanently alters their transport properties such as their permeability or selectivity. The foulants could be inorganic, organic, or biological substances depending on the application the membrane is used for. They not only physically interact with the membrane surface but also chemically degrade the membrane material sometimes [13]. In the case of membranes for biomedical applications we talk more about biofouling by biological substances like proteins, human cells, bacteria, etc.

For such applications synthetic hydrophobic materials are often chosen as matrix materials but their hydrophobicity makes them prone to biofouling, which in turn limits their application [14].

Membrane fouling and surface modification

Chapter

I

This phenomenon could be minimized by periodical washing with chemicals, but it results in the simultaneous degradation of the membrane material's lifetime, and it could involve additional environmental and economic costs.

The management of fouling involves different strategies: improvement of pre-treatment processes, optimisation of cleaning protocols, amelioration of membrane module design, and development and modification of membranes taking into consideration different parameters such as hydrophilicity, charge and the roughness of the surface [13].

Since the separation process by membrane is essentially a surface phenomenon, it is a natural consequence to modify membrane surface for reducing the fouling. Surface modification of membranes is thought to be equally important to the membrane industry as membrane material and process development [15].

In membrane manufacturing, surface functionalization of membranes has already become a key technology. The aims of surface modification of a membrane are essentially two-fold [16].

First, improvement of the selectivity as shown in Figure 1. 2.B, or even the formation of entirely novel separation functions. The second fold is the minimization of undesired interactions (adsorption or adhesion, or in more general terms membrane fouling) that reduce the performance as illustrated in Figure 1. 2.B. This can be achieved via the introduction of additional (tailored) interactions (affinity and responsiveness). Novel membranes with a high selectivity, e.g. for special biomolecules are in high demand. Consequently, particular attention is paid to truly molecule-selective separations, i.e. advanced nanofiltration and ultrafiltration membranes [17]. In addition, a membrane selectivity that can be switched by an external stimulus or can adapt to the environment/ process conditions would be an important feature.

Membrane fouling and surface modification

Chapter

I

In the overall process of membrane modification, many factors need to be considered, such as uniformity, reproducibility, stability, process control, and reasonable cost, together with precise control over functional groups, which is a big challenge [12]. Among the surface modification techniques developed to date, grafting and coating are common processes as it allows control of grafting or coating density, thickness of the modifying layer, and chemistry (through polymer material choice) without significantly changing the structure of the membrane.

Grafting processes establish covalent bonding between the surface modifier and the membrane surface through "grafting from" or "grafting to" techniques, illustrated by the scheme in Figure 1. 3 adopted from [18]. It creates stronger interactions resulting in better stability than coating processes. In grafting from, polymer grows from the membrane surface by putting the membrane in contact with a monomer solution given that there is additional energy supplied to initiate the polymerization and to activate the membrane. While in grafting to, a known composition of polymer covalently bonds to the surface activated membrane. Both grafting methods require additional energy and time-consuming multiple step process which generates high costs. In addition, membrane pretreatment with alkali solution or irradiation can damage the properties and structure of the membrane, thereby affecting the membrane's performance.

Figure 1. 3. Scheme depicting the two processes "grafting from" or "grafting to" used for surface modification [18].

Conversely, coating is readily achieved through immersion, spray coating, dip-coating, and spin coating making it suitable for large-scale production at a reduced cost. In this process, surface-modifying molecules self-assemble on the surface of membrane establishing noncovalent (hydrophobic, electrostatic, etc.) interactions [11]. For example, it could be a functional copolymer with hydrophobic groups forming a strong affinity with a hydrophobic 4. Membrane surface functionalization by polymers adsorption Chapter I membrane. Since surface modifiers interact with the membrane through non-covalent interactions, it may not be stable resulting in detachment from the membrane surface. Thus, the success of the coating depends on the extent of interaction between the surface modifier and the membrane surface.

Membrane surface functionalization by polymers adsorption

The adsorption of soluble polymers at solid interfaces is a highly complex phenomenon with a vast number of possible configurations of the molecules at the surface. The driving forces of adsorption processes are numerous including covalent or chemical bonding forces, interactions between polar molecules, electrostatic interactions, hydrogen binding, hydrophobic and hydrophilic interactions, etc. [19]. As illustrated in Figure 1. 4, depending on the nature of these interactions we distinguish between chemisorption that involve covalent bonding formation, and physisorption related to van der Waals forces between dipoles, to electrostatic attractions or to hydrophobic interactions, etc. [20]. This latter does not imply a significant change in the electronic orbital patterns of the species involved in the adsorption. It is a reversible phenomenon that involves low adsorption energies of the order of a few kilocalories per mole, and it is a process generally favoured by low temperatures.

Adsorption isotherms

A multiplicity of adsorption isotherms that describe the adsorption of solved substances at solid surfaces has been reported in the literature [21]. They were classified by Giles and al, and later a theoretical basis was given to the classification adopted [22]. Thus, the several equations that have been proposed to model isotherms have either theoretical or empirical foundations [23]. The most common isotherm model is the L class described by Langmuir equation, where interactions between adsorbate molecules are considered as negligible. This model describes the monolayer adsorption and it assumes that the surface is energetically homogeneous, that no interactions between the adsorbed molecules take place, that the molecules remain fixed once adsorbed, and that the thickness of the monolayer corresponds to that of a molecule [21].

If the driving forces for adsorption are strong (e.g. electrostatic interactions in the case of polyelectrolytes), if the behaviour of the adsorbate is not ideal, e.g. multilayer adsorption, or if equilibrium conditions are not reached during measurement, deviations from this isotherm may happen [19]. Overall, Langmuir law is considered as applicable in the case of physisorption only where the driving force is van der Waals interactions. A special variant of the L class is the H class that represents high interaction forces between adsorbate molecules. In this case, we speak about cooperative adsorption.

Isotherms of the C class are characterised by a constant partition of the solute between the solid surface and the solution and it can be observed in the case of penetration of adsorbate molecules in the surface region of the solid. Linearity means that new sites are created during adsorption. This is interpreted by the fact that the initially adsorbed molecules have caused the expansion of the pores of the solid, which allows other molecules to penetrate.

In the case of long-chain molecules like polymers, effects from the adsorbed molecules must be observed including configuration effects [24], aggregation effects, micelles formation, multilayer adsorption, molar weight, chain stiffness [25].

Interactions of functionalized membrane with proteins

Polymer-coated surfaces have been widely studied to control material interactions with the biological environment. Thereby, the understanding of bio-physicochemical interactions at the nano-biointerface and the driving forces is of paramount importance. Wherefore, several studies have sought to investigate the forces leading to these interactions [26], [27]. It has been reported in many studies that among all possible parameters, water-mediated hydrophobic and hydration forces are considered to be the major players in protein adsorption [26]. Thus, it has been disclosed that proteins should not be able to displace water from the surface and adsorb on hydrophilic surfaces, whereas the opposite behaviour is expected for hydrophobic surfaces.

However, this concept was not enough to explain all experimental results, where several drawbacks remained, as the involvement of these forces in causing adsorption in the case of small proteins [28], [29]. Besides, this concept does not consider the competitive adsorption of different mutually interacting proteins.

Further studies recognized that protein adsorption was governed by various polar and nonpolar interplay mechanisms. The latter include nonspecific electrostatic and ionic screening effects as well as more local noncovalent interactions such as salt bridges or hydrogen bonds.

The polar interactions can be manipulated by a change in pH or salt concentration. Nonpolar interactions include van der Waals and solvent-mediated phenomena such as hydrophobic attraction [28] or hydration repulsion [29].

Nel et al. [27] have listed the main possible forces involved in proteins adsorption from biological systems, and they explain their origin and nature, as well as the possible impact it could have on the interface as shown in Table 1

Methods for characterization of functionalized membranes and protein adsorption

The surface modification efficiency could be judged only by assessing the performance of the membrane regarding the purpose for which the modification was performed. Thus, it is essential to determine the modified membrane surface properties (chemical composition, surface wettability, modification layer density, and morphology) through surface characterization to understand its performance and stability. There are numerous characterization methods and they can be thermodynamic, spectroscopic or imaging methods [19]. Among thermodynamic techniques the following ones can be listed [19]: calorimetry, volumetry, gravimetry, inverse gas chromatography, electrokinetics and wetting measurements.

The spectroscopic techniques are often used for surface chemical analysis, and it includes mainly infrared spectroscopy (ATR-FTIR), surface plasmon resonance spectroscopy, fluorescence spectroscopy, X-ray photoelectron spectroscopy, Raman spectroscopic and energy dispersive X-ray spectroscopy.

Other methods such as contact angle measurements can be used for determination of membrane wettability, microscopy techniques for analysis of surface morphology, and characterization of surface charge.

Furthermore, the evaluation of the membrane permeability to the solvent and its retention rate during filtration process are among methods that allow to assess the performance of the membrane. The methodology to assess these parameters is explicitly detailed in chapter IV where dead-end filtration experiments are discussed. Thus, as stated in section 3.2 of chapter IV, the permeability can be calculated using the Darcy law given by equation 4.1, while the retention could be evaluated using the equation 4.3.

Surface chemical analysis

Determination of the membrane surface chemical composition is essential to verify the presence of coating and adsorbed foulants. Their amount can be correlated to assess the modified membrane's performance and stability. As stated above, there are several characterization techniques for this purpose, and the most frequently used according to the literature are summarized in Table 1. 2. However, it should be considered that membranes are generally used in their wet state while some characterization techniques only allow analysis using samples in its dry state. Thus, it is important to keep in mind that membranes could behave differently depending on the characterization technique used. 1. 2. Spectroscopic methods used in membrane surface characterization [30].

In infrared spectroscopy (IR), the sample is irradiated with infrared light. When the frequency of the incident light matches the vibration of the molecules in the sample, the molecules absorb the radiation. The remaining transmitted light is detected and converted to a spectrum showing the changes in the infrared intensity as a function of frequency (or wavenumber) [30]. The peaks in the spectrum correspond to bond stretching or deformation specific to functional groups present in the sample and its intensity could be correlated to the amount.

Raman spectroscopy uses a monochromatic light source that can be in UV, visible, or nearinfrared regions. This technique uses the concept of Raman scattering to generate a spectrum with intensity of the scattered light as a function of Raman shift (energy difference between the incident and Raman scattered photons). Groups with double and triple C-C bonds, disulfide bonds, and C-H bonds are detected easily with Raman spectrometer, while it is the opposite for water [30]. Consequently, this is interesting for membrane characterization as it allows analysis of wet samples. Raman spectroscopy is complementary to FTIR (Fourier Transform Infrared spectroscopy) since bands that are strong in infrared spectrum are weak in Raman spectrum and vice versa. In X-ray Photoelectron Spectroscopy (XPS), the sample is placed under ultrahigh vacuum and is irradiated with focused X-rays resulting in photoemissions. The emitted electrons and their binding energies unique for each element will be detected allowing identification of the elements present in the sample. Results are presented as spectrum with intensity as a function of binding energy. In addition, the peak intensity is proportional to the quantity of the element in the sample. This method is non-destructive and non-conducting samples, such as polymeric membranes, can be analysed without conductive coating. However, the membranes should be analysed in their dry state, and the analysis time should be kept short since increased exposure period could lead to sample deterioration [30].

Energy-dispersive X-ray Spectroscopy (EDS) is coupled with an electron microscope where the sample is irradiated with an electron beam followed by X-ray generation which is unique for each element [31]. However, this technique requires that the sample chamber is under vacuum condition, thus, membranes must be analysed in their dry state. In addition, nonconducting samples, such as polymeric membranes, should be coated with a conductive material prior to the analysis. There is also a possibility of alteration in the composition of polymeric samples upon exposure to the electron beam.

Fluorescence microscopy could be useful when it comes to studying the presence of foulants such as proteins on a membrane surface. Indeed, when multiple proteins are present on a sample surface, FTIR technique may not be able to distinguish them from one another due to their almost similar composition. Fluorescence microscopy uses laser of a specific wavelength as the incident light source that will excite the fluorescent molecules or fluorophores in the sample.

The sample will then emit fluorescent light of another wavelength in which the observer can view through the fluorescent microscope. This method can visualize auto-fluorescent specimens on a surface [32].

Membranes wettability

Surface wetting mostly governs the interactions between solid surfaces and fluids. Thus, this phenomenon is essential when investigating membrane processes since it influences flux, rejection, and fouling. The most common method to determine the degree of wetting is through the measurement of the contact angle formed by a droplet of liquid on membrane surface (Figure 1. 5). It depends on the interfacial tensions of all the interfaces involved. For instance, when a drop of water is put on a solid surface under air, the shape of the drop changes under the pressure of the different surface-interfacial tensions until equilibrium is reached [30]. A hydrophilic surface will favour the interaction with water, thus, the water droplet spreads on the surface and the measured contact angle is small. Conversely, a hydrophobic surface will repel water, thus, to minimize their interaction, the contact angle formed will be high.

It is important to note that on porous substrates such as porous membrane, the effect of some factors such as liquid absorption, pore structure, surface roughness and drop size should be considered [33]. It should also be considered that the contact angle of a sample could change depending on the environment implying that measured contact angles could be different for dry and wet states. For instance, polymeric membranes tend to reorganize their structure to adapt to the surrounding medium [34]. Consequently, its surface properties will vary depending on the environment it is currently in. Since membranes are generally used in their wet state, it would be more interesting to observe its surface wetting properties in an environment similar to its working condition that is their wet state. Moreover, the contact angle measurement requires a number of precautions to be used effectively, including surface cleanliness, liquid purity, temperature control, multiple measurements, etc.

Contact angle analysis is a convenient tool to characterize grafted polymer surfaces, because contact angles are very sensitive to changes in surface chemistry [31]. Eventually, there are several methods to measure contact anglesessile drop method, captive bubble/drop method, and Wilhelmy plate method [30]. Several studies quantified the amount of the deposited brushes on a membrane surface using the difference of mass [35]. It is done by measuring the weight difference between the modified (WM), and unmodified (WD), dry membranes with respect to membrane surface area (Amembrane) (equation 1.1).

𝐶𝑜𝑎𝑡𝑖𝑛𝑔 𝑑𝑒𝑛𝑠𝑖𝑡𝑦 = 𝑊 𝑀 -𝑊 𝐷 𝐴 𝑚𝑒𝑚𝑏𝑟𝑎𝑛𝑒 (1.1)
Although this method is fast and simple, it is not a very sensitive method. In fact, a previous study revealed that coating density started to reach a plateau at copolymer concentrations beyond 3-5 mg•mL -1 when FTIR imaging showed that there was still an observable difference between 5 and 10 mg•mL -1 concentration [35]. Thus, this method exhibits some inaccuracies due to the non-consideration of the humidity during measurement. Besides. Besides, it cannot verify the homogeneity of the coating. It does not consider the contribution of the pores to the surface area and the amount of modifying functions that penetrate the pores during modification process. Lastly, membranes could potentially absorb moisture when exposed to air, which could lead to overestimation of the measured weights. Thus, it is recommended to consider the coating density trend instead of the actual value [35].

Mass balance in solution using UV Visible spectroscopy

UV Visible spectroscopy is an analytical technique where a sample is irradiated with light of different wavelengths in the visible, ultraviolet, and near-infrared regions [32]. Depending on the content of the sample, the light is partially absorbed. The remaining transmitted light will be detected and recorded as a function of wavelength creating the sample's UV-visible spectrum, which can be used to identify or quantify the content of the sample. Generally, the results are presented as absorbance as a function of wavelength since the intensity of the absorbance is directly proportional to the concentration of the analyte. Also, the concentration can be obtained quantitatively by measuring absorptions of multiple standard solutions with known concentration followed by the application of Beer-Lambert's Law.

Since the initial copolymer concentration is known, then the difference in concentration before and after the immersion is the amount adsorbed on the surface. Some researchers used UV-visible spectrophotometer to quantify the adsorbed amount of BSA and lysozyme on PVDF membranes. The remaining concentration of the protein on the solution after the immersion process was determined by measuring the absorbance at 280 nm [36]. However, this method does not consider the amount of coating that penetrated the pores.

Characterization of protein adsorption in solution

A way to better understand proteins adsorption on membrane surfaces is to study in solution the interactions with the polymers or the functional groups used for the membrane surface functionalization. Another method that was revealed to be effective for proteins adsorption study in solution was the use of colloidal core-shell particles that exhibit a large surface to allow the adsorption of a high amount of protein in small volumes [37].

Thus, a wide variety of methods can be added to those listed previously to characterize proteins adsorption [26]. Among these methods calorimetry utilizes the enthalpic effect generated by the binding of proteins to polymers in solution to provide direct thermodynamic information that is not available on planar surfaces [38].

If the concentrations are not too high, spectroscopic techniques such as fluorescence spectroscopy can be applied to monitor adsorption of fluorescent-labeled proteins [37]. Another spectroscopic technique is FTIR, that can be used to determine the secondary structure of adsorbed proteins with great precision, because it is not hampered by the turbidity of these aqueous suspensions [26].

Scattering methods as small-angle X-ray and neutron scattering (SAXS, SANS), have been also extensively used for the analysis of proteins uptake by polyelectrolyte brushes in solution [37]. Proteins have a strong X-ray contrast in water, which leads to a marked change of the scattering curves caused by their adsorption or uptake by other solutes. Besides, investigation of scattering data could furnish information about the radial distribution of the adsorbed proteins within the layer and the total amount of adsorbed protein.

Added to those stated above, others techniques can be used to characterize proteins-polymers systems in solution. For instance, dynamic light scattering (DLS) is used for the evaluation of the hydrodynamic diameter of the proteins-polymers system, size exclusion chromatography (SEC) determines the molecular weight, while UV-visible spectroscopy allows to estimate the amount of adsorbed protein [26]. Blood is a fluid transported in the vascular system through veins and arteries that supply nutrients and oxygen to different parts of the body, which is necessary for its survival and development while carrying away metabolic waste. It is 55% plasma (91% water and 9% proteins, ions, nutrients, wastes, etc.) and 45% erythrocytes (red blood cells), leukocytes (white blood cells) and thrombocytes (platelets). Erythrocytes carry the oxygen, leukocytes are for immune response, and thrombocytes' main function is to stop bleeding through blood clot formation after adhesion to the wall of the damaged vessel [39].

Whole blood can be delivered directly to patients suffering from active bleeding after major trauma without undergoing separation [40]. However, it can also be separated into different specific fractions (e.g. red blood cells concentrates and platelets concentrates), some of which can address direct blood conditions.

Red blood cells concentrates are essential to patients who suffer from severe bleeding or during surgical operations [41]. They are also commonly provided to premature infants to address symptoms of anemia [42]. The importance of red blood cells in oncology has also been shown recently, as membrane-modified red blood cells could act as natural chemotherapy drug carriers to specifically target cancer cells [43].

As for platelets, their transfusion is necessary for patients undergoing intensive medication (chemotherapy), hematological disease, and surgery [44]. In addition, the platelets concentrate or usually called Platelets Rich Plasma (PRP) play an important role in many processes such as coagulation, immune response, angiogenesis and the healing of damaged tissues. That's because platelets, are an important reservoir of proteins called growth factors that regulate cell growth and division. Those proteins contained in the granules of platelets include plateletderived growth factor (PDGF), transforming growth factor (TGF), platelet factor interleukin (IL), platelet-derived angiogenesis factor (PDAF), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), insulin-like growth factor IGF and fibronectin [45].

From above it can be concluded that separation of blood components, and the use of each fraction alone could be better for more targeted applications.

The separation of blood components is often done by centrifugation, a fast technique but energy demanding. It uses the action of gravitational force to separate blood into three major a mixture of leukocytes and thrombocytes (platelets) in between [46]. Thus, centrifugation remains ineffective to separate white cells from platelets because they have almost the same density. Adding to that, if it is not done in proper conditions, it can lead to hemolysis of red blood cells (RBC) resulting in contamination of plasma [47].

A slower alternative technology but less energy demanding, is separation by porous membranes in which the separation is primarily based on size differences. But after membrane functionalization by introducing some smart groups into the surface to control interactions with different components, both the particle size and the particle's affinity for the membrane material are ruling criteria for the separation process [5].

Globally, blood filtration can be conducted without necessarily designing smart membranes, but by controlling the structure of the membrane and its wetting properties, as it has been done for many years with dialysis membranes aimed at purifying blood [48]. However, when it comes to separating blood components from whole blood, the introduction of some smart functions into the surface of the membrane could permit to control specific interactions with the desired blood component that has to be retained.

Concept of smart membranes

The supporting layer of membranes used for blood separation are now mostly synthetic hydrophobic polymers such as PVDF. They are chosen for their outstanding bulk properties that prevent membrane structure damages during the manufacture or sterilization steps.

However, being hydrophobic makes them easy to interact with plasma proteins partly hydrophobic leading to some unwanted phenomena as platelet activation [5]. Thus, to enhance their hemocompatibility it is necessary to introduce some smart functions onto these membranes surface that can improve their resistance to nonspecific proteins adsorption and prevent or reduce the biofouling blood cells. Among materials minimizing the interactions with blood components, the neutral poly(ethylene glycol) (PEG) and its derivatives such as poly(ethylene glycol methacrylate) (PEGMA) have been proved to do it well [49].

Another route was proved to improve the hemocompatibility of hydrophobic membrane, it consists on coating or grafting charged moieties either positively and negatively in the surface which forms the so called zwitterionic systems [50]. In addition to hydrophobic nature of polymeric membranes leading to biofouling, this phenomenon could be also due to electrostatic interactions in the case of membranes with surface charge deviating from electroneutrality. This could happen due to the local charge density bared by the cells and proteins that is likely different from zero because they carry both positively and negatively charged functional groups. Furthermore, if the pH of the surrounding medium in which blood flows is different of the isoelectric point of the proteins, coated and grafted membranes, that is, membranes with a local charge bias, will undergo nonspecific proteins adsorption [5]. This issue can be turned into an advantage in the development of smart biomedical membranes. Indeed, those charged materials could be used to interact specifically with some blood cells and proteins, while being totally blind to others. The cells separation in this case will be no longer based on their size but rather on their surface charge.

The stimuli-responsive materials are among the functions that could be also incorporated onto the membranes surfaces. The physical behaviour of this variety of brushes in solution can be controlled by altering the environmental conditions. A typical example of stimuli-responsive materials is poly(N-isopropylacrylamide), which can expand or shrink by tuning the temperature, thereby affecting the membranes' pore size [51].

Only one of these interactions is not enough to ensure an interesting membrane property in regard to the complexity and the multiple components of the blood. Thus, the combination of two or more functional groups among those mentioned above could be promising for the development of smart biomedical membranes for blood separation (Figure 1. 6). Indeed, it can provide a more refined control of interactions between blood cells and membranes materials.

While one functional group establishes electrostatic-based interactions with the targeted cells, the other functional group prevents the adhesion of the plasma proteins and other cells contained in the blood. The adhesion could be then reversible using stimuli-responsive brushes to release for example the cells already captured before.

PVDF as supporting material for blood separation systems

Membranes are widely used as supporting materials for separation processes in biomedical applications and research on their use in blood-contacting devices is growing steadily. For instance, their application in the separation of plasma and viruses from whole blood, the concentration and de-mineralization of blood plasma using ultrafiltration membranes have been under investigation [49]. However, when the blood is in contact with the material outside the human body, some biological responses are likely to happen such as the activation of blood coagulation system at the blood/material interface. Then a response of the immune system arising from the blood/material contact and other tissues responses occurs [49]. Therefore, the choice of materials that can be used to design membranes for blood contacting devices is limited by their lack of hemocompatibility and biofouling resistance.

Synthetic polymeric membranes, such as polyvinylidene fluoride (PVDF) among others, are usually chosen for blood separation research [52]. The main advantages of PVDF are sufficient thermal and mechanical resistance preventing damages during the manufacture and sterilization steps [5]. In addition, some studies have confirmed that PVDF porous membranes can be used in biomedical application such as artificial blood vessel, tissue engineering and blood purification [53]. Unfortunately, PVDF is a hydrophobic material, with a relatively low surface energy and high water contact angle, which make it easy to adsorb blood plasma proteins and blood cells thus triggering coagulation, hemolysis and other immune responses [53], so that one may assume that it cannot be used as a hemocompatible material. Yet, modifying the surface of PVDF membrane directly in contact with blood might be an interesting path to take advantage of the excellent bulk properties of this material.

In order to capitalize on the usefulness of PVDF membranes in filtration operations, many studies have investigated (surface) modification of this material to make it polar and less hydrophobic [14], [17]. Many of the modification methods are aiming to structure a hydration layer to improve the anti-hemolysis property of PVDF membrane or to establish a heparin/heparin-like anticoagulant layer to prolong the blood coagulation time when blood contact membrane materials [8].

Chang et al. modified commercial PVDF membranes by grafting zwitterionic polymer, and unveiled that blood compatibility was obtained when overall charge neutrality of the hydrated layer was achieved [START_REF] Chang | Hemocompatibility of Poly(vinylidene fluoride) Membrane Grafted with Network-Like and Brush-Like Antifouling Layer Controlled via Plasma-Induced Surface PEGylation[END_REF]. They also used poly(ethylene glycol) methacrylate (PEGMA) to modify the surface of PVDF membranes and proved an increase of the resistance to proteins adsorption or blood cells adhesion. Other studies proposed a surface glycosylation of PVDF microporous membranes to improve their anti-biofouling properties along with their resistance to platelet adhesion [START_REF] Yuan | Facile surface glycosylation of PVDF microporous membrane via direct surface-initiated AGET ATRP and improvement of antifouling property and biocompatibility[END_REF]. Hydrophilic polymer polyethylene glycol (PEG), called also polyethylene oxide (PEO) when it is large (> 20 kDa), and its derivatives are one of the most studied polymers to prevent adsorption of blood proteins, blood cells, and bacteria. PEGylated copolymers on membranes enhance the trapping of water at its surface through hydrogen bonding leading to the formation of hydration layer which gives a physical and energetic barrier against protein, cells, and bacteria [5]. Consequently, biofouling on the membrane is reduced. To ensure the stability of these polymers on the membrane surface, they are usually combined with a hydrophobic block that will interact with the hydrophobic membrane surface. In some studies the polystyrene (PS)

block was used to anchor the hydrophilic poly(ethylene glycol) methacrylate (PEGMA) block to the hydrophobic PVDF surface which made the membrane resistant to bovine serum albumin (BSA) adsorption [START_REF] Chiag | Biofouling Resistance of Ultrafiltration Membranes Controlled by Surface Self-Assembled Coating with PEGylated Copolymers[END_REF]. Other studies have shown that PVDF membrane modified with PS/PEGMA block copolymer reduces the adhesion of platelets, erythrocytes, leukocytes, and fibrinogen [49]. Tuning the amount of the polymer blocks coated on the membrane surface allows control of the hydrophobic/hydrophilic balance resulting in tunable fouling resistance.

Poly(N-isopropylacrylamide) (PNIPAAM) is a temperature-responsive polymer that undergoes a rapid transition from a hydrophilic to a hydrophobic structure in water at its lower critical solution temperature (LCST) between 30 and 35ᵒC [51]. It swells (hydrophilic) at temperatures below its LCST due to hydrogen bonding between the water and PNIPAAM molecules, and it collapses (hydrophobic) at temperatures above its LCST due to preferred intramolecular interaction among PNIPAAM molecules. This means that its hydrophilicity and consequently, cell, protein, and bacterial adhesion behaviour can be regulated with temperature which can be further controlled through the incorporation of other anti-fouling functional groups as demonstrated by several studies [START_REF] Venault | Stimuli-Responsive and Hemocompatible Pseudozwitterionic Interfaces[END_REF].

Poly(acrylic acid) (PAA) is a negatively charged bioactive polymer in aqueous medium which is widely used to modify polymeric surfaces for drug release and protein immobilization [START_REF] Zhang | Functionalized polypropylene non-woven fabric membrane with bovine serum albumin and its hemocompatibility enhancement[END_REF]. AA is a hydrophilic monomer that allows to introduce carboxyl groups (-COOH) which react with the amino groups from proteins. It has been shown that this polymer binds to blood proteins at some specific conditions of pH and ionic strength. Other studies demonstrated that albumin polymer could be conjugated with fibrinogen and successfully attached to a plateletimmobilized surface [START_REF] Takeoka | Fibrinogen-Conjugated Albumin Polymers and Their Interaction with Platelets under Flow Conditions[END_REF]. Thus, it would be interesting to investigate the interactions of blood proteins (HSA, fibrinogen, and globulin) with PAA and verify the potential use of protein modified PAA to selectively interact with platelets.

6. PhD strategy and positioning 6.1. Context and objectives

Chapter I

The variety of properties of functional groups may provide an opportunity to develop new membrane materials with specific affinities and interactions with complex biological fluids like blood.

PhD strategy and positioning

The PhD thesis has been conducted as part of the MOSAIC-3D project, which is an international collaboration between Laboratoire de Génie Chimique (UMR 5503, Université de

Context and objectives

This project aims at designing and developing smart membrane technologies to address several critical needs related to blood cells screening and the concentration of a growth factor stored inside the granules of platelets and expressed after their activation. This growth factor namely platelet-derived growth factor (PDGF) is a mitogen among those numerous growth factors mentioned earlier in section 5.1. It is used particularly to help heal diabetic ulcers, in orthopedic surgery and periodontics as an alternative to bone autograft to stimulate bone regeneration and repair. To allow that to happen, thrombocytes (platelets) need first to be separated from other blood cells.

The current PDGF extraction and purification methods are centrifugation [5] and chromatography [START_REF] Antoniades | Purification of human platelet-derived growth factor[END_REF]. Centrifugation is fast but energy demanding. Besides, it does not allow to separate thrombocytes from leukocytes. Chromatography is effective on separation, but its discontinuous mode and its low yield and productivity hamper its use on a large scale production [START_REF] Coskun | Separation techniques: Chromatography[END_REF]. Wherefore, this project proposes to use smart membranes tailored at the nanoscale as an efficient alternative to facilitate the reversible adhesion and activation of Thus, the main challenges of this project are:

 The design of tailored functional groups (brushes of copolymer to be grafted onto the membrane surface) to have switchable control between attachment and detachment of proteins and cells.

 Control of the membrane architecture through surface modification with the aim to enhance its selectivity or non-adhesive properties.

 Tuning of operating conditions by the analysis and the control of interface properties during filtration to have the best performance of the developed membranes.

Strategy

The strategy is based on designing and testing different membrane systems in order to (1) purify platelets from whole blood (separation of blood components) and ( 2

Contribution of PhD thesis

The research target is the preparation of an interface/membrane that can selectively capture platelets from human whole blood (Figure 1. 9). This design represents the ultimate design goal and it involves the grafting of three different functional groups: one type of functional group for the selection of platelets, one functional group to provide bio-inert properties to the system, and the last group is to release platelets. Thus, there should be specific biofouling of the membrane system. These very preliminary results confirmed the potential of the polymers to influence the adhesion of platelets, though a lot remained to be understood before getting full control of this capture mechanism.

In particular, the efficacy of a surface modification may be jeopardized if the functionalized surface is poisoned by unexpected interactions with other solutes or colloids than the target ones. In the specific case of the extraction of platelets from blood, the presence of various proteins especially those at high concentration such as human serum albumin (HSA) which is well known to adsorb on a wide variety of surfaces, maybe an issue. A comprehensive understanding of the interactions between blood proteins and the polymers candidate for surface modification was therefore considered as a prerequisite, in the project strategy. From that arises the objectives of this PhD thesis given below and illustrated in Figure 1. 11.

-Understand and control the local interactions (electrostatic, hydrophobic …) between the selected moieties (PAA, PEG) with major human blood proteins (albumin, ɣ-globulin, and fibrinogen). That's why, the second chapter of this manuscript is dedicated to present an explicit study of these interactions in batch in different physicochemical conditions using mostly Small Angle X-ray Scattering (SAXS) as a tool of investigation. -Study the functionalization of PVDF membranes using the copolymers mentioned previously, and then investigate the static adhesion of human blood proteins on the copolymer. For that, the third chapter discuss the optimization of the modification process for each copolymer. It also presents the different interactions that could take place but at the modified interface this time.

-Assess the performance of the functionalized membranes for filtration processes. This point is detailed in the fourth chapter that presents all the experiments related to filtration tests. Indeed, the filtration of pure water was first performed to validate the operating conditions of the modification process discussed in the third chapter. Then, dead-end filtrations of protein solutions were conducted to investigate the adhesion of blood proteins on modified surfaces but this time in dynamic mode. As stated in the previous chapter, the development and surface modification of biomaterials used for blood purification requires the understanding and the control of interaction between blood proteins and the modifying polymers [1], [2]. Wherefore, this chapter presents an explicit study of the interaction in solution between some abundant proteins in human plasma (HSA and IgG) and some polymers chosen for surface modification later such as poly acrylic acid (PAA) and polyethylene glycol (PEG).

In addition to biomaterials and medical devices, interactions between proteins and polymeric materials are involved in many other processes in several fields [3], [4]. Thus, the study of these interactions could be relevant for versatile application, as protein separation [5], [6], drugs delivery [7], [8], enzyme immobilization [9], among many other applications in chemistry and biology [10].

In this study, tailored membranes are proposed for blood cells screening and the concentration of growth factors as we report in the previous chapter. For that, the architecture of membrane biointerface at the nanoscale will be customized by coating on it some functional groups such as PAA and PEG. PAA is a negatively charged bioactive polyelectrolyte in aqueous medium suggested to promote platelets adhesion, especially when it is conjugated to proteins such as fibrinogen. But it can also be used to prevent platelets adhesion when it is conjugated to albumin [11]. Thus, nonspecific interactions between other blood components and polymers used for interface modification could hamper the capture of platelets. PEG which is an electrically neutral polymer known for its antifouling properties is often used to reduce these nonspecific interactions [12], [13]. Among those blood components able to interfere, HSA is the most abundant plasma protein ([HSA]≈42 mg.mL -1 in blood [14]) that maintains the blood pH and osmotic pressure and transports some molecules in the blood. This protein is able to decrease platelet adhesion and activation on a solid surface by being adsorbed onto that particular surface [11]. Therefore, the control of these nonspecific interactions that could take place between the modifying polymer and blood proteins is required.

According to the literature, a number of protein-polyelectrolyte complexes have been studied in a variety of physicochemical conditions via several analytical techniques [15], [16].

Concerning complexes of serum albumin and polymers, the interaction of bovine serum albumin (BSA) with spherical polyelectrolyte brushes in solution was evidenced using Small Angle X-ray and neutron scattering (SAXS and SANS) [17], [18]. Interactions between BSA Chapter II and planar synthetized poly(acrylic acid) were also the object of a dedicated work [19].

Furthermore, binding of HSA with small polyelectrolyte brushes of PAA (with 25 repeating units only) [20], and with dendritic polyglycerol sulfate [21], has also been discussed by isothermal calorimetry (ITC). In fact, it has been proved that only one HSA is bound per chain of PAA 1.8 kDa. Then, for a pH maintained at 7.2, a study on the effect of salt concentration revealed that this binding was dominated by an attractive electrostatic potential between the two solutes [20]. Using Total Internal Reflection Fluorescence (TIRF) spectroscopy, it also has been proven that negatively charged BSA is strongly adsorbed on planar PAA (278 AA units ≈20 kDa) at low ionic strength and pH > 5 due to electrostatic interactions [22]. However, the current knowledge does not permit to give the exact stoichiometry of the polyelectrolyte-protein complexes for larger polymer, and if it is correlated to the size of the polyelectrolyte.

In the present study, we used SAXS combined to chromatography to assess the affinity of HSA to bind to a larger PAA (100 kDa) to check out the formation of multiprotein-polymer complexes compared to the 1HSA-1PAA binding reported in the literature using smaller polyelectrolyte [20]. First, as a starting point for further experiment with polymers, the structure of native HSA is examined in phosphate buffer solution. Then, the HSA/PAA interaction is studied in media with different ionic strengths or pH in order to determine the conditions that could lead to an attractive interaction (section 4.3). After that, a detailed study modeling the Porod volume is conducted to determine the stoichiometry of the (nHSA-PAA) complex found to be formed. For this purpose, the influence of the HSA/PAA molar ratio on the complex size (radius of gyration and Porod volume) is appraised (section 4.4). Eventually, a modelling study is conducted to investigate the protein and complex structures. Thus, a compatible model is calculated using BUNCH program [23] to fit the HSA-PAA complex scattering data. It allowed us to propose an all atoms pearl necklace model that can describe the structure of complex (section 4.5.3). Subsequently, this complexation was investigated using different sizes of PAA at the different physicochemical conditions outlined in the preceding sections and the stoichiometry was assessed (section 4.6). After that, these interactions were studied with another blood protein less abundant than HSA but larger, which is human γ-globulin (IgG). In the end, the interaction of both proteins (HSA and IgG) were tested with PEG instead of PAA.

Many results obtained from the studies reported above have been the subject of a scientific article published in Soft Matter [24], and they have also been discussed in different national and international conferences listed in Appendix D. For the investigation of native HSA structure in solution and HSA-PAA interaction, the concentration of HSA was set at 5 g•L -1 to avoid repulsion between particles which can distort the measured intensity in small angles. The pH effect was studied using different buffer solutions at 0.1 M (TRIS base pH 8, HEPES pH 7, MES pH 6, mixture of sodium citrate dihydrate and citric acid at pH 4-5). To study the ionic strength influence, the salts concentration was adjusted by adding the needed amounts of 4 M NaCl solution.

Materials and samples preparation

Then, all samples, whether protein free, polymer free or a mixture of both, were stirred using a Thermo Scientific mini centrifuge (mySPIN 12) at 10 000 rpm for 2 minutes at least, to ensure good dissolution of the solutes, before being pipetted into the samples holder for the SAXS measurement. Out of use, the samples were stored at 4°C in a refrigerator.

Small angle X-ray scattering (SAXS) measurement

SAXS is a powerful analytical tool for structural analysis, that has become very popular in the biological community with many substantial advancements made over the last few decades, particularly in data collection and analysis [25]. The major advantage of this method is its ability to obtain structural information from macromolecules in solution. It provides detailed structural analysis and physical information by characterizing the sizes and shapes of proteins and complexes in a broad range of molecular sizes from a few kDa to GDa [26]. SAXS also allows to study the behaviour in solution of native particles in conditions close to their physiological environment, and to evaluate the structural variations linked to a modification of the external conditions. Advances in SAXS instrumentation allowed not only to determine a few simple geometric parameters of flexible ensembles, but also to assess their probable configurations in solution [23].

3. Methods 3.2. Small angle X-ray scattering (SAXS) measurement

Chapter II

In a SAXS experiment, the sample is exposed to X-rays of a specific wavelength, and the intensity of the scattered X-rays is recorded by a two-dimensional X-ray detector ( Figure 2. 3).

As the macromolecules are randomly oriented, the resulting scattering intensity folds into a one-dimensional signal varying with the modulus (q in A -1 ) of the scattering vector calculated as follows [26]:

𝑞 = 4𝜋 𝑠𝑖𝑛(𝜃) 𝜆 (2.1)
where λ is the X-rays wavelength, and 2θ denotes the scattering angle. The scattering pattern of the buffer is carefully collected as well by an independent measurement in the same measuring cell, and subtracted from the sample solution scattering leaving only the signal from the particles of interest. The final curve denoted I(q) is the averaged contribution of the structures from all the macromolecules contained in the sample as illustrated in Figure 2. 3. This latter is based on the crystal particles present in the sample, and it is related to the overall shape and size of these particles under investigation. However, it could be altered by various parameters such as contrast (electron density), sample to detector distance, resolution, and beam collimation [27].

The final intensity distribution provides significant information regarding the size, shape, and general structure of the sample. Indeed, several parameters of the investigated sample can be extracted from the experimental scattering pattern including molecular weight, excluded particle volume, maximum dimension Dmax and the radius of gyration Rg. These parameters directly reflect the overall characteristics of the particles.

3. Methods 3.2. Small angle X-ray scattering (SAXS) measurement

Chapter II

In the case of non-ideal (i.e. real) solutions representing the interacting systems, the scattering is proportional to the namely concentration dependent structure factor S(q) of the solution that takes into account the interactions between solute particles [28]. The structure factor can readily be obtained from the ratio of the experimental intensity that obtained by extrapolation to infinite dilution were all correlations between particles have vanished [29].

Thus, at infinite dilution S(q)=1 and it is thereby advisable to extrapolate scattering patterns collected at several concentrations to zero concentration to obtain an undistorted pattern in the low angle region.

Information provided by SAXS

It is important to mention that a SAXS profile has distinct regions from which the information may be extracted: Guinier, Fourier, and Porod [27]. In the Guinier region, the classical and the most known parameter directly extracted from the SAXS data, Rg may be evaluated using the following Guinier approximation:

lim 𝑞→0 𝐼 = 𝐼 0 𝑒𝑥𝑝 (- 1 3 𝑞 2 𝑅 𝑔 2 ) (2.2)
In principle, the extrapolated forward scattering I(0) and Rg can be extracted from the y-axis intercept and the slope of the linear fit of the Guinier plot which is equivalent of a linear variation of Ln I(q) vs q 2 . The Rg is the average root-mean-square distance to the center of density in the molecule weighted by the scattering length density, and it provides a measure of the overall size of the macromolecule. It is greatly affected by aggregation of particles, polydispersity, and improper subtraction of the background or buffer. As for the determination of I(0), it is particularly interesting because this value allows to calculate the molecular mass. This is based on knowledge of the forward scattering intensities and concentrations of both the macromolecule of interest and a standard such as bovine serum albumin (BSA) [30].

It should be noted that Guinier's law is only valid at small angles and that a deviation from linearity may reflect a non-ideality of the solution (presence of attractive or repulsive interactions). In fact, attractive interactions between particles and nonspecific aggregation result in overestimation of both Rg and I(0), whereas repulsive interactions lead to an underestimation of Rg and I(0) [26].

3. Methods 3.2. Small angle X-ray scattering (SAXS) measurement 44

Chapter

II

The information deduced from the analysis at small angles remains rather limited (size, molecular mass, interactions...). If one considers the information contained at the larger angles (higher resolution), it is then possible to get more information on the studied system, by measuring the I(q) function over a wider range of angles. In the Fourier region, the pair distribution function p(r), which is a histogram of distances between all possible pairs of atoms within a particle, may be determined by an indirect Fourier transformation of the experimental scattering pattern using the following equation [30]:

𝑝(𝑟) = 𝑟 2 2𝜋 2 ∫ 𝑞 2 𝐼(𝑞) sin(𝑞𝑟) 𝑞𝑟 ∞ 0 𝑑𝑞 (2.3)
In some cases, it is more intuitive to interpret the structural properties analyzing the p(r) rather than the scattering data itself. The radius of gyration can be calculated from the p(r) function which often yields a more reliable estimate compared to the Guinier approximation, in particular for unstructured systems, and that by using the following equation [26]:

𝑅 𝑔 2 = ∫ 𝑟 2 𝑝(𝑟) 𝑑(𝑟) 𝑑 𝑚𝑎𝑥 0 2 ∫ 𝑝(𝑟) 𝑑(𝑟) 𝑑 𝑚𝑎𝑥 0 (2.4)
This autocorrelation function p(r) is a real space representation of the scattering data and allows one to graphically display the peculiarities of the particle shape [29]. All information listed above are obtained at small and medium angles, while at high angle is found the third region of a SAXS profile which is the Porod region, and that provides other information than those extracted from the Guinier and the Fourier regions. For instance, the Porod invariant Q may be determined, providing information on surface, volume and the internal organization of the studied particles. In fact, from the Porod invariant Q it is possible to obtain the Porod volume of an object which is nominally the excluded volume of the macromolecule in solution [31] :

𝑉 𝑝 = 2𝜋 2 𝐼(0) 𝑄 * 1 -𝜙 𝑆(0) (2.5)
When the dispersion is diluted the volume fraction 𝜙 → 0, 𝑆(0) → 1 and the Porod volume can be determined by knowing the zero angle intensity I(0) and Q.

Q is called the Porod invariant because its value does not depend on the structure but on the volume fraction and the contrast between two phases Δ𝜌 :

3. Methods 3.2. Small angle X-ray scattering (SAXS) measurement

Chapter II 𝑄 = ∫ 𝑞 2 (𝐼(𝑞) -𝐾)𝑑𝑞 ∞ 0 = 2𝜋 2 𝜙 (1 -𝜙) 𝑟 𝑒 2 (Δ𝜌) 2 (2.6)
Where K is a constant defined to ensure the asymptotical intensity decay proportional to q -4 at higher angles [32].

The value of Q can be roughly obtained from the integration of the Kratky plot (classically with a limited integration boundary at q=0.25) by considering K=0:

𝑄 = ∫ 𝑞 2 𝐼(𝑞) 𝑑𝑞 0.25 0 (2.7)
When the intensity decreases too slowly at large q (less than q -3 ) as for polymeric systems [33], it is well more accurate to integrate the original equation after determining the constant K.

Whereby the constant K is calculated from the Porod asymptote I(q). q 4 versus q 4 at higher angles.

In this Chapter, the asymptote has been always determined for the same range of q between 0.3 and 0.445 with a homemade Python code to keep the control of the calculation. The code is given with the plots that show the determination of K in Appendix B, as well as the corrected dimensionless Kratky plot obtained by subtracting K from I(q). An example that illustrates the invariant Q calculation and the estimation of the constant K is shown in Figure 2. 6. It can be seen that the subtraction of the asymptote K leads to a curve that tends to zero for high q that then permit a good integration of the invariant Q (right-bottom subfigure in Figure 2. 6). In direct injection mode, that is, without size exclusion chromatography step, 50 µL of sample are pipetted from the samples holder maintained at a constant temperature (20 °C) using a circulating water bath, and are injected to the measurement cell placed under vacuum to limit air absorption. Data were collected on a 150x150 mm area DECTRIS detector (Pilatus 1M) with a sample to detector distance of 1216 mm. The detector is highly sensitive, making it ideal for detecting low scattering samples, and can be shifted along the beam, which increases the angular range of measurement (objects with dimensions between 0.5 and 15nm).

The experimental scattering intensity I(q) is expressed as a function of the scattering vector q (Å -1 ). Each scattering curve reported in this manuscript is an average of at least 6 measurements on the same sample, and each SAXS measure corresponds to a data collection time of 1800s. Scattering curves were recorded within the range 0.005 < q < 0.5 Å -1 . Finally, actual solutes scattering intensity I(q) was obtained by subtracting the buffer solution contribution from the total SAXS signal. For a given protein, several concentration ranges must be tested. Low concentrations limit the aggregation phenomena observed at small angles, but the signal is very noisy at high angles.

The information at high angles can be improved by increasing the concentration of the sample, thus favoring the aggregation phenomena and the loss of information at small angles [30]. It is possible then to group all the high and low concentration data on the same diffusion curve in order to limit the problems of background and aggregation. To do this, the curves must have sufficient overlap, as this type of data manipulation can introduce a significant bias that will affect the envelope and structure calculations.

SAXS-SEC combination

It has been reported in the literature that combining SAXS to the size exclusion chromatography (SEC) could be a very promising and interesting technique to optimize data acquisition [25]. Thus, it allows to improve the information that could be extracted from the scattering pattern.

3. Methods 3.2. Small angle X-ray scattering (SAXS) measurement 49

Chapter

II

In fact, data obtained from the SAXS measurement of the elution peaks are combined to those obtained from direct measurement on the same samples without going through chromatography before. In fact, chromatography allows to eliminate aggregates since acquisitions are done on the elution peaks representing the main fraction of the studied particles only. Meanwhile, a direct injection mode permits to improve information at high angles by minimizing the noise at this q-range. Thus, scattering data at small angles obtained from acquisitions on the elution peaks are merged with those at high angles obtained from direct injection mode provided that the two curves overlap sufficiently. All data processing operations concerning buffer subtraction, averaging of diffusion curves and other processing (scaling, subtraction or division by constants) are done using Foxtrot software [34], or the graphical interface Primus [31]. Once the data have been processed, it is possible to calculate the p(r) with the program GNOM [35].

Computation of scattering from molecular model

Calculation of scattering curves from atomic models, and the comparison of these predicted curves with the experimentally determined SAXS profiles is a method for rapid characterization of proteins and complexes if high-resolution structures or homology models are known. In fact, the availability of a fast, robust and easy-to-use software to compute scattering patterns from atomic coordinates revolutionized the field by providing the link between SAXS data and atomic scale molecular structures [30]. Given a model, the theoretical scattering curve can be computed and fitted to the measured data, and the model that provides a good fit to the data is From a molecular model and its atomic coordinates, the CRYSOL code [36] calculates the radius of gyration and an X-ray scattering curve either in vacuum or in a contrasting solvent.

From the atomic coordinates, CRYSOL generates an electron density map, which, by Fourier CRYSOL employs a Gaussian sphere approximation for the calculation of the excluded volume and uses spherical harmonics to calculate an envelope at the surface of the atomic model to approximate the hydration layer.

Modelling in simple geometrical bodies using Sasview

According to the form of the experimentally determined SAXS pattern, it is sometimes possible to find simple geometrical bodies that can describe the investigated macromolecules.

In fact, the theoretical scattering intensities of many known geometrical bodies have been Sasview is a free software [37] among those that can perform the fitting process of the SAXS data to a suitable model, which can provide information about the structures present in a sample.

It also allows to keep control on the parameters of the used model. In fact, this is done by selecting every time a viable model with which to fit the data and then attempt to get a good fit.

If a good fit can't be achieved, another model is selected and attempted to be fitted to. If this fails, the same models can be attempted but the polydispersity of different parameters can be varied. It is important to undertake this time-consuming and thorough approach to ensure the data is fitted as well as possible.

However, to get a good fit in Sasview data should be attempted to be fitted to as few parameters as possible. Using more complex models with fewer parameters runs the risk of over-fitting the data, in which the obtained values lose meaning. Besides, just because a data 3. Methods 3.2. Small angle X-ray scattering (SAXS) measurement 52

Chapter II set has been fitted to a structural model it doesn't necessarily mean the structure is exactly the dimensions that your fit suggests.

In addition, with Sasview a data set may be sufficiently fitted to two or more different models. In this instance, either both are presented or there may be reason to suggest one is favoured over the other (such as a slightly lower fitting error).

Rigid body modeling from SAXS profile

While I(q) can be recreated rapidly from the structural models of the macromolecules or complexes present in the sample, the reverse problem consisting on deducing the possible structure(s) from a scattering profile is harder to do. In fact, it is possible to infer a possible structure for a macromolecule and for the assembly of macromolecular complexes through direct refinement against the scattering data. However, this is possible only with strong external constraints which vary according to the amount of available structural information [30].

In approaches where a shape for the macromolecule is tentatively extracted from the experimental curve and usually represented by bead models [38], very general constraints of connectivity or looseness are used. As for the macromolecular complexes, they are difficult to study because of their large size, internal flexibility and transient nature. However, a large number of individual components structures have been produced [25], so the challenge is to rebuild the macromolecular complexes that these different subunits form. A number of approaches have been developed using SAXS to determine the positions and orientations of subunits within macromolecular complexes [23], [39]. In the rigid-body fitting approach, the integrity of the domains with known atomic structures is maintained, while their position and orientation is varied. Indeed, starting from an arbitrary positioning of subunits, random rigid body movements and rotations (Figure 2. 11.A) are conducted to search for a best fit of the computed complex scattering to the experimental data. The sequence and specific connectivity of the missing residues constitute the additional constraints [25]. In addition, the use of symmetry, inter-residue contacts and inter-subunit distances are fully supported [30].

The automated program BUNCH [40] is among the tools that allow to conduct rigid body (red curve). The inset represents the Guinier plot (Ln I(q) vs q 2 in red line) with the residual (green curve) that allow to determine the radius gyration using Primus. The HSA 3D structure shown in cartoon representation using PyMOL software.

Figure 2. 12 shows that the experimental SAXS spectrum from purified commercial HSA (blue line) matches very well the calculated intensity from the crystal structure (red line), evidencing that the HSA used for this study is correctly folded at 75 µM in the PBS buffer condition. These preliminary results validate the use of HSA as a template to study protein polymer interaction. In these conditions we can expect that the SAXS will be sensitive enough to sense an interaction between HSA and PAA. The HSA radius of gyration Rg was then evaluated by fits using Foxtrot or Primus. Both software consider the Guinier approximation based on equation 2.2. It allows to determine Rg from the slope of the linear fit at low q of I(q) Å) in a SAXS study for HSA with the same purity >96% [14].

Likewise, the Porod volume and molecular weight were estimated using Primus that consider the Porod law based on the calculation of the Porod invariant Q as explained in the methods section. Thus, Vp and Mw were found to be around 100,000 Å 3 and 64.5 kDa respectively. These results seem to be consistent with the molecular weight of HSA (66.5 kDa), which validates the use of Primus to evaluate such parameters in further experiments.

It is also important to point out that if the HSA molecule is assimilated to a sphere with the radius being Rg obtained previously, we obtain a volume around 115,000 Å 3 which is close to of the HSA Porod volume estimated above using Primus.

Study of HSA-PAA interaction

Variation of PAA concentration

The characterization of single protein was then followed by assessing the interactions between HSA and the polyelectrolyte chains using direct SAXS measurement. For that, we first studied the effect of an increase in PAA concentration on the SAXS scattering intensity for an HSA/PAA molar ratio ranging from 0 (protein alone as a reference) to a molar ratio of 3/2. in the signal at small angles is observed, which could be due to the formation of an HSA-PAA complex. While in the intermediate and high q-range the SAXS curves remain almost unchanged by the complexation. Indeed, the evolution of the slope P of the corresponding power law I(q) = q -P shows that at intermediate angles (0.1 to 0.2 Å -1 ), P slightly varies from For the first three curves, the data were processed with GNOM to determine biophysical parameters such as the radius of gyration Rg, the maximal distance Dmax using the autocorrelation function p(r). This function was calculated by an indirect Fourier transformation applied to the scattering intensity I(q) using the equation (2.3). The pair distribution function refers to the distribution of electrons averaged over a radius r. Thus, as stated in the materials section, it entirely depends on the shape and the size of the particle and vanishes at their maximum electron pair distance (Dmax). The radius of gyration was then derived from p(r) using the equation (2.4).

This alternative estimate of Rg makes use of the whole scattering curve, and is much less sensitive to interactions or to the presence of a small fraction of oligomers. Chapter II 108.9 Å for the complex). The Porod volumes (Vp) for the HSA and the complex were also evaluated. For a flexible system such as polymer chains in solution it is difficult to evaluate the exact Vp. All parameters are reported in the table attached to Autocorrelation function determined with GNOM of the complex and table summarizing the biophysical parameters extracted from the complexes' SAXS data.

As observed in Figure 2. 14.A, the SAXS curve of HSA is consistent with a 3D object with a large plateau at small angles and a typical decrease at intermediate angles, whereas PAA exhibits a behavior of a flexible polymer in solution (large decrease following a power law I(q)= q -P with P close to 1.7 which corresponds to polymers with local rigidity) [42]. The SAXS curve obtained for the complex presents a combination of properties of HSA and PAA. It shows a constant decrease at small angles and the same signal of HSA starting from 0.05 Å -1 . The direct comparison with a linear combination of HSA and PAA SAXS curves (sum curve in determined with p(r) calculation. One can assume that the complex of HSA-PAA100 kDa exhibits a behaviour in solution equivalent to the one of a solid cylinder measuring around 400 Å in length and 78 Å in diameter.

To validate this hypothesis predicting a formation of a defined object instead of amorphous aggregates, solutions of HSA-PAA were flowed in various mobile phases through a size exclusion chromatography column. The chromatographic profiles were then compared to that of the single HSA detected mainly at 7.74 min and as classically with a small amount of dimers at 6.9 min (Figure 2. 15).

In fact, in a PBS mobile phase (without PAA) at pH 7.4, a peak at 7.7 min of retention time was obtained, corresponding to the molecular weight of HSA alone. This experiment shows that the complex can be dissociated in a medium not containing PAA, illustrating the reversibility of the complex formation. On the other hand, if 50 µM of PAA is added to the mobile phase, the pH decreases to 5.2 and the elution profile is modified with a strong shift of the elution peak towards high molecular weights (Figure 2. 15). This observation proves that the protein is no longer single in the solution but bound to the PAA chains. The Gaussian shape of the peak eluted at 5.8 min corresponds to a profile of unique object in solution and not to a continuous distribution of mass such as in aggregates. These experiments were run at PBS ionic strength (0.137 M) and at pH controlled by the PAA concentration, ranging from 5.2 to 7.4 as mentioned above (Figure 2. 13). Since the pH remains higher than the HSA isoelectric point (4.7) and knowing that PAA exhibits a pKa of 4.5, we expect repulsive forces that keep them away from each other. It is because they both theoretically bear negative charges. Yet, the adsorption of HSA molecules on PAA chains is found to happen. Attractive interactions between a polyelectrolyte and a protein having the same net charge were reported by several authors [16], [43]- [45]. Since the proteins can carry both positive and negative charges, this binding found to occur in our study could be due to the attractive electrostatic potential between the positive charged patches on the surface of HSA and the negatively charged chains of PAA, which should be the operative force that overcome the repulsion forces. A detailed ITC study performed on the binding between HSA with negative overall charge and a negatively charged PAA [20], shows the same behaviour between the two solutes, where each HSA molecule has found to bind to one PAA chain causing an increase in the system entropy. It has been explained by the action of the HSA surface positive charges as a multivalent counter ion for the polyelectrolyte as illustrated in Figure 2. 16.

.2. Variation of HSA concentration

In order to check if the concentration of protein also plays a key role in the HSA-PAA interactions, it was varied from 75 to 600 µM for a polymer concentration ranging from 10 to 50 µM. slope. This translation is necessarily due to the increase of the protein amount, which could be observed in Figure 2. 17 when single HSA concentration was increased (without PAA).

The SAXS scattering data (Figure 2. 17 For a free polymer solution, Rg has found to decrease with the increase of PAA concentration as it can be noticed in Table 2. 1. This could be arising out the compaction of the flexible chains of polymer due to electrostatic repulsion of the PAA chains having the same charge. The same way, if we look at this for a fixed protein concentration this time and an increasing polymer concentration from 10 to 30 µM, we notice an increase of Rg remains more or less significant at greater HSA concentration. Indeed, for 75 µM of HSA and going from 2 to 30 µM of PAA, the radius of gyration passes from 30.4 to 44.3 Å, while for 600 µM of HSA, the Rg still around 30 Å, because the protein molar concentration is at least 20 times greater than that of the polymer. Since the radius of gyration obtained here by fitting is an average of the Rg of HSA that yields 30 Å, and the one of PAA that happened to be around 110 Å, it makes sense that the average will be proportional to the amount of each solute. Previously, we stated that the adsorption of protein on polyelectrolyte was substantiated by an electrostatic attraction between positive charges carried on the HSA surface and the negative PAA chains. This finding make it obvious that this attraction could not be the only operative force that overcome the strong repulsion forces inducted by the fact that both solutes have the same overall charge. Indeed, these last results disclose either a steric hindrance effect involved in the HSA-PAA binding phenomenon. This means that increasing the polymer concentration allows the PAA chains to be close enough to HSA molecules to initiate the attractive electrostatic force between protein positive patches and the polyelectrolyte negatively charged.

Or, it could be correlated to the pH of the medium found to be reduced with the increase of PAA amount. Thus, the overall negative charge of the protein is reduced which weakens the repulsive forces between HSA and PAA, and allow the electrostatic attraction to overcome.

Ionic strength and pH effect on HSA/PAA complexation

In the previous section, we suggested that HSA-PAA complexation was due to electrostatic interactions and that it was potentially correlated to the pH of the medium. Hence, the influence of the ionic strength and pH was investigated in this section.

For the ionic strength study, the Subsequently, SAXS measurements on the HSA-PAA mixture for a matrix of conditions were performed where the ionic strength was varied from 0.05 to 0.75 M for each pH 5, 6 and 7, in order to determine the best conditions of complex formation. As the major effects are observable at small angles due to the increase in size when the complex appears, we decided to probe the changes by evaluating the radius of gyration determined from Guinier law for each condition. Although the PAA and the complex have close Rg values (Rg  110 Å), the average value of Rg was found around  50 Å when the HSA and the PAA do not interact, which is much lower than that of the complex. The data are plotted in Figure 2. 21. One can notice that the radius of gyration of free PAA or of single HSA found to be around 110 Å and 30 Å respectively, do not depend on the pH. Besides, pH in the range 6-7 does not affect the radius of gyration of the mixture HSA+PAA, found to be approximately 50 Å for all salt concentrations investigated. This observation highlights the fact that HSA-PAA binding does not take place in these conditions. As stated previously, the radius of gyration obtained here by fits is an average of the Rg of HSA and of the one of PAA, which makes sense to obtain an average proportional to the amount of each solute in the mixture (protein/polymer ratio of 3/2).

A different behaviour is observed at pH 5. In fact, increasing the ionic strength from 0.05 to 0.75 M has led to decrease the Rg from 110 Å, (which is almost the polymer radius of gyration), to 69.8 Å. This is due to the fact that beyond 0.15 M of ionic strength the HSA-PAA complex It can be concluded from this study that the complex is stable at pH  5 and a salt concentration below 0.15 M. This is partially inconsistent with what has been reported in the literature where the HSA has been reported to form complexes with short PAA (2 kDa) at pH of 7 [20]. This could be due to the fact that PAA in our study is much larger, and so it bears higher negative charge which leads to stronger electrostatic repulsions with the protein. Thus, the decrease in pH allowed to reduce the negative charge of PAA until it initiate the electrostatic attraction with the positive charges on HSA surface.

We henceforth know the conditions that lead to the assembly of HSA-PAA complexes in bulk. Therefore, we can imagine that this study constitutes a first knowledge brick to tune these reversible interactions between long PAA grafted on a membrane surface and HSA. Controlling the ionic strength and the pH should enable to regulate these interactions, promoting or preventing their happening. Thus, the effect of the pH and the ionic strength will be again From the values of Rg reported in Table 2. 2, it can be concluded that the attachment of proteins to the polymer chains has taken place on all the polyelectrolyte chains available for HSA/PAA molar ratio ranging between 3.6 and 4.5.

To support this hypothesis, the Porod volume was evaluated for the different HSA/PAA molar ratios. As stated in the materials section, from SAXS experiments it is possible to obtain the Porod volume of an object from the Porod invariant Q. (Plots given in section 2 of the

Appendix B).

The calculated Porod volumes (reported in Table 2. 2) exhibit the same behaviour as that of the radius of gyration. For a HSA/PAA molar ratio of 3.6, the Porod volume is found to be around 500. Where n is the number of HSA in each complex particle.

Thus, for a given initial [HSA]0/[PAA]0 molar ratio, it is possible to define [HSA], [PAA] and [c] as the molar concentrations in the mixture during the complexation progress of HSA, PAA and the complex, respectively. Thus, these concentrations can be calculated as follows:

[𝑃𝐴𝐴] = [𝑃𝐴𝐴] 0 -[𝑐] (2.8) [𝐻𝑆𝐴] = [𝐻𝑆𝐴] 0 -(𝑛 × [𝑐]) (2.9)
If the PAA is on excess ([HSA]0 < n[PAA]0), the composition in the mixture is:

[𝑐] = [𝐻𝑆𝐴] 0 𝑛 (2.10) [𝑃𝐴𝐴] = [𝑃𝐴𝐴] 0 - [𝐻𝑆𝐴] 0 𝑛 (2.11)
If HSA is on excess ([HSA]0 > n[PAA]0), the composition in the mixture is:

[𝑐] = [𝑃𝐴𝐴] 0 (2.12)

[𝐻𝑆𝐴] = [𝐻𝑆𝐴] 0 -(𝑛 × [𝑃𝐴𝐴] 0 ) (2.

13)

For known [HSA]0/[PAA]0 molar ratio, it is then possible to determine the relative volume fraction of each solute i in the mixture as follows:

𝜑 𝑖 = 𝑁 𝐴𝑣𝑜 • [𝑖] • 𝑉 𝑖 ϕ (2.14)
Where ϕ is the volume fraction of the mixture of solutes in solution and it is expressed as:

𝜙 = 𝑁 𝐴𝑣𝑜 ([𝑐] • 𝑉 𝑐 + [𝐻𝑆𝐴] • 𝑉 𝐻𝑆𝐴 + [𝑃𝐴𝐴] • 𝑉 𝑃𝐴𝐴 ) (2.15)
Where Vc, VHSA, VPAA are the volume of a molecule of the complex, HSA and PAA respectively. These volumes were estimated using Primus from SAXS spectra of single solutes and found to be: 500.10 3 , 100.10 3 and 50.10 3 Å 3 for the complex, HSA and PAA, respectively.

Therefore, assuming a stoichiometry of complexation can allow to evaluate the relative volume fraction of HSA, PAA and the complex using the complexation equilibrium suggested previously (Figure 2. 23). for a dilute dispersion S (0) =1, the average volume can be written as follows (O. Spalla eq. 54)

[33]:

𝑉 𝑀 = ∑ 𝑁 𝑖 • 𝑉 𝑖 2 𝑛 𝑖 ∑ 𝑁 𝑖 • 𝑉 𝑖 𝑛 𝑖 (2.16)
The Porod volume of the mixture can be written then as a function of the relative volume

fraction of solute, 𝜑 𝑖 𝑉 𝑀 = ∑ 𝜑 𝑖 • 𝑉 𝑖 𝑛 𝑖 ∑ 𝜑 𝑖 𝑛 𝑖 = ∑ 𝜑 𝑖 • 𝑉 𝑖 𝑛 𝑖 (2.17) 𝑉 𝑀 = 𝜑 𝑐 • 𝑉 𝑐 + 𝜑 𝐻𝑆𝐴 • 𝑉 𝐻𝑆𝐴 + 𝜑 𝑃𝐴𝐴 • 𝑉 𝑃𝐴𝐴 (2.18)
Porod volume calculations whether from SAXS experiments or from the theoretical model as well as the relative volume fraction were performed using a code written in Python (given in

Appendix B).

Knowing Vc, VHSA, VPAA estimated previously using Primus, the equilibrium suggested previously can be then used to evaluate the Porod volume by assuming a stoichiometry, n, of complexation. In order to facilitate data processing in further modelling of the structure of HSA-PAA complex, that of native HSA was first investigated to find a simple geometrical model that can describe this structure. For that, simple geometrical bodies were tested using the program Sasview [37]. As exposed in Figure 2. 25, the best fitting curve (plotted as a red line) to the experimental I(q) (plotted as blue line), corresponds to a cylinder (Disk) model with a radius of 37 Å and a length of 31 Å. It is important to note that using these dimensions a volume of cylinder that yields 133.10 3 Å 3 is obtained, which is higher than the Porod volume of HSA found to be around 100.10 3 Å 3 . This could mean that the HSA does not completely fill the cylinder as it could be seen in Figure 2. 25. Based on what has been done for the protein alone and the stoichiometry proposed for number of HSA molecules that could be bound on each PAA 100 kDa, geometric models were also tested to describe the HSA-PAA complex by fitting the experimental I(q) using Sasview program.

The first suitable shape found to fit well the SAXS experimental data corresponds to a cylinder (Radius=22 Å; Length=380 Å) plotted as yellow line in Figure 2. 26. This is consistent with the primary analyses of the p(r) function done previously and given in Figure 2. 14.B.

Indeed, this latter has revealed that the HSA-PAA complex exhibits a cylindrical form of a length around 400 Å but with a radius that yields 40 Å.

It can be noticed that the obtained radius is also smaller compared to that found for the protein. This could be due to the fact that HSA molecules are only linked to certain areas of the polymer chain, while for the remaining parts of the chain the radius has been evaluated to be 8 Å, so the average radius was around 22 Å. As stated previously, for such geometrical bodies the length is theoretically expressed as follows:

𝐿 2 12
= 𝑅 𝑔 2 + 𝑅 𝑐 2 . Thus, knowing that Rg was found to be around 110 Å and the crosssection radius Rc, is estimated to be 22 Å, it can be then concluded that both shapes are consistent with the theoretical model.

The same way the structure of PAA was investigated by fits using Sasview and found to be like a flexible cylinder with radius of 8 Å and a contour length of around 570 Å. Taking this under consideration, the polymer and the complex could be designed as illustrated above (Figure 2. 26).

Modelling of all-atoms structure of HSA-PAA complex

As stated previously, the analysis of the autocorrelation function p(r) has revealed that we can fit the data at small and medium q (less than 0.2 Å -1 ) using a cylinder of axial length close to 400 Å and diameter equal to 78 Å. Thus, the cylinder body was tested to fit the HSA-PAA complex experimental I(q) using Sasview and found to do it well as shown in the previous section. This model can provide information on the global shape of the complex, but provides no structural information on the internal organization of the complex. After that, size exclusion chromatography was used to investigate this complexation that has been revealed to be correlated to the PAA size. In fact, for every PAA size, HSA-PAA complex solution was flowed in a mobile phase having the same concentration of the studied PAA that has been used to prepare the complex sample. These experiments were performed by setting HSA concentration at 225 µM to avoid depletion of complex solution from HSA due to the dilution once in the column. The concentration of PAA was then chosen for every size of this latter by respecting the stoichiometry of complexation revealed previously.

The peaks of elution obtained with the different PAA sizes were then compared to each other and to the peak of single HSA illustrated in Figure 2. 30. Results disclosed a shifted peak of elution to higher molecular weights when increasing the size of PAA. This suggests that the size of the formed complex is bigger when the PAA is larger, which confirms that the complexation stoichiometry is effectively related to the PAA size. Afterwards, a SAXS samplings of the chromatography elution peaks were performed to obtain the scattering intensity I(q) of the complex fraction only, and to remove the contribution of aggregates. However, since the signal are very noisy at high angles, acquisitions on the same samples with the same concentrations were performed in direct injection mode without passing through the chromatography to improve the information at high angles. Subsequently, the autocorrelation function corresponding to every scattering pattern was calculated using Primus program, and it is shown in Figure 2. 31. The primary analysis of p(r)

displays an increasingly skewed distribution with the PAA size increase, and with a clear maximum at small distances corresponding to the radius of the cross-section Rc. This supports the hypothesis of the formation of more elongated HSA-PAA complex when the PAA is larger.

Various parameters were then extracted from these SAXS data (whether I(q) or p(r)) such as Rg, Vp, Rc and Dmax, and they are summarized in Table 2

Chapter II

As for the radius of the cross-section Rc, it did not vary too much compared to the Dmax, which indicates that the complex is only becoming more elongated with the increase in PAA size without big changes in size of the section. After that, the molecular weights of the HSA-PAAs complexes were evaluated from the chromatography data using the calibration curve given in Figure 2. 2. The calculated Mw were then compared to those estimated from SAXS data using Primus program. The obtained results

(quoted in Table 2. 3) disclosed the same trend of evolution with both methods with some differences between the two as exposed in Figure 2. 32.

Knowing the Mw of HSA-PAAs complexes and the theoretical Mw of both protein (≈ 67 kDa)

and PAAs, it was then possible to estimate the number of HSA molecules that could be bound to the chains of every PAA size. For instance, the evaluated Mw of HSA-PAA5kDa complex suggests that only one HSA molecule is bound to the PAA 5 kDa, while for HSA-PAA30kDa complex, the Mw suggests that one to two HSA molecules could be attached to every chain of PAA 30 kDa as illustrated in Figure 2. 32. This is consistent with the stoichiometry of complexation proposed previously based on the analysis of the other parameters.

4. Results and discussion 4.6. Influence of PAA size on HSA-PAA complexation

Chapter II

As for the HSA-PAA50kDa complex, the estimated Mw supposes that every complex particle contains 3 to 4 HSA molecules and one chain of PAA 50 kDa. Finally, the Mw of the complex HSA-PAA100kDa assumes that this latter could be formed from 4 to 5 HSA molecules with every chain of PAA 100 kDa. Thus, the stoichiometry that we can obtain from the Mw assessment for these two polymers (PAA 50 and 100 kDa), is a bit different compared to that suggested previously. In fact, it has been assumed that a maximum of 3 and 4 HSA molecules could be bound to the chains of PAA 50 and 100 kDa respectively. This outcome could indicate that these stoichiometries are just an average, and that not all the complexes contain the same number of HSA molecules. This could be due to the polydipersity of the polymer, which means that from the beginning not all polymers chains exhibit the same size, which can explain deviation on the stoichiometry of complexation. However, it is not possible to conclude on this because we do not have information on the polydipersity of polymers. From these results, it can be also noticed that the stoichiometry of complexation was not proportional to the PAA size. This could be arising out of the electrostatic interactions that could take place between two HSA molecules, and between HSA and PAA. In fact, HSA-PAA binding is supposed to be due to the attractive potential between positives patches carried on the surface of HSA and the negatively charged PAA. At the same time, two protein molecules will repel each other because of their global negative charge. This suggests that a minimal distance is needed between two HSA molecules attached to the same PAA chain, so that the HSA-PAA attractive potential can overcome the HSA-HSA repulsion.

Study of HSA-PAAs complexation reversibility

It has been revealed previously in this chapter (section 4.2.1) using chromatography that the HSA-PAA100kDa complex tend to dissociate in medium with pH higher than 5. In this section, the reversibility of HSA-PAA complexation was investigated for different sizes of PAA in mobile phases with pH maintained at 7. In order to assess the role of PAA presence on keeping the stability of complex, this study was also performed in mobile phases at pH 5 either containing PAA or without PAA. Results shown in Figure 2. 33 reveal different behaviours of HSA-PAA complex depending on the mobile phase and on the PAA size. First, it can be noticed that at pH 5 and in presence of PAA in the mobile phase, the shift of HSA-PAA5kDa complex peak (orange peak) is not that remarkable, which is understandable as the chains of PAA 5 kDa are supposed to be bound to only one protein. Despite of that, it was possible to see that the peaks are shifted back to that of 4. Results and discussion 4.8. Study of interaction between PAA and IgG Chapter II single protein in the absence of PAA from the mobile phase (black peak) or when its pH was maintained at 7 (green peak). This indicates that the complex has been dissociated and that the protein is no longer bound to the PAA in these conditions.

When it comes to the other sizes of PAA (30, 50 and 100 kDa), the shift of HSA-PAA complex peaks (orange peaks) is more obvious, which is necessarily due to the higher stoichiometry of complexation. Then, it is possible to notice the reversibility of complexation (dissociation of the complex) even in the presence of PAA in the mobile phase when the pH is maintained at 7 (green peaks) with all PAA sizes. This dissociation of the HSA-PAA complex can also be seen in a mobile phase with pH 5, but not containing PAA (black peaks). However, in this case the dissociation seems to be not complete and various distributions of size could be observed from Figure 2. 33 (black peaks). Indeed, the black peaks are broader showing that a large part of aggregates are still in there, with a part of complex and the detached single HSA, which could be illustrated by Figure 2. 1. In addition, the dissociation seems to be harder in these conditions when the PAA is larger, where less protein is detached which is reflected by smaller peak of single HSA every time the PAA is larger.

Study of interaction between PAA and IgG

In the previous sections, the HSA-PAA complexation has been proved to take place using several methods. This binding was revealed to be correlated to the pH of the medium and the stoichiometry of complex was proved to be related to the PAA size. In order to check if the HSA-PAA binding was specific of these two solutes, or it can be reproducible with another protein, the interaction of IgG with the same polyelectrolyte was investigated in this section.

For that, the experiments were performed using two sizes of PAA (30 and

Chapter II

It can also be seen from the obtained SAXS patterns that I(q) has been found to increase when 50 µM of PAA 30 kDa were added to the protein at pH 5 and 7. Indeed, the radius of gyration of the mixture was found to be around 80 and 75 Å respectively. The same thing was noticed when adding 50 µM of PAA 100 kDa at pH 5 and 7, but the increase of the intensity was a bit higher. However, the curves of I(q) did not display a difference at pH 5 or 7 as it was the case with PAA 30 kDa, which was reflected by a values of Rg closer found to be around 91 and 89 Å respectively. Since IgG has an isoelectric point around 7.2, it bears a positive overall charge at pH lower than 7.2. Thus, we can imagine that at this range of pH, electrostatic attractions could take place between IgG and the negatively charged chains of PAA, leading to the formation of complex which results in increase of I(q) at small angles. However, the overall charge of IgG at pH 7 is supposed to be almost neutral, and despite of that an increase in intensity was observed. This outcome indicates that the increase of I(q) reflecting an increase of the global size of the investigated macromolecules inside the sample may not be due to binding between IgG and Since experiments were performed at I = 0.15 M, we can think that IgG unfolding and aggregation hypothesis would rather be more consistent with our study. In fact, at a pH below 7, the overall charge of IgG becomes positive which could result in electrostatic attraction with the PAA chains causing partial unfolding of IgG as suggested in the study mentioned above [51]. But this could also bring the protein molecules closer to each other, which could lead to their intermolecular interaction, and so aggregation via Coulomb and hydrophobic interaction.

In order to validate that, chromatographic runs were conducted, where samples of IgG-PAA mixture were flowed in mobile phases with pH maintained at 5, 7 and 8 and containing the studied PAA as explained previously. The chromatographic pattern of IgG at different pH were drawn together, and then for each pH the patterns for the mixture with PAA 30 and 100 kDa were compared to that of IgG. The obtained results exposed in Figure 2. 35 confirm first that actually single IgG does not exhibit structural changes due to pH change. Indeed, it can be noticed that the elution peaks of the protein at different pH are perfectly superimposed. However, from these profiles it seems that IgG has more aggregates than HSA did.

After that, different behaviours were observed depending on the pH of the mobile phase when PAA was added to the protein. In fact, no clear shift was observed at pH 7 and 8 after the addition of PAA 30 kDa or PAA 100 kDa, although the SAXS results showed an increase in intensity at small angles at pH 7. This could be due to a greater contribution of aggregates that seems to be somewhat larger after the addition of PAA. As for pH 5, a shift of the elution peak towards higher molecular weights with a greater amount of aggregates is clearly observed. This shift in the elution peak was found to be the same with both PAA 30 and 100 kDa, but the rate of aggregates was higher with the PAA 100 kDa. In addition, the peaks are less resolute, where aggregates are not well separated from the main peak of the protein, which explains the increase of Rg. This upshot supports the hypothesis suggested previously and assuming that a partial aggregation of IgG is taking place after the addition of PAA.

Study of interaction between PEG and blood protein (HSA and IgG)

In has been revealed in the previous sections that HSA and IgG blood proteins exhibit different behaviours towards a negatively charged polyelectrolyte namely PAA. In fact, this bioactive polymer suggested for the capture of platelets has been found to form complexes with the most abundant protein in plasma (HSA) in some specific conditions of pH an ionic strength.

Meanwhile, it did not display a specific interaction with the IgG in these same physicochemical conditions, except for a partial aggregation of this latter.

In this section, the interactions of these blood proteins (HSA and IgG) with a neutral polymer namely PEG known for its resistance to proteins adsorption was investigated. For that, SAXS measurements were performed on the mixture of 10 g•L -1 of protein (which makes 150 µM for HSA and 65 µM for IgG) + 500 µM PEG. The experiments were conducted using three sizes of PEG (2, 8 and 20 kDa) with an ionic strength maintained at I = 0.15 M and pH of 5, 7 and 8.

The experimental scattering patterns of proteins at different pH were drawn together as reference to show the stability of the proteins structures with the pH change. Then for every pH we displayed I(q) of the protein-PEG mixtures with different PEG sizes compared to that of single protein. both HSA and IgG do not undergo through interaction with PEG in these conditions as it was the case with PAA. This could be arising out of the hydrophilicity of PEG which prevents its interaction with proteins through hydrophobic interactions. Besides, PEG is not ionised nor polar in solution and it does not bear neither positive or negative charges, which means that it cannot have electrostatic interactions with the charged proteins.

Some previous studies have attributed the resistance of PEG to protein interaction to its flexibility and to the hydrophilicity of its end group (-OH) [52]. However, other studies reveal that PEG could eventually exhibit weak hydrophobic interactions with proteins in aqueous solutions [53]. Indeed, it has been shown that the hydrophobic CH2-CH2 groups of PEG 20kDa could interact with the hydrophobic sites of BSA. It has been stated that such interactions increase with PEG concentration is due to the increased hydrophobicity of the longer PEG chain, and so multiple and steady contact points with proteins [53].

4. Results and discussion 4.9. Study of interaction between PEG and blood protein (HSA and IgG) 89

Chapter II However, nothing of that has been observed in our study even though we used a PEG 2 kDa (PEG methyl ether methacrylate) that has a CH2=CH-CH3 hydrophobic end group, which could be due to its small size. But even with the same PEG used in the study mentioned above (PEG 20 kDa), no changes in the scattering intensities was noticed. This indicates that either interactions between PEG and proteins are so week that it is not possible to sense them using SAXS, or no interactions are taking place at all. SAXS combined to chromatography has been revealed to be a promising tool to probe not only protein behaviour and structure, but even proteins-polymers complexes and interactions.

The study of the HSA-PAA100kDa interactions revealed a complexation phenomenon of protein molecules with the polymer chains. Tests on the effect of the ionic strength showed that this HSA-PAA binding is chiefly an electrostatic interaction, where attractive and repulsive interactions compete. Decreasing the pH to 5 allowed the negative overall charge of the protein to be reduced until the electrostatic attraction between the PAA chains and the positive patches on the protein dominated. The evaluation of the complex size suggests the attachment of several proteins to each polymer chain. Thus, the effect of the ratio of the protein concentration to the polymer concentration was also explored, which revealed that 4 HSA molecules could potentially be bound to each PAA100kDa chain. The complexation model that we proposed, allowed us then to describe the Porod volume of the mixture for different HSA/PAA ratios, which corroborates the stoichiometry of complexation assumed previously.

Besides, this complexation was found to be reversible where the HSA-PAA complex tended to dissociate once put in a diluted medium (without PAA) or at a pH greater than 5. That known, we can imagine that it is now possible to control the interactions between HSA and PAA at the membrane interface by changing the physicochemical conditions (pH, ionic strength).

The SAXS modelling study conducted using Sasview and aimed at describing the complex structure disclosed that the cylinder body (L ≈ 400 Å) fits well the experimental SAXS data giving an idea of the global shape of the complex, but without providing any structural information. Then, an all-atom model that can describe the final structure of the complex was sought using BUNCH program. Interestingly, the all-atom model found was consistent with that suggested using Sasview. In fact, the complex has been revealed to form an elongated pearlnecklace structure as a cylinder of around 400 Å of length with polymer pellets between the HSA molecules and a non-specific orientation of the HSA.

The HSA-PAA binding as well as the dissociation were found to be reproducible with smaller PAA sizes (5, 30 and 50 kDa) at the same physicochemical conditions of pH and ionic strength outlined previously. However, the complexation stoichiometry has been found to be related to the size of PAA. Indeed, a maximum of 3 HSA molecules has been assumed to be bound to each chains of PAA50kDa, 2 with each chain of PAA30kDa, while 1 HSA was the maximum to bind with PAA5kDa chains.

Introduction

It has been shown in the previous chapter that in some specific physicochemical conditions, the polymers suggested for the membrane functionalization such as PAA exhibit a specific interaction or not in solution with some blood proteins such as HSA and IgG. However, this does not mean that the interactions between these proteins and the same polymer fixed at the interface of a membrane would be similar to those revealed in solution. Thus, the reproducibility of these interactions on a functionalized membrane surface was investigated in this chapter, but since the modification of the membrane surface is an essential step, it was first explicitly explored.

Many studies have investigated surface modification of polymeric membranes in order to capitalize on their usefulness in filtration processes [1], [2]. Depending on the application, some physical, chemical or biological characteristics are incorporated into the surface to enhance a specific affinity with a target solute or prevent an unwanted phenomenon. Wherefore, the appropriate modification technique to use among many others is often explored to achieve excellent hydrophilicity and biocompatibility of the membrane. Overall, the modification technique can be either physical or chemical depending on whether or not a chemical reaction takes place during the modification process [3]. Although chemical methods allow to control better the deposition of the modifying layer, the physical modification is simpler, cost effective, and environmentally friendly [1]. One of the most common techniques based on physical processes is coating via physisorption. It is a simple method wherein a material is deposited on the membrane to form a thin layer and non-covalently adheres to the surface mainly because of van der Waals attractive interactions [4].

In this chapter, the functionalization of hydrophobic PVDF membranes surfaces is studied using coating by immersion, which involves the physical adsorption, an interesting membrane surface modification technique due to its simplicity and scalability. It only involves two steps:

1) immersion of the membrane in the coating bath (copolymer solution) for a specific time followed by 2) drying to remove residual solvent for the characterization of the membrane. The coating is performed using different copolymers as surface modifiers such as polystyreneblock-poly(acrylic acid) (PS-b-PAA) and polystyrene-block-poly(ethylene oxide) (PS-b-PEO).

Since coating via physisorption relies on non-covalent interactions between the coating layer and the membrane surface, the coating amount depends on the extent of interaction between the two. That can be influenced by the nature of the coating copolymer (polymer chain length,

Chapter

III

hydrophobic/hydrophilic block ratio, configuration), of the substrate and process conditions.

Thus, to get the best yield of this modification, the optimization of the process was conducted at room temperature by tuning different operating conditions (coating time, copolymer concentration, hydrophobic/hydrophilic chain length …etc), and it is discussed later in this chapter.

After that, the adsorption of blood proteins (HSA mostly and IgG) on the modified PVDF membranes was investigated using mainly Fourier-transform infrared spectroscopy (FTIR). As stated earlier, it allows to compare the interactions that could take place on a modified interface to those revealed in the previous chapter to happen in solution between blood proteins (HSA and IgG) and polymers (PAA and PEG) at some specific physicochemical conditions of pH and ionic strength. Thus, the effect of these conditions (pH and ionic strength) found to be decisive on the happening or not of the protein-polymer interactions was assessed.

As stated in chapter one, a training on the copolymers synthesis and membrane surface modification was carried out during a 6-weeks stay in the Research and Development Center for Membrane Technology (Chung Yuan Christian University, ChungLi, Taiwan). In addition, the adsorption of platelets on polystyrene well plates modified with some PS-PAA and PS-PEG copolymers was tested, and results are given in Appendix A.

It is important to note that two master students (Abigail Rosales and Sharath Jayakumar) hired to work on the project for 6-months each one have contributed to conduct some experiments reported in this chapter.

Effect of coating time

Optimization of coating time is rarely discussed in the literature related to membrane coating. Researchers usually specify coating time without further explanation as to why that time was used. Intuitively, an increase in coating time should lead to more coating since the copolymers are given enough time to assemble on the surface. This was confirmed in the literature when PVDF membranes were coated with PS-PEGMA copolymers [5]. Besides, a study investigating the variation of the coating time from 2 to 12h showed an increase in deposition degree of tannic acid and tetraethylenepentamine on PVDF membranes until it reached a plateau at 7h [6]. Similarly, an increasing trend with plateau was also observed when PVDF membrane was coated with polystyrene-poly(sulfobetaine methacrylate) (PS-r-PSBMA) [7]. These studies suggest that the adsorption process reaches a saturation point at longer coating time. That's because after certain time of coating the copolymers in the solution need to overcome the barrier created by the already attached copolymers. Consequently, coating becomes less and less favorable as coating time increases. Thus, optimum coating time depends on the studied copolymer/substrate system and should be established for every system.

The results obtained from these studies display some adsorption kinetics that could be described by the adsorption models reported in section 4.1 of chapter I such as Langmuir model that assumes the monolayer adsorption. However as stated in this same section, deviations from the model could be observed in the case of copolymer adsorption related to the copolymer specific properties such as, configuration, size, hydrophobic/hydrophilic ratio, etc.

Effect of copolymer concentration

Copolymer concentration is a parameter to be considered in optimizing the coating process.

Studies have shown that coating density increases with the copolymer concentration until it reaches a plateau regardless of the copolymer configuration [5], [8], which could be described as stated above by the well-known adsorption models such as Langmuir isotherm. However, as stated in next paragraph, the hydrophobic/hydrophilic segment ratio also plays a significant role in coating concentration efficiency [9], which leads to deviation from the adsorption model.

According to the literature, varying the concentration of poly(styrene-r-4-vinylpyridine) coated on PVDF membranes showed an increase in coating density with copolymer concentration [10]. Results exhibit a plateau reached at larger concentrations (10 mg•mL -1 ) which could be an indication of the saturation of the interface. The results of these studies suggest that the occurrence of plateau or saturation is common.

This is because at higher concentration, the surface may be fully covered, and the copolymers already attached to the surface may prevent the copolymers in the solution to reach the surface and be attached to it. Thus, copolymer concentration should be optimized when designing a coating process to avoid the excess use of coating material.

Effect of Hydrophobic/hydrophilic chain length

The adsorption of a copolymer on a membrane surface can be optimized by controlling the copolymer hydrophobic/hydrophilic block ratio and the block molecular size or chain length. the chain length of the hydrophilic PEGMA blocks reduces the copolymer adhesion [9], [12].

An increase in chain length of PEGMA while keeping the chain length of PS anchoring block constant (also decrease in PS/PEGMA ratio) decreased the adsorbed coating and required higher copolymer concentration to reach the plateau (Figure 3. 2) [9]. This means that larger PEGMA moieties introduced increased steric hindrance blocking the access of PS moieties to the PVDF surface. The same behaviour is reported in a study aiming at improving the biocompatibility of PVDF membranes by coating them with PPO-b-PSBMA [11]. PPO was used as an anchoring block since it has a high tendency to establish hydrophobic interactions with any hydrophobic membrane material. PPO block length was kept constant while PSBMA chain length was varied. The highest coating density was obtained at the lowest PSBMA/PPO ratio (0.5) where PSBMA had the shortest length (Figure 3. 1), which was attributed to two factors. First, when the PSBMA segment is relatively short, hydrophobic interactions between the PVDF membrane and PS dominate over hydrophilic interactions between the solvent and the PSBMA segment.

Conversely, when the PSBMA segment is long (PSBMA/PPO ratio=1 and 2), hydrophilic interactions become stronger since more PSBMA is available. As a result, it diminishes the coating efficiency driven by hydrophobic interactions. Second, similar to the previous study, longer hydrophilic PSBMA chains length increase steric hindrance. In addition, it can be observed that the effect of PSBMA chain length on coating density is not linear (Figure 3. 1).

2. Literature review on parameters affecting coating 2.4. Effect of copolymer configuration 104

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Another example of the hydrophobic/hydrophilic chain length effect is shown in a study mentioned before, where PVDF membrane surface was modified with a random copolymer of PS and PSBMA (PS-r-PSBMA) [7]. In this study, the adsorption of fibrinogen was found to be higher for bigger hydrophilic block amounts (70 % wt. PSBMA). This could mean that there was an insufficient coating amount due to larger PSBMA segment implying that it was difficult to establish hydrophobic interactions between the PS block and PVDF surface. These studies

show that an increase in the hydrophilic blocks size does not improve the coating density. Thus, to achieve good coating coverage, the hydrophilic interactions between the solvent and copolymer, and steric hindrance caused by hydrophilic block must be minimized.

Effect of copolymer configuration

Copolymer configuration also plays a role in coating adhesion. This was proved in a previous study that investigated the effect of varying PS-PEGMA copolymer configuration (diblock, triblock, random) with similar copolymer molecular weights and hydrophilic/hydrophobic block (PS/PEGMA) ratio on coating density [8]. The random copolymer has the highest coating density, followed by diblock, then triblock. This was attributed to the random arrangement of the styrene units in the random copolymer as illustrated in Figure 3. 3. The latter provided the most anchoring configurations to the PS surface than diblock and triblock copolymers assuming there are no chain repulsion and minor steric hindrance (Figure 3. 3). Also, it is possible to have multilayer coating with random copolymers since the hydrophobic blocks are not completely in contact with the PS surface. In fact, the available PS blocks can have intermolecular interactions leading to multilayer coating [8].

2. Literature review on parameters affecting coating 2.4. Effect of copolymer configuration 105

Chapter III

A similar trend in coating density was found when various PS-PEGMA copolymer configurations were used to coat PVDF membranes [12]. The random copolymer has a higher coating density than di-block copolymer whether it has a higher or a lower molecular weight (Figure 3. 4). andc) PS/PEGMA ratio = 3.7-3.9 [12].

Another study using FTIR mapping to identify PS-PEGMA coating presence and to analyze its heterogeneity on PVDF membranes showed a different trend than the previous studies [5].

Results showed that the diblock copolymer had a better coating coverage level than the random copolymer at lower copolymer concentrations (1 and 5 mg•mL -1 ) but similar at a higher concentration (10 mg•mL -1 ). In addition, the diblock copolymer had slightly better antiadsorption properties towards BSA protein than the random one at lower concentration (1 mg•mL -1 ) but similar at 5 and 10 mg•mL -1 . The difference in the trend from the first study could be due to different surfaces used. The first study used PS surfaces, which have stronger interaction with the copolymer than PVDF surfaces due to hydrophobic interactions between two groups of the same chemical nature. The second study used PEGMA blocks that were shorter or almost similar chain length with PS blocks (Figure 3. 4) while the last one used a PEGMA block twice longer than the PS block (PS53-b-PEGMA124 and PS61-r-PEGMA121). As mentioned earlier, long PEGMA brushes could introduce steric hindrance minimizing its coating efficiency.

2. Literature review on parameters affecting coating 2.5. Summary of different parameters effect on coating 106 Chapter III

Summary of different parameters effect on coating

The table below (Table 3. 1) summarizes the main trends that can be observed regarding the effect of the different parameters reported in this section on coating density evolution, as well as the deviations from Langmuir adsorption model related to each parameter.

Table 3. 1. Effects of the different parameters reported in this section on the coating density evolution, as well as the deviations from Langmuir adsorption model related to each parameter.

Parameter Effect on coating density Comparison to Langmuir adsorption model

Coating time

The coating density increases

Materials

Commercial polyvinylidene fluoride (PVDF) microporous membranes (VVHP, Millipore

Co.) with an average pore size of 0.1 μm and a thickness of 125 μm were used as-received for the whole study. The specific properties of this membrane are summarized in Table 3. 2 below.

𝑃𝐷𝐼 = 𝑀 𝑤 𝑀 𝑛 (3.1)
Where Mw is the weight average molecular weight and Mn is the number average molecular weight. The size of the copolymers used in this study has been evaluated using dynamic light scattering (DLS). After that, the evaluated hydrodynamic diameter (Dh) was used to estimate the copolymers radius of gyration using the following equation [13]: Where Rh=Dh/2 is the hydrodynamic radius and Rg is the radius of gyration.

All the copolymers used in the study with their average molecular weight, PDI, molecular structure and the evaluated hydrodynamic diameter and radius of gyration are summarized in the Table 3. 3.

Absolute ethanol (EtOHabs) and tetrahydrofuran (THF) used to solubilize the copolymer were purchased from VWR Chemicals Avantor® and Acros Organics, respectively. As for phosphate buffered saline (PBS) solution 10X (BP399, Fisher BioReagents), it was diluted to PBS 1X (pH=7.4; I=137mM) using ultrapure water before use. Ultrapure water was purified using ELGA PURELAB Prima purification system with an ELGA PURELAB Classic water purification system (final minimum resistivity of 18 MΩcm).

As for blood proteins used for the proteins adsorption study, ɣ-globulins from human blood (G4386), and albumin from human serum (A1653) were purchased from Sigma-Aldrich.

Coating of PVDF membranes

Operating process, coating time, copolymer concentration, and the hydrophobic/hydrophilic chain length were investigated to optimize the coating procedure using the different copolymers mentioned in the materials paragraph as surface modifiers. The experiments were performed at an average room temperature measured to be around 22 ± 3 °C.

The copolymer solutions were prepared by dissolving the desired weight of copolymer in 50% (v/v) EtOHabs-THF solvent. An area of 0.5 cm 2 of PVDF membrane was then immersed in 1 mL of the copolymer solution at the desired concentration and for the required time. For each set of experiments, a pristine membrane was taken as blank and was treated the same way as the coated ones but using the EtOHabs-THF solvent instead of copolymer solution.

For the change in process conditions, two operating processes were tested (Figure 3. 5) with the following order of procedure: 1) Immersion -Washing -Drying (IWD) and 2) Immersion -Drying -Washing (IDW). It is important to note that in the second case the membrane was drained and left to air for one minute before it was put in a Petri dish and placed in the oven to dry. In order to check the stability of coating when washing before and after drying, these two procedures were compared to a third one consisting of two steps only, Immersion and Drying (ID). For all experiments in this part drying time was 2h at 40ᵒC while washing was done three time using PBS 1X solution to remove the non-adsorbed copolymer. 3. Materials and methods

Methods for experiments

Chapter

III

As for the optimization of coating time, PVDF membranes have been immersed for 1h, 2h, 4h, and 6 h using 5 mg•mL -1 copolymer solution. These experiments have been conducted using two copolymers, PS30-b-PAA5 and PS30-b-PAA8, and followed with only one variable changing for the other copolymers. That variable is the copolymer concentration in solution, which was varied from 1 to 10 mg•mL -1 while fixing the coating time at 2 hours.

The other optimization condition was to check the copolymer hydrophobic/hydrophilic chain length effect on the coating ability of the copolymer over the membrane surface. Thus, to assess first the hydrophilic part effect, the length of the hydrophobic group was fixed using three copolymers with the same PS size and different PAA sizes. Then, the size of the hydrophobic block was also varied by changing the length of PS using both PS-b-PAA and PS-b-PEO. The experiments were conducted using a copolymer concentration of 5 mg•mL -1 and a coating time of 2 hours.

Static adsorption of blood proteins

The protein solutions were prepared by dissolving 1 mg of the desired protein (HSA or IgG) in 1 mL of PBS 1X. They were prepared the day before the adsorption experiment and kept refrigerated. The membrane was modified according to the optimum conditions fixed as mentioned previously. It was then immersed in 1 mL of PBS 1X overnight to hydrate the copolymer structure. Afterwards, PBS was replaced by 1 mg•mL -1 of protein solution for 2h at room temperature. Then, it was rinsed three times with PBS to remove the non-adsorbed proteins, and finally the membrane was dried in the oven for 2h at 35°C for further characterization. Figure 3. 6 illustrates the procedure of the protein adsorption on PVDF coated membrane. For each set of experiments, the adsorption of protein on the pristine membrane was performed as reference to compare it to its adsorption on the modified one. ELMER. The same procedure is implemented for the protein adsorption study. Some solutions of copolymer whether before or after coating have been diluted 4 to 5 times depending on the initial concentrations before the measurement because of the absorbance limit of the spectrometer according to which the calibration curves were also established.

The spectrometer used in this study is fitted with a beam splitter that allows dividing the coming beams in two (Figure 3. 7), one going through the reference cuvette when the other one is going through the sample cuvette. Therefore, for each measurement two quartz cells are used, one filled with 3 mL of solvent as reference, and the second one is filled with the same volume but of copolymer solution. The scans were carried out each time in a wavelength λ interval between 1000-100 nm, and the absorbance value was taken at 261 nm for the copolymer and at 280 nm for the proteins, where their peaks appear respectively. In ATR sampling, an infrared beam is directed onto an optically dense crystal with a high refractive index. The infrared beam reflects from the internal surface of the crystal and creates the so-called evanescent wave that is projected towards the sample, which is in contact with the crystal (Figure 3. 8). Some energy from the wave is adsorbed by the sample and the rest is transferred to the detector [14]. In regions of the infrared spectrum where the sample absorbs energy, the evanescent wave will be attenuated or altered. The attenuated energy from each evanescent wave is passed back to the IR beam, which then exits the opposite end of the crystal and is passed to the detector in the IR spectrometer. The system then generates an infrared spectrum. The detection and quantification of certain functional groups can be done by combining FTIR with ATR and it was found to be a high quality analysis tool for determining the chemical groups of complex samples [15]. Initially the sample holder was cleaned with ethanol and the background spectrum was collected using air to ensure there was no disturbance in the spectra due to the presence of impurities.

The membrane was then placed over the sample holder and was tightened to the point where it was in contact with the diamond crystal, and the sample spectrum was collected. All spectra were recorded over the wavenumber range from 4000 to 400 cm -1 , and they were unprocessed except for baseline alignment to qualitatively compare absorbance peak heights. Beer-Lambert Law (equation 3.3) states that absorbance is proportional to concentration. Thus, when comparing similar material, a higher absorbance peak means higher concentration on the surface.

Fourier transform infrared spectroscopy (FTIR)

FTIR is a technique that is used to obtain an infrared spectrum of absorption or emission of a solid, liquid and gas. It can be used for microscopy and imaging to characterize interfacial modifications. The instrument that determines the absorption spectrum for a compound is called a spectrophotometer, which is schematically illustrated in Figure 3. 9. transformation computer software to get finally the desirable spectrum [17].

FTIR spectroscopy is one of the most powerful tools for the determination of functional groups in a membrane together with possible molecular bonds between chemical compounds of membrane. Understanding of positions of IR absorption bands in the spectrum as wave numbers could be used for identification of the various chemical components (e.g., aromatic, amides). Basically, transition energies corresponding to changes in vibrational energy state for many functional groups are located in the mid-IR region (4000 -400 cm -1 ). Hence, the appearance of an absorption band in this region can be used to determine whether specific functional groups exist within the molecule [17]. Typically, a single bond such as O-H, C-H, and N-H is detectable in higher wavenumber (2500-4000 cm -1 ). Whereas, the triple bond and double bond are detectable in the middle wavenumber region 2000-2500 cm -1 and 1500-2000 cm -1 respectively. Additionally, the vibration of the molecule as a whole gives rise to a complex pattern of vibrations at low wavenumber region 650-1500 cm -1 that are characteristic of the molecule as a whole and hence can be used for the identification.

In FTIR, the absorption intensity relies on the molecule's change in dipole moment as a result of the absorption. Consequently, functional groups containing polar bonds, such as O-H, C=O, and N-H, are detected easily with FTIR, while detection of nonpolar groups can be difficult [18]. However, membrane samples should be analysed in its dry state since the OH group absorbs strongly in the infrared region and could obscure other interesting bands.

FTIR can be combined with attenuated total reflectance (ATR) technique, which is useful for surface characterization due to its access to the surface vibrational frequencies. In this mode, the sample is placed in contact with a crystal made from high refractive index transparent material as stated in the previous paragraph. The incident infrared light is focused on one end of the crystal at a certain angle and it undergoes several total internal reflections in the crystal until it reaches the detector (Figure 3. 10). The incident beam penetrates slightly onto the sample at each reflection, which results in a spectrum describing the surface chemical composition of the sample. However, the use of rough and highly porous membranes decreases the contact between the membrane and the crystal which could disturb the measurements [18].

FTIR is used in this study due to its ease of operation and simplicity. The membrane surfaces were scanned with an infrared spectrometer (IN10MX Thermo Scientific) under ATR mode using a germanium crystal with 25° incident angle. The spectrometer is equipped with a detector MCT-A cooled with liquid nitrogen, and a KBr beam splitter. The spectral resolution is 8 cm -1 , and 16 scans are acquired on each measurement point. 50x50 points were measured (with step size of 100 µm between 2 points), which makes a total area of 25 mm 2 that was used for surface chemical mapping to determine the coverage of the coating and the protein adsorption. The sample was prepared by fixing the dried membranes to the glass slide with an adhesive tape and ensured that the surface was flat to obtain proper results. The glass slide was fixed to the sample holder. The focus was adjusted such that the light falls on the center point of the membrane.

The sample was set at the required position and the ATR tip was fixed to the microscope to perform mapping after being cleaned to ensure there was no impurities.

Files obtained from the FTIR analysis were first processed with the OMNIC Software Suite (OMNIC Atlµs v.9.2, Thermo Fisher Scientific) which allowed the scanned maps to be opened.

To generate the chemical maps, peaks of interest that confirm the presence of the coating and proteins were first identified using ATR. Peaks with the highest absorbance and unique to the material of interest are chosen for the chemical mapping. Then, the peak area was measured to generate the chemical maps, which are color-coded according to absorption peak intensity from blue (lowest intensity) to red (highest intensity). A higher peak intensity indicates more coating or protein presence on the surface. All maps generated are unprocessed except for the atmospheric and the baseline corrections.

It is important to note that experiments of membrane coating with copolymer and protein adsorption on the membrane were repeated from 2 to 3 times, and so the mapping to confirm the reproducibility of the results.

Determination of copolymers and proteins characteristic peaks

Since FTIR mapping is the major technique used to characterize the modified membrane in this part, the spectra of the PVDF pristine membrane and the pure copolymers were determined first using ATR. These spectra were then compared to differentiate the peaks representing the chemical structure of the copolymer that are used after that to generate the chemical color-coded Based on the molecular structure of the copolymer given in Table 3. 3, and the ATR spectra shown in Figure 3. 11 it can be noticed that the PS group and the copolymer backbone can be easily seen for all PS-b-PAA copolymers. In fact, whether C-H deformation and C=C stretching of the aromatic in PS, =C-H stretching in PS, or -C-H stretching in the copolymer backbone, they are all observable in the ATR spectra and their peaks are summarized in Table 3. 4. As for the hydrophilic group, ideally, the spectrum of carboxylic acid in PAA should have strong absorption for O-H stretching at 3300-2500 cm -1 (broad peak), and for C=O stretching at 1700 cm -1 . However, not all the peaks are observable for all the PS-b-PAA copolymers. As a matter of fact, C=O stretching is seen for all of them except for PS30-b-PAA2, which could be due to The same procedure has been done for the PS-b-PEO to determine the peaks representing its chemical structure. From the ATR spectra given in section 2 of the Appendix C, it can be noticed that the PS group and the copolymer backbone are easy to observe as it was for the PSb-PAA. Whereas, the absorbance of C-O stretching in PEO could be seen at 1150-1085 cm -1 , but it overlaps a bit with peaks of the PVDF membrane.

Therefore, for most of FTIR mapping results, absorption peak at 700 cm -1 was chosen to analyse the presence of copolymer over the membrane surface as it had strong absorbance and it did not overlap with the peaks of the membrane material (PVDF).

As for the human blood proteins (HSA and IgG), ATR spectra of their powder form were also obtained and are given in Appendix C. Their general structure is intricate, but it is known that they are made of amino acids connected by peptide bonds (amide) [19]. Therefore, notable peaks are observed in their spectra at 3500-3070 cm -1 attributed to the amide N-H stretching vibration, and at 1680-1630 cm -1 due to the amide C=O stretching as shown in Table 3. 4.

4. Methodology for the analysis of ATR-FTIR results 4.2. FTIR maps processing 120 Chapter III

FTIR maps processing

The areas of the characteristic peaks of the copolymers and those of the proteins determined using ATR were then measured using OMNIC Software Suite (OMNIC Atlµs v.9.2, Thermo Fisher Scientific) to generate the color-coded maps from FTIR mapping. The obtained maps display the qualitative distribution of the copolymer or the protein (depending of the chosen peak of interest) over the membrane surface. However, FTIR mapping was used to assess the variation of different conditions (coating time, concentration, etc), which results in different intensity colour scales (rainbow scales) for the maps obtained under different conditions, and thereby these maps cannot be compared.

For that, the color-coded maps with different rainbow scales (using the full scale value given by the OMNIC software) were converted to maps coded with the same grey scale from black (lowest intensity) to white (highest intensity) using a Python code given in section 3 of the Appendix C. The code allows then to evaluate the grey value all over the analysed area to calculate an intensity average value for the area of the peak of interest (that should be correlated to the adsorbed quantity). Besides, the code evaluates the standard deviation around the average value that could estimate the heterogeneity of coating or that of the protein distribution.

Below in Figure 3. 12 is shown an example of conversion of a color-coded map generated for the peak at 700 cm -1 for the coating at different concentrations of PS30-b-PAA2 as compared to a pristine membrane (PM), to a map coded with a grey scale. Thus all the maps latter in this chapter were converted using this same grey scale, so they can be compared.

Results and discussions

In this section we will be discussing the results we obtained as part of the coating procedure optimization of PVDF membranes with the different copolymers. Besides, the adsorption of blood proteins on PS-b-PAA coated membrane is explored under different conditions to investigate the selectivity of PAA towards HSA revealed in the previous chapter in solution.

The adsorption of HSA on membrane coated with PS-b-PEO is also studied to test the efficiency of PEO on improving the resistance of PVDF membranes to fouling.

Variation of the coating operating process

In this part of the optimization study, the influence of the coating procedure (illustrated in procedure determined from this study will be used for the coating of the other copolymers. A copolymer concentration of 5 mg•mL -1 and a coating time of 2 hours are used in this study. The influence of these two parameters on the coating density will be analysed latter in this chapter (section 5.2 and 5.4).

As stated previously, the PS segment is the anchoring block to the PVDF membrane surface as a result of their hydrophobic/hydrophobic interactions, while the PAA block forms a hydrophilic layer. That implies that the stability of coating depends on the strength of the interactions between the PS segment and the membrane surface. Thus, the main reason for checking the various operating processes is to analyse the effect of washing on the stability of the copolymer coated over the membrane surface before and after the membrane is dried.

Wherefore, the coating was performed by either Immersion -Washing -Drying (IWD) and Immersion -Drying -Washing (IDW), and the results are compared to a procedure consisting only on Immersion and Drying (ID). The three samples are dried at 40°C before being then compared with the pristine PVDF membrane (PM) as reference using FTIR mapping. The FTIR maps were generated in this section using three peaks (2920 cm -1 , 1715 cm -1 and 700 cm -1 )

assessing respectively the presence of the copolymer backbone, the PAA chains and the PS block. It was done by measuring the area of each peak alone to see qualitatively the distribution of the corresponding functional group over the membrane surface as shown in Figure 3. First, differences in the intensity of absorbance of the different peaks are noticeable from the obtained maps. In fact, for the 700 cm -1 and the 2920 cm -1 peaks, the intensity of absorbance is higher than that of the peak at 1715 cm -1 , which makes it easier to assess the presence of the PS and the copolymers backbone than the PAA. That confirms what has been figured out previously from the ATR spectra. Despite of that, the maps show that both copolymers adhered to the membrane surface, and almost the same trend of coating is observed for both of them using the three peaks. Indeed, more copolymer presence is observed with ID and IDW than IWD as it can be seen from Figure 3. 13 and Figure 3. 14. This suggests that washing after drying results in better copolymer deposition than washing immediately after immersion. The latter involves the presence of both the solvent used for coating and the PBS used for washing in the environment. This could mean that the interactions between the copolymer and the surrounding medium is slightly stronger than the hydrophobic interactions between the copolymer and the membrane resulting in partial removal of the coating. Otherwise, this could be owing to the fact that some copolymer chains were deposited but not adsorbed onto the 5. Results and discussions 5.1. Variation of the coating operating process 123 Chapter III membrane surface and they were then removed during rinsing step. Meanwhile, when washing was done after drying, the solvent had already dried out in between the copolymer brushes before washing. This could ensure stronger hydrophobic interactions leading to better copolymer adhesion, and so higher copolymer amount over the membrane surface. Besides, this could increase the deposition of the copolymer chains not initially adsorbed onto the membrane surface.

From the FTIR maps, it is also possible to observe that the distribution of the copolymer over the membrane surface was not always homogeneous, and especially with PS30-b-PAA8.

This could be due to heterogeneities of the membrane morphology and on the distribution of the membrane pores. length of PS block supposed to ensure the hydrophobic interactions with the PVDF hydrophobic membrane. However, this is still an observation and not a conclusion at this stage that needs to be studied with different sizes of copolymer, which is discussed explicitly later in this chapter (section 5.3). Now, if the difference between IWD and IDW processes was clear enough by a simple visual analysis of the maps, the comparison between IDW and ID processes is harder this way. For that, the color-coded maps with different rainbow scales were converted to maps with the same grey scale as explained in section 4.2. Since the C-H stretching in PS showed the highest intensity of absorbance at 700 cm -1 , the map generated for this peak area was chosen to conduct these processing. From the converted maps in the grey scale exposed in Figure 3. 15, we can notice that the coating is more heterogeneous for IWD than IDW and ID, which is the same thing that has been previously observed from the color-coded FTIR maps. This is consistent with the fact that less copolymer is present on the surface for the IWD process after the nonhomogeneous removal of the adsorbed copolymer during washing step. From these image analyses, the average intensity of the peak area and the standard deviation (represented by error bars) are quantitatively evaluated for each copolymer and for the different operating conditions as shown in Figure 3. 16. Eventually, the mean value of the peak area for the ID process is a bit higher than that of the IDW for the coating of both copolymers as shown in Figure 3. 16. Despite the difference in intensity is not that great, these results suggest that washing is still removing a small part of coating even after drying. This could mean that the PS/PVDF hydrophobic interactions of this removed part of the copolymer are weaker than the 5. Results and discussions 5.1. Variation of the coating operating process 125 Chapter III interactions with the PBS used for washing. Otherwise, it could mean that the removed part was only deposited and not adsorbed, and even after the membrane was dried this part was not attached to the surface. The results display also a higher standard deviation for the IWD process implying bigger heterogeneity, which confirms what has been noticed previously from FTIR maps and the converted maps. The obtained mean intensities plotted in Figure 3. 16 confirm also the observation made previously from the FTIR maps regarding the comparison of the copolymer adhesion to the membrane surface. In fact, a higher intensity was obtained for the coating of PS30-b-PAA5 than that of PS30-b-PAA8 involving a bigger amount of the first copolymer attached to the membrane surface.

At this stage, the optimization study aims to covering the membrane surface with the copolymer in order to investigate then the copolymer interactions with the human blood proteins. Therefore, the ID process was chosen to conduct the experiments on the other parameters explored later in this chapter.

5. Results and discussions 5.2. Variation of coating time 126 Chapter III

Variation of coating time

As stated in the literature review of this chapter, not much attention has been drawn to the fact that the coating density may be dependent on the time that the membrane has been left in contact with the copolymer solution. Therefore, the effect of the coating time on the coating density is evaluated in this section. For that, the membranes were immersed for 1, 2, 4 and 6 hours in a 5 mg•mL -1 copolymer solution (PS30-b-PAA5 and PS30-b-PAA8) at room temperature (22 ± 3 °C). The samples were then prepared for FTIR mapping as explained previously, and the maps were generated for the peak at 700 cm -1 . The obtained results exposed in Figure 3. 17 display clearly an increase in the copolymer presence, so of the coating density when the coating time increases. However, this increase was quickly not that notable after 2 hours of immersion. In fact, the maps at 2, 4 and 6 hours evince the presence of the copolymer all over the analysed area, which could involve almost a complete coverage of the membrane with the copolymer. Eventually, this could mean that the copolymers the standard deviation to the obtained average coating density. Even known, the initial concentrations have been checked to consider the deviations to the calibration curve that could be due to the solutions preparation and the dilutions done before each measurement. As a matter of fact, the measured initial concentrations have been found to be 5.06 and 5.2 mg•mL -1 for PS30-b-PAA5 and PS30-b-PAA8 respectively, which is higher than 5 mg•mL -1 . As exposed in Table 3. 5, this deviation is not negligible compared to the final concentration obtained after immersion and needs to be considered. Thus, the measured concentrations were used to calculate the difference of mass instead of 5 mg•mL -1 , since after immersion the same solutions are used for the UV-visible spectroscopy measurement. It has to be mentioned that since the coating was performed by immersion, the calculated coating density corresponds to the attachment of copolymer on both sides of the membranes, each one has an area of 0.5 cm 2 , which makes a total area of 1 cm 2 . In addition, the adsorbed amount estimated this way includes the copolymer that penetrated into the pores during coating and about which we do not have any clear information. Despite of that, the obtained results exhibited almost the same trend whether shown by the FTIR maps or by the calculation of the mean intensity of the peak area. Indeed, the coating density has been found to increase when the immersion time increases. Even after 2 hours a slight increase is observed where the coating 5. Results and discussions 5.2. Variation of coating time 129 Chapter III density of the PS30-b-PAA5 copolymer is raised from 0.44 mg•cm -2 at 2 hours to 0.48 mg•cm -2 at 4 hours, then 0.46 mg•cm -2 at 6 hours. This increase is greater in the case of PS30-b-PAA8, where the coating density raised from 0.31 mg•cm -2 at 2 hours to 0.38 mg•cm -2 and then 0.4 mg•cm -2 at 4 and 6 hours respectively. This could suggest that the copolymer is still able to adhere to some areas of the membranes not visible on the mapping since the analysed area is very small (25 mm 2 for each sample) compared to the total coated one (0.5 cm 2 ). It could also be due to the fact that the copolymer is still diffusing inside the pores of the membranes even after the saturation of the surface, which is not possible to see with FTIR mapping or the calculation of the intensity because both techniques are related to the surface only. Indeed, DLS measurement proved that all the copolymers exhibit a very small size as shown in Table 3. 3 compared to the average pore size (0.1 µm) of the membrane.

Table 3. 5. The calculated coating density and the measured mean intensity of the peak area (700 cm -1 ) with their standard deviations for different coating times. Now, considering the relatively high standard deviations to the average of the calculated coating density shown in Table 3. 5, it is difficult to conclude on the differences between 2, 4 and 6 hours. However, at this stage the study aims at covering the membrane surface with the copolymer layer, so it will be possible then to investigate the interactions with human blood proteins. Therefore, we estimated that 2 hours of immersion was enough to do that and is faster to perform experiments, thus it was chosen as the optimum coating time for further experiments.

Variation of hydrophobic/hydrophilic chain length

The copolymer hydrophobic/hydrophilic block ratio and the chain length have a substantial role in the adsorption of the copolymers onto the membrane surface. In fact, the primary results Results from the first mapping (Figure 3. 20) sustain what has been noticed in the previous sections. In fact, for a same hydrophobic chain length, the copolymer with the shortest hydrophilic block adheres better to the membrane surface. This means that hydrophobic interactions between the PVDF membrane and PS block are diminished by stronger hydrophilic interactions between the solvent and PAA segment due to the increase of PAA size. The decrease in adhesion propensity could be also due to the fact that larger PAA moieties introduced increased steric hindrance blocking the access of PS moieties to the PVDF surface.

This outcome is consistent with what has been reported in the literature [9], [11] and stated in the review section of this chapter (section 2.3).

5. Results and discussions 5.3. Variation of hydrophobic/hydrophilic chain length 131

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From the second mapping it can be noticed that the higher coating is obtained for the copolymer PS100-b-PAA107 (PAA/PS ratio ≈ 1), which makes sense because the two other copolymers are of a PAA/PS ratio around 2 and 3. This suggests that copolymers with smaller hydrophilic/hydrophobic blocks ratio can be deposited better onto the membrane surface due to stronger hydrophobic interactions with the PVDF membrane. However, PS26-b-PAA76

(PAA/PS ratio ≈ 3) has been found to adhere more to the membrane surface than PS13-b-PAA22

(PAA/PS ratio ≈ 2) although the first copolymer has higher hydrophilic/hydrophobic ratio. This means that the block's ratio is not the only parameter involved in the coating of the copolymer, a minimum of PS length is also required to have sufficient hydrophobic interactions with the PVDF membrane. As a matter of fact, the presence of PS13-b-PAA22 is almost not seen in the map shown in Figure 3. 20, which is probably due to the small size of PS, and so weaker hydrophobic interactions with the membrane. These results came first to sustain what has been noticed from the FTIR maps, but it also allows to compare the coating ability of all copolymers. In fact, it confirms that copolymers with PS block size less than 20 kDa show a very weak propensity to deposit compared to the other copolymers. However, the most remarkable thing from Figure 3. 21 is that the difference of intensity is more obvious when the hydrophilic chain is small as can be seen for a PAA segment of 2, 5 and 8 kDa with a PS block of 30 kDa. When the hydrophilic chain is larger (PAA of 76 kDa), the difference is lower even though the PS block size (26 kDa) is close to that of the previous copolymers. Then, increasing the PS block size from around 30 to 100 kDa, 5. Results and discussions 5.4. Variation of the copolymer concentration 133 Chapter III the increase in intensity is not that big compared to the increase in size, which is maybe due to the steric hindrance introduced by the size of PAA. This suggests that from a certain length of hydrophilic segment, the steric hindrance dominates over the hydrophobic interactions of PS block with the PVDF membrane. However, since we are looking for the peak at 700 cm -1 representing the PS group, the latter could be hidden by the largest PAA segments which form the top layer, whereas the PS is attached to the membrane. This depends on how the copolymer chains are arranged on the membrane surface, something that we do not know in this case.

From Figure 3. 21, it can also be observed that PS26-b-PAA76 copolymer has higher intensity than PS28-b-PEO13, even though the former has a larger hydrophilic chain. This means that the strength of interactions between the hydrophilic block and the solvent depends on the hydrophilic segment nature. The latter could also have a role on how the copolymer is attached to the membrane, which affects the ability to see the PS group using FTIR mapping.

Variation of the copolymer concentration

Effect of PS-b-PAA concentration on HSA adsorption

One of the parameters usually explored in membrane surface modification study is the concentration of the modifier substance. Classically, in coating studies using copolymers as surface modifiers, the coated amount is evaluated as a function of the copolymer concentration in the coating bath by calculating coating densities [9], [12]. In this section, the effect of this latter on the coverage of the membrane surface is first investigated by evaluating the mean intensities from FTIR maps as explained previously (section 4.2). The coating was performed with PS30-b-PAA5, PS30-b-PAA8, PS26-b-PAA76 and PS100-b-PAA107, and that by using ID process, 2 hours of immersion and a copolymers concentration of 5 mg•mL -1 . The FTIR maps were generated this time for the peak at 700 cm -1 representing the PS group, and the one at 1715 cm -1 representing the PAA segment. That was done to check what has been discussed in the previous section about the ability of seeing the PS block using FTIR mapping depending on the hydrophilic and the hydrophobic blocks lengths. It also allowed to show the presence of both PS and PAA over the membrane surface, and to have an idea about the deposition way of the copolymer onto.

Subsequently, the effect of the copolymer concentration on the adsorption of HSA, the most abundant protein in human plasma, is discussed using FTIR mapping. The adsorption of HSA on the coated membranes was conducted using a protein concentration of 1 mg•mL -1 at a pH around 7. The FTIR maps were generated for the peak at 1660 cm -1 to check the presence of FTIR results exposed in Figure 3. 22 have all displayed the same trend of coating with the four PS-b-PAA copolymers. Indeed, whether for the presence of PS, PAA or HSA, an increase in intensity is observed by increasing the copolymers concentration until 5 mg•mL -1 . Beyond 5. Results and discussions 5.4. Variation of the copolymer concentration 135 Chapter III this concentration, the maps do not show clear visual differences. In fact, almost the same coverage of the surface by the copolymers is observed at 5 and 10 mg•mL -1 implying a saturation of the membrane surface at 5 mg•mL -1 . In contrast, what has been clearly visible from the maps is that the PAA is not necessarily visible in the same locations over the membrane surface as the PS block.

Subsequently, the effect of the copolymer concentration on the adsorption of HSA, is discussed using FTIR mapping. The adsorption of HSA on the coated membranes was conducted using a protein concentration of 1 mg•mL -1 at a pH solution around 7. The FTIR maps were generated for the peak at 1660 cm -1 to check the presence of HSA over the modified In addition, the evaluated intensities validate that a copolymer concentration of 5 mg•mL -1 sufficiently covers the membrane surface and using higher concentration would be excessive.

From the FTIR maps, it can also be seen that the HSA presence increased with the concentration of copolymer until 5 mg•mL -1 . But, it seems that this increase is not correlated to the copolymer size since the same scale of intensity was obtained except for PS30-b-PAA8. The evaluated mean intensities (Figure 3. 24) confirm that the HSA adsorption reached the maximum for a copolymer concentration of 5 mg•mL -1 . Beyond this copolymer concentration, the mean intensities are almost unchanged for all the copolymers, despite some differences of HSA adsorption are disclosed on membranes coated with different copolymers, especially on PS30-b-PAA8 coated membrane.

These results (Figure 3. 24) expressed first the ability of the PVDF pristine membrane (PM)

to adsorb HSA, but the adsorption has been found to vary considerably between the different samples. This adsorption could be due to the hydrophobic interactions between the membrane and the hydrophobic parts of the HSA [20]. However, it has to be noted that the variabilities of HSA adsorption displayed on the PM (first points in Figure 3. 24) could induce some inaccuracies on HSA adsorption on membranes coated the different copolymers.

In addition, results evince that the distribution of the protein was not homogeneous, which is shown in Figure 3. 24 by the standard deviations to the average value of the intensity. This is 5. Results and discussions 5.4. Variation of the copolymer concentration 137

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probably due to heterogeneities on the morphology of the membrane implying variation of the hydrophobic interactions strength over the membrane surface.

After that, results displayed greater ability of PS-b-PAA coated membrane than the PM to adsorb HSA. Indeed, the HSA adsorption increased with the presence of the copolymer until reaching a plateau at a copolymer concentration of 5 mg•mL -1 . Since the experiment was carried out at pH 7, this could not be a complexation between PAA and HSA as observed in solution in the previous chapter at pH 5 only and not at 7. Two explanations could be brought to this behaviour. For long PAA chains, the adsorption of HSA on the coated membrane could be due to the steric hindrance that retains the protein in between the brushes of copolymers. For smaller PAA chain, it could also be due to the hydrophobic interactions between the hydrophobic parts of the protein and the accessible PS block on the surface, which is also possible to happen in the case of large PAA chains. As a matter of fact, if the distance between two PAA moieties is large enough, that increases the chances for the HSA molecules to be in contact with the PS segments horizontally attached to the membrane surface regardless of the size of PAA. Thus, the electrostatic repulsions between HSA and PAA will be reduced and the hydrophobic interactions between the protein and the coated membrane will be stronger. This would be consistent with what is reported in the literature regarding the adsorption of HSA on PS surfaces. Indeed, it has been shown using UV spectroscopy that HSA adsorbs strongly onto polystyrene plates and onto polystyrene latex [21]. That could be also the reason why the presence of HSA on the membrane surface was not correlated to the length of the PAA segment. However, this does not explain the increase of HSA adsorption on coated membranes than on PM if in both cases the adsorption is driven by hydrophobic interactions.

Thus, we can imagine that these hydrophobic interactions are stronger between the protein and PS than those between the protein and the pristine PVDF. Otherwise, the adsorption on membrane coated with PS-b-PAA may be driven by the combination of both hydrophobic interactions between HSA and the visible PS blocks, and the electrostatic attractions that could take place between positive patches carried on HSA and the negatively charged PAA.

Effect of PS-b-PEO concentration on HSA adsorption

As a water-soluble and electrically neutral polymer, poly(ethylene oxide) (PEO) is wellknown for its excellent resistance to the nonspecific adsorption of proteins [22]. Wherefore, it's sometimes used as copolymer when conjugated to a hydrophobic group such as PS for the modification of biomaterials interfaces to prevent cells adhesion [23]. However, its resistance to the non-specific adsorption of proteins strongly depends on having a good coating layer of PEO onto the material surface. For that, the effect of PS30-b-PEO13 concentration on HSA adsorption is investigated in this section using ID process and 2 hours of coating time. The copolymer PS30-b-PEO13 was chosen to perform these experiments because it was the one that showed the best ability of adhesion to the membrane surface in the previous section. The maps were generated for the peaks at 700 cm -1 (copolymer) and at 1660 cm -1 (HSA), and the mean intensities for these peaks areas were evaluated from the converted maps using the grey scale.

Results shown in Figure 3. 25 evince the reaching of a plateau at a copolymer concentration of 5 mg.mL -1 , implying almost a complete occupation of the available sites for HSA adsorption.

As for HSA adsorption, it was found to be reduced with the increase in copolymer concentration, which confirms the antifouling properties of PEO. The exact behaviour has been proven to take place in a previous study, where the rejection of HSA was found to increase with increased grafting density of PEG of molecular weight 5kDa [24].

This resistance to protein adsorption is probably due the strong water affinity of PEO, its large excluded volume and strong coordination capability to water molecules [25]. Many studies have demonstrated that PEO chains physically or chemically fixed on the material surface form a surface hydration layer and thus resist the nonspecific adsorption of foreign substances [26]. for the peaks area at 700 cm -1 and 1660 cm -1 ; [HSA]= 1 mg•mL -1 .

HSA adsorption on membrane coated with both PS-b-PAA and PS-b-PEO

The previous results have evinced the adsorption of HSA on PS-b-PAA coated membranes.

Meanwhile, the adsorption of the same protein on membrane coated with PS-b-PEO was proved to be reduced. In this section, the adsorption of HSA is tested on PVDF membranes coated with both copolymers at the same time, and it was compared to that on each copolymer alone. The coating was performed using PS26-b-PAA76 and PS28-b-PEO13, two copolymers that have

shown almost similar abilities of PS adhesion to the membrane surface. The copolymer concentration used for coating was 5 mg•mL -1 each one alone, and 5 mg•mL -1 of PS26-b-PAA76 + 5 mg•mL -1 of PS28-b-PEO13 in the mixture. Samples were characterized using FTIR mapping and maps were generated for the peaks area at 700 cm -1 and 1660 cm -1 to check the presence of copolymers and HSA respectively. FTIR maps and the evaluated mean intensities, shown in Figure 3. 26, confirm the presence of both copolymers on the same surface. Indeed, the coverage of the membrane coated with the mixture of both copolymers was found to be larger than that with each copolymer alone at the same concentration. As for the HSA adsorption, results proved once again that it increased in the presence of PS-b-PAA, and that it was reduced in the presence of PS-b-PEO. When both copolymers are present, the adsorption of HSA seems to be less compared to that on PS-b-PAA alone but higher compared to that on PM and on PS-b-PEO alone. This suggests that the interactions between HSA and the visible segments of PS dominate against those between HSA and PEO. This could probably be due to the random arrangement of the copolymer brushes on the membrane surface, which creates heterogeneity on the distribution of the PEO chains and on the spaces between the copolymer moieties. In the previous section, the adsorption of HSA on PVDF membranes coated with PS-b-PAA was observed at pH 7, which was probably due to its hydrophobic interaction with the PS blocks combined to some electrostatic attractions with the negatively charged PAA. Meanwhile, it has been proven explicitly that at this same pH, the HSA does not show any affinity with PAA chains in solution. In fact, the binding between the two was found to happen only at pH 5 forming HSA-PAA complexes. Moreover, the number of HSA bound to each PAA chain has been proven to be correlated to the PAA chain length. In order to check the reproducibility of these same findings, but with a PAA fixed onto a surface, the adsorption of 1 mg•mL The FTIR maps were generated first for the peak area at 700 cm -1 to confirm that the copolymer was correctly coated onto the membrane surface and then for the peak area at 1660 cm -1 to assess the presence of HSA. The obtained results (Figure 3. 27) confirm that both copolymers were correctly coated and show their presence all over the membrane surface.

Indeed, for the coating of both copolymers, the evaluated mean intensities (Figure 3. 28) of the peak area at 700 cm -1 were almost the same for the different coated samples. That allowed us to compare then the adsorption of HSA at different pH on these same samples.

The maps evince also the presence of HSA over the whole range of studied pH, but its adsorption has been revealed to be greater for a pH of 5. The evaluation of the mean intensities of the peak area at 1660 cm -1 (Figure 3. 28) has affirmed that, where an intensity of 0.58 was obtained at pH 5 against 0.37 at pH 7 and 8 on membranes coated with PS30-b-PAA5 copolymer.

As for membranes coated with PS100-b-PAA107 copolymer, the intensity yields 0.64 at pH 5 against 0.41 and 0.40 at pH 7 and 8 respectively. It is important to remember that the protein and the PAA chains are both negatively charged because the HSA has an isoelectric point around 4.7, and the PAA has a pKa that yields 4.5.

However, even though the overall charge of the protein is negative this latter carried positives patches on its surface [27] as shown in Figure 3. 29.

Thus, repulsive electrostatic interactions are supposed to happen because of the negative overall charges that both HSA and PAA bear, whereas the adsorption of HSA was found to take place. As stated previously, this behaviour is probably due to hydrophobic interactions between HSA and the visible PS segments in the spaces between PAA brushes, in addition to electrostatic attractions that could take place between the negatively charged PAA and the positive patches on HSA surface (Figure 3. 29). Thereby, the increase in adsorption at pH 5 could be due to the reduction in the negative overall charge of the protein, which reduces the electrostatic repulsions and allows HSA molecules to get closer to PAA chains. Thus, it could increase the HSA adsorption driven by the attractive electrostatic interactions with PAA, and so increases the total amount of HSA adsorbed onto the membrane surface. These results have proved also that the adsorption was not correlated to the PAA size as it was the case in the previous chapter (section 4.6). This could be due to the fact that the protein molecules may not have access to the full length of the PAA chains linked from one end to the PS block which is attached to the membrane in a random way. This suggests that not all the length of the PAA segment is in contact with the protein molecules, which makes it difficult to imagine the same interactions as in solution.

In order to support these findings, the adsorbed amount of HSA has been estimated by difference of mass before and after immersion of the coated membranes in the protein solution 5. Results and discussions 5.7. Ionic strength effect on HSA adsorption onto membrane coated with PS-b-PAA 144 Chapter III using the UV-visible spectroscopy. As previously explained, the initial concentrations were evaluated also for more reliable measurement. Since the adsorption was carried out by immersion, the adsorbed amount of HSA was calculated by considering the area of both sides of the membrane (1 cm 2 ). Even though the adsorbed amounts of HSA are estimated for both sides of the membrane, results summarized in Table 3. 6 confirms the adsorption of HSA onto the coated membranes with higher amount at pH 5. This outcome corroborates the hypothesis suggesting that HSA adsorption at pH 5 is controlled by two forces, the hydrophobic interactions with the PS, and an increased electrostatic attraction with the PAA due to the reduction of the negative overall charge of HSA. The HSA-PAA interaction evidenced in solution at pH 5 is here responsible of an increase in adsorbed HSA by c.a. 53%.

Ionic strength effect on HSA adsorption onto membrane coated with PS-b-PAA

Since we suggested that the increase in HSA adsorption at pH 5 could be due to the increased attractive electrostatic interactions, we varied in this section the salts concentration by adding the desired amount of NaCl to the protein solutions in order to check that. Therefore, the ionic strength was increased from 0 M of added salts (0.05 M provided by the buffer) to 0.5 M final concentration, and the pH was set at 5 using a mixture of sodium citrate di-hydrate and citric As previously, the coating was performed for PS30-b-PAA5 and PS100-b-PAA107 using ID process, 2 hours of coating time and a copolymer concentration of 5 mg•mL -1 . After that, the modified membranes were immersed in 1 mg•mL -1 HSA solution, before being dried and characterized using FTIR mapping. The maps were then treated the same way and the mean intensities of the peaks area at 700 cm -1 and 1660 cm -1 were evaluated.

Whether from FTIR maps exposed in This outcome is consistent with what has been proven in the previous chapter (section 4.3),

where the interactions between HSA and PAA were found to take place only at ionic strength ≤ 0.15 M. This confirms in some way the attractive electrostatic nature of the interactions between the protein and the PAA layer negatively charged. This could be coherent with the results of a study reported in the literature regarding interactions between HSA and PAA [27].

In this latter, simulations have showed that PAA can bind to some preferential positives patches on HSA because of specific electrostatic interactions.

In fact, in presence of attractive interactions, adding salt leads to screen, and thus to reduce the range of the attractive interactions. Thereby, the attraction between positive patches carried on the HSA surface and the PAA brushes should then be less efficient to induce adsorption.

Therefore, the electrostatic repulsion due to negative overall charge of both overcomes the attractive forces and reduces the adsorption (as shown at I=0.5 M in Figure 3. 31). Indeed, the intensity was around 0.3 at I=0.5 M even though the pH was 5, which is lower than the intensity found to be around 0.4 at pH 7 and I=0.15 M. This confirms that the adsorption of HSA at pH 7 on a PVDF membrane coated with PS-b-PAA was not due only to hydrophobic interactions.

It proved that electrostatic attraction between PAA and positive patches carried by HSA plays effectively a role even at pH 7 and this role became bigger at pH 5 due to stronger electrostatic attractive forces.

Eventually, these results confirm once again that, unlike interactions in solution with free PAA chains in the medium, those between HSA and a fixed PAA are not correlated to the PAA chain length. against intensities around 0.55 and 0.18 previously. This could be due to the large size of IgG (~150 kDa), which makes the copolymer less visible after the adsorption of the protein onto the surface. This also suggests that the IgG adsorption is due to the hydrophobic interactions between the PS segments of the copolymer brushes and the hydrophobic parts of the protein.

Thereby, a large part of the PS blocks is hidden by the protein presence on it. Therefore, IgG does not exhibit specific interactions with PAA chains as it did for HSA. That could also be the reason why no difference was observed between the adsorption of IgG on coated membranes and on PM. Thus, HSA is much more soluble than IgG in PBS which means that the latter protein has weaker interactions with the solvent which increases its relative affinity for hydrophobic surfaces such as PVDF and PS. This is consistent with the behaviour of IgG already reported in the literature, where it was proved to be adsorbed strongly whether on PVDF [20], [28], or on PS surfaces [29]. These results proved that IgG can be adsorbed on membranes coated with PS-b-PAA even if the protein has no affinity with the hydrophilic PAA chains, something that has been proved in the previous chapter.

Chapter III

Conclusions

FTIR mapping is a powerful tool that allowed to assess the presence of different components with different chemical structure qualitatively on the membrane surface. It permitted us to investigate the distribution of the copolymers and proteins on the same surfaces.

The processing of FTIR maps using the Python code that allowed to evaluate the mean intensities of the peaks absorbance has strengthened the reliability of the mapping results. It gave the quantitative aspect to the analyses of the copolymer attachment and the protein adsorption.

The evaluation of the coating density by mass balance using the UV-visible spectroscopy sustained the FTIR results, despite the high standard deviation to the values and the nonconsideration of the copolymer that penetrated into the membrane pores.

Coating via physisorption has been proved to be an effective technique for the modification of PVDF membranes even if it does not allow to control the distribution of the copolymer brushes over the membrane surface. In addition, drying immediately after the immersion was found to strengthen the attachment of the copolymer layer to the membrane surface, and make it difficult to remove with washing. Meanwhile, washing immediately after immersion and before drying removed a big part of this layer.

Two hours of immersion and a copolymer concentration of 5 mg•mL The adsorption of HSA on PS-b-PAA coated membranes has been revealed to be correlated to the pH and the ionic strength as it was the case for the HSA-PAA complexation in solution.

That involved the electrostatic attraction role between HSA and PAA on this adsorption, but also the hydrophobic interactions between HSA and PS blocks.

The coating of PS-b-PEO over the membrane surface increased the rejection of HSA and enhanced the resistance of the membrane to the nonspecific adsorption of proteins. The coating of both copolymers (PS-b-PAA and PS-b-PEO) onto the same membrane surface has been found to be possible, where each copolymer has brought its contribution to the HSA adsorption.

In fact, the protein adsorption on membrane coated with both copolymers was lower than on PS-b-PAA alone but greater than that on PS-b-PEO alone.

Conclusions

Chapter

III

The IgG has shown a different behaviour on the PS-b-PAA coated membrane than that shown by HSA. Indeed, it exhibited the same ability of adsorption whether on a PVDF pristine membrane or on a coated one.

Chapter IV

Introduction

As reported in the previous chapter, the modification of PVDF membrane was successfully performed by coating PS-b-PAA and PS-b-PEO onto the surface. Besides, these copolymers were either found to selectively attach to blood proteins in the case of PS-b-PAA or to enhance the resistance to their adsorption in the case of PS-b-PEO. In this chapter, the modification was repeated on larger membrane surfaces, and the dynamic (i.e. in filtration mode) adsorption of HSA was investigated via its dead-end filtration through the modified membrane. This could help analyzing the extent to which blood proteins are adsorbed to the membrane surface in dynamic mode since in long terms our system is meant to capture of platelets when blood is passed as feed through the filtration system.

However, there are certain aspects that need to be considered during the filtration process.

Especially during microfiltration, pore narrowing or pore blocking can reduce the membrane permeability and prevent the transmission of certain components of blood cells through the membrane. Pore blocking can be caused by both blood proteins and by the copolymers used for surface modification and that penetrate into the pores. Blood proteins tend to foul the surface whereas the pores are narrowed down by aggregates formed by the protein [1]. The aggregates formation can be due to the pressure, temperature, pumping, stirring and also due to electrostatic forces and hydrophobic interactions [2]. Another important phenomenon that affects the filtration performance is concentration polarization. It is a function of hydrodynamic conditions along the membrane and is independent of the membrane physical properties. It can create in some cases a new phase at the interface such as a protein gel layer, which reduces the permeate flux by offering a hydraulic resistance to the flow of solvent and by causing osmotic back pressure [3]. Moving on to certain important characteristic properties of membrane filtration are pH and ionic strength. It has been reported in the literature that at low pH, there was more selectivity in separating proteins like IgG and BSA, which was attributed to electrostatic interactions [4]. This is something that we also probed in the previous chapters, whether in solution using SAXS [5] or on the membrane surface using FTIR. It is to be noted that electrostatic interactions between the charged groups of the protein and the polymeric groups of the membrane play a major role in the aggregation or deposition [6]. Apart from electrostatic interactions, protein transport performance also depends upon operating conditions of the membrane used for filtration like the pore morphology, and surface physicochemical properties [7].

Chapter IV

Hence, the effect of surface modification on the membrane permeability is first assessed in this chapter. Then, the deposition of HSA on PVDF membrane coated with different sizes of PS-b-PAA is explored during dead-end filtration experiments. Besides, the influence of HSA retention on the membrane transport properties is investigated by following the flux evolution during the dead-end filtration of HAS through the membrane. The permeability measurement is also performed on PS-b-PEO coated membrane, and the HSA filtration through was then carried out.

Eventually, the filtration is performed on PS-b-PAA coated membrane at pH 5, at which the adsorption has been revealed to be increased in the previous chapters whether at the interface of a modified membrane in static mode, or in solution.

It is important to note that the experiments presented in this chapter were performed with the help of a master student (Sharath JAYAKUMAR) hired for 6 months to work on this project.

Materials

The membranes used in this chapter are the same as those used in chapter III. It is a commercial polyvinylidene fluoride (PVDF) microporous membrane (VVHP, Millipore Co.)

with an average pore size of 0.1 μm and a thickness of 125 μm. However, a larger area of membrane (3.8 cm 2 ) was used here compared to 0.5 cm 2 used previously. The different properties of this membrane are given in Table 3. 2 (section 3.1 of chapter III).

The copolymers used for surface modification are chosen among those which exhibited a good propensity of adhesion to the membrane surface in the previous chapter and that also The filtration experiments were performed with either ultrapure water or solutions of albumin from human serum (A1653) purchased from Sigma-Aldrich, and that by using a pressurized stirred Amicon cell (Series 8010, Merck Millipore).

Copolymers were dissolved in 50% (v/v) absolute ethanol (EtOHabs) and tetrahydrofuran (THF) purchased from VWR Chemicals Avantor® and Acros Organics, respectively. While HSA was dissolved in PBS 1X (pH=7.4; I=0.137M) diluted from phosphate buffer saline (PBS) solution 10X (BP399, Fisher BioReagents) using ultrapure water. Ultrapure water was purified was obtained from an ELGA PURELAB Prima purification system combined to an ELGA PURELAB Classic water purification system (final minimum resistivity of 18 MΩcm).

Methods

Permeability measurement and membrane coating

Before the filtration experiments are conducted, precautions should be taken to obtain accurate data. First, the hydrophobicity of the membrane could result in low transport characteristics. To overcome this and to help wetting the membrane, it was first immersed in absolute ethanol for a while before starting its compaction. Membrane compaction was a second step, which is the compression of the membrane due to applied pressure resulting in the unsteady and declining transport properties. The primary reason to perform membrane compaction is to reach steady transport characteristics.

In order to perform compaction, dead-end filtration of ultrapure water was conducted through the membrane placed in the Amicon cell (Series 8010, Merck Millipore). For that, the pressure was increased between 0.2 and 1 bar (by steps of 0.2 bar), and for each pressure the After that, the pressure was decreased in the same range and the permeability was determined from the slope of the curve plotting flux vs pressure as stated by the following Darcy Law:

𝐽 𝑤 = 𝐿 𝑝 𝜇 ∆𝑃 (4.1)
Where Jw is the water flux (m 3 •m -2 •s -1 ), Lp is the permeability (m), μ is the viscosity of the fluid (Pa•s) and ΔP is the transmembrane pressure (Pa).

After the evaluation of the pristine membrane permeability, its coating was performed. For this purpose, the cell was emptied of water without removing or touching the compacted membrane and the copolymer solution was then added. The membrane was left in contact of 4 mL of 5 mg•mL -1 copolymer solution during 2 hours. Subsequently, the copolymer solution was removed from the cell and the permeability of the coated membrane was determined the same way as for the pristine membrane. The filtration of ultrapure water through the coated membrane was performed by varying the pressure between 0.2 bar and 1 bar. For each pressure, the weight of the permeate was then collected every 5 min during 20 min, which was sufficient to reach almost a steady flux. The flux Jw was then calculated (in L•m -2 •h -1 ) by relating the weighted mass of permeate to the time and the membrane area (3.8 cm 2 ). It is important to mention that all experiments were performed at a room temperature of 22 ± 3 °C.

Dead-end filtration of HSA

The dead-end filtration of HSA was performed on membranes coated with the different copolymers mentioned in the previous section and the HSA retention was compared to that of the pristine membrane. This experiment was carried out by filling the Amicon cell with 10 mL of protein solution with a concentration of 1 mg•mL -1 that was filtered through the membrane.

The pressure was set at 1 bar and the weight of permeate was noted down every minute until almost 3 mL was left in the retentate. The volume reduction factor (VRF), that can be calculated using the formula below would be equal to 3.3.

𝑉𝑅𝐹 = 𝑉

0 𝑉 𝑅 = 𝑉 0 𝑉 0 -𝑉 𝑃 (4.2)
3. Methods 3.2. Dead-end filtration of HSA 161

Chapter

IV

Where V0 is the initial volume of HSA solution, while VP and VR are the volume of permeate and retentate, respectively. The dead volume that may remain between the membrane and the support and in the permeate pipe was collected with permeate.

The permeate and retentate were then collected and the absorbance of the samples was measured at 280 nm using UV-vis spectroscopy as explained in section 3.3.1 of chapter III. The absorbance value was noted down and the concentrations of the permeate and retentate were then calculated using the calibration curve (given in section 1 of the Appendix C) obtained with absorbance of HSA at known concentrations. Then the retention was calculated using the following equation:

𝑅𝑒𝑡𝑒𝑛𝑡𝑖𝑜𝑛 (𝑅) = 1 - 𝐶 𝑃 𝐶 𝑅 (4.3)
Where CP and CR are the HSA concentration in permeate and retentate, respectively, at the end of the dead end filtration (i.e for a VRF=3.3).

Besides, the concentration factor (FC) can be then defined as follows:

𝐹𝐶 = 𝐶 𝑅 𝐶 0 (4.4)
In order to analyse the extent of membrane fouling during dead-end filtration of protein solution, the flux reduction was calculated using the following formula:

𝐹𝑙𝑢𝑥 𝑟𝑒𝑑𝑢𝑐𝑡𝑖𝑜𝑛 = 𝐽 𝐽 𝑤 (4.5)
Where J is the final flux at 1 bar during filtration of protein solution and Jw is the flux at 1 bar during water filtration.

Further, mass balance can also be used during filtration of protein solution to calculate the amount of protein adsorbed on the membrane surface using the following mass balance equation:

𝑉 𝑖 𝐶 𝑖 = 𝑉 𝑃 • 𝐶 𝑃 + 𝑉 𝑅 • 𝐶 𝑅 + 𝑚 𝑎𝑑𝑠 (4.6)
It is important to mention that all experiments from membrane compaction, to its coating and then dead-end filtration of HSA, were performed at a room temperature of 22 ± 3 °C.

Experiments have been repeated at least twice to confirm the obtained results.

FTIR and ATR analysis

As stated in the previous chapter, FTIR was used to assess qualitatively the presence of the copolymer and the protein over the membrane surface. Thus, the membrane surfaces were scanned with an infrared spectrometer (IN10MX Thermo Scientific) equipped with a detector MCT-A cooled with liquid nitrogen, and a KBr beam splitter. FTIR mapping was performed under ATR mode using a germanium crystal with 25° incident angle where 16 scans are acquired on each measurement point with a spectral resolution of 8 cm -1 . A total membrane area of 25 mm 2 was covered during each mapping where 50x50 points were measured (with step size of 100 µm between 2 points). Since the analysed area (0.25 cm 2 ) is too small compared to that coated initially (3.8 cm 2 ), simple ATR measurements were carried out on the membrane area not assessed by FTIR mapping. For that, ATR spectra are measured by collecting 16 scans for each point in five different locations of the membranes with a spectral resolution of 4 cm -1 using a Nicolet 6700, Thermo Scientific instrument. The five ATR spectra obtained from the five different locations were then averaged to get a spectrum that represents the chemical composition of the membrane surface.

FTIR analysis were processed with the OMNIC Software Suite (OMNIC Atlµs v.9.2, Thermo Fisher Scientific) which allowed the scanned maps to be displayed. The maps were generated at 700 cm -1 and 1720 cm -1 to evaluate the PS-b-PAA distribution over the membrane surface, and at 1660 cm -1 and 3300 cm -1 to assess that of HSA.

The average mean intensities from FTIR maps are evaluated as explained in section 4.2 of chapter III to compare qualitatively the presence of HSA over the membrane surface.

It is important to mention that FTIR and ATR analysis were carried out only after finishing all the steps of the filtration experiment, going through membrane compaction, permeability measurement, membrane coating and finally protein filtration. Besides, all samples were dried in the oven at 35 °C during 2 hours before ATR and FTIR measurement.

Water contact angle

Water contact angle is used to study the wettability of the modified membrane and compare it to that of the pristine one (principle explained in section 4.3.3.2 of chapter I). Since PVDF membranes were coated using copolymers of different hydrophobic/hydrophilic blocks sizes, the contact angle is expected to change by changing the block ratio. That was studied for various copolymers among those mentioned in section 2. Results are discussed later here in this chapter.

Accordingly, contact angle meter was used to determine the hydrophobicity of the membrane before and after coating, and then after HSA dead-end filtration. Initialization of the device was carried out by checking the base level of the contact angle meter to ensure that the sample holder was flat. The method used to determine the contact angle of the sample was by deposit drop method. The microscope attached to the device was tilted downwards between 0° and 1° at a position between 5 and 6 mm. The syringe used for measuring the contact angle was SY20 (28944) that was specifically used for the deposit drop method. The syringe was cleaned with pure water twice to ensure removal of impurities. Then it was pressed gently to remove any air gap. The substance that was used to measure the contact angle was water and the phase environment was set to air. The sample was prepared in such a way that the membrane was fixed tightly to the glass plate with an adhesive tape and ensured that the membrane was flat to prevent errors during measurement of contact angle. Then the glass plate was placed on the sample holder. The light was kept at high intensity to obtain a clear image of the measurement.

The magnification of the microscope was set to 2.5X to obtain a precise image of the measurement. The adjustment knobs were used to position the sample at the center of the screen and the baseline was corrected. Then, the measurement was taken by bringing the needle closer to the sample and running the procedure. Once the water droplet (4 µL) falls on the sample, the syringe was moved away, and the measurement was saved. In such a way, five measurements were taken for each sample and the same procedure was followed for different samples. All samples were dried in the oven at 35 °C during 2 hours before contact angle measurement. Accordingly, the membrane was every time wet in ethanol and then compacted before the coating by copolymers or HSA filtration was performed. Thus, any further deviation of the transport properties would not be due to the compression of the membrane or to the solvent exchange, but to coating or fouling.

Effect of the copolymer size on the permeability of coated membranes

Once the compaction had been carried out, the Amicon cell was emptied from water to add then the copolymer solution in order to perform the coating of the compressed membrane (single contact of 4 mL of 5 mg•mL -1 copolymer solution during 2 hours). These experiments were conducted using PS30-b-PAA5, PS26-b-PAA76 and PS100-b-PAA107 in order to explore the copolymer size influence on the permeability of the membrane. Wherefore, permeability measurements were performed for membranes coated with each copolymer mentioned above.

Eventually, membranes were dried and FTIR mapping was performed to check the coating presence and that the copolymer layer had not been removed during the permeability measurement i.e. when the water was passed through. FTIR maps were generated for the peaks at 700 cm -1 and 1715 cm -1 to assess respectively the presence of PS and that of PAA. For each peak, the maps are taken using the same scale for different copolymers, so that their coating over the membrane surface can be qualitatively compared. although their distribution over the membrane surface was heterogeneous. This means that physical adsorption of the copolymer onto the membrane surface has taken place and it is stable even if water was passed through the membrane immediately after coating for permeability measurement. This is probably due to the fact that the copolymer is not soluble in aqueous solutions which reduces its interaction with water and favors its stability onto the membrane.

4. Results and discussions 4.2. Effect of the copolymer size on the permeability of coated membranes 166

Chapter IV However, it can also be noticed in Figure 4. 2 that the PS seems to be more present for the coating of PS30-b-PAA5 than for PS100-b-PAA107 coating and then PS26-b-PAA76 that showed the lowest intensity of coating. This would confirm hypothesis reported in the literature that longer hydrophilic chain could introduce increased steric hindrance blocking the access of PS moieties to the PVDF surface [8]. Besides, larger PAA necessarily have stronger interactions with the solvent [9], which reduce the copolymer adhesion into the membrane surface.

However, longer hydrophilic segments could hide some parts of the PS blocks from being visible in FTIR. In fact, PAA seems to be logically more visible for the coating of PS100-b-PAA107 than the coating of PS26-b-PAA76 and then that of PS30-b-PAA5. Hence, if larger PAA hide some PS segments from being seen in FTIR maps, it is not possible to say which copolymer is more or less present but it is more accurate to say which part of each one is more visible. This From Figure 4. 3, it can be noticed that the permeability decreases with the increase in size of the copolymer coated over the membrane surface as evidenced by the permeability decline due to coating (Table 4. 2). Indeed, the permeability was found to be reduced by around 31% after the coating of PS30-b-PAA5 and by 48 % after that of PS26-b-PAA76 whereas the decline was of 65 % after PS100-b-PAA107 coating. This could be arising from the fact that larger chains length of the copolymer block the passage of water through the membrane by creating additional resistance to the system thereby reducing its permeability. Results evince first the adsorption of HSA on the pristine membrane, which is probably due to hydrophobic interactions between the hydrophobic parts of protein and the PVDF membrane.

The deposition of HSA seems to be then greater on coated membrane. Besides, a visual analysis of the FTIR maps discloses that it is slightly increasing with the increase in the copolymer size.

However, since the visual difference is slight and the protein distribution over the membrane surface is heterogeneous, it is difficult to conclude on that by saying on which case the HSA deposition would be higher.

It is important to remember that the pH of HSA solution in this case was around 7 (pH of PBS). Thus, the HSA deposition would be due first to the adsorption driven by the combination of the hydrophobic interactions of the protein with the visible PS blocks on the membrane surface and the electrostatic attraction between the positive patches carried on HSA surface and ) and proved by the investigation of the ionic strength. This could also explain why the HSA adsorption is higher on modified membranes than on pristine membranes (PM) where it is probably caused by only hydrophobic interactions. However, the HSA deposition is taking place during dead-end filtration which is operated under pressure. Thus, the increase of HSA deposition could also be related to the accumulation of HSA once all the sites of adsorption are saturated, which could lead to the formation of a protein gel on the surface.

Otherwise, we can think that the greater adsorption of HSA on coated membrane than on PM could be due to stronger hydrophobic interactions between the protein and PS blocks than between the protein and the PVDF membrane. One can imagine that this could also be due to the fact that proteins are trapped between the polymer brushes in the case of coated membranes.

Thereby, their removal when water is passed through the membrane would be easier on a pristine membrane than on a coated one, and it would be difficult when the copolymer brushes are larger.

ATR measurements were also performed on the remaining surface of the membrane, which was not analysed by FTIR. As for the contact angle measurement, ATR spectra were taken in five different locations of the membrane surface, before being averaged then to obtain the final spectrum that can give information about the chemical composition of the membrane surface. First, the characteristic peaks of the copolymer at 700 cm -1 for PS and at 1720 cm -1 for PAA appear on these spectra (Figure 4. 5.A) which supports the results discussed in the previous section confirming the presence of the copolymer all over the membrane surface. Then, it is also possible to observe on the same spectra the characteristic peaks of HSA at 1660 cm -1 and at 3300 cm -1 , which affirms its presence too. However, while this outcome with the FTIR maps shown previously confirm the presence of the copolymer and the protein all over the membrane surface, it does not address the issue of their heterogeneous distribution.

For a more quantitative comparison, the spectra were superimposed to compare their peaks height, and so the amount of HSA adsorbed onto the surface. In fact, as stated in the previous chapter (section 3.3.2) and according to the Beer-Lambert law (equation 3.3), a higher absorbance peak means higher concentration on the surface. Therefore, the comparison of the spectra shown in Figure 4. 5.B indicates that more HSA was found on the membrane coated with PS30-b-PAA5 than on the PM since the characteristic peaks of protein (1660 cm -1 and 3300 cm -1 ) are higher in the first case. Compared to this latter case, these peaks are then even higher for membranes coated with PS26-b-PAA76 and PS100-b-PAA107, whereas no difference is shown between these two copolymers, as their peaks whether at 1660 cm -1 or 3300 cm -1 overlap almost entirely. These results are consistent with what has been observed from FTIR mapping.

However, the trend observed here is partially different compared to that obtained in the previous chapter (section 5.4.1), where the adsorption of HSA was found to be not related to the copolymer size. This could be arising out of the fact that the pressure applied during filtration helps to trap more HSA between longer PAA brushes. In addition, it could enhance electrostatic interactions between the proteins and larger PAA chains by pushing HSA in contact with bigger part of PAA brushes. In other words, a longer part of the PAA chains becomes accessible to the HSA molecules to interact with it.

After dead-end filtration of HSA through modified membranes, the contact angle was also measured as previously explained. The obtained average values were compared to that obtained on the pristine membrane and on the modified membranes without HSA on the surface i.e. before the protein filtration is performed (Table 4. 1). seem to change much. This could mean that the contact angle technique is not that sensitive to slight differences of HSA adsorption previously observed from FTIR and ATR measurements.

HSA retention influence on membrane transport properties

As stated in section 3.2, during dead-end filtration, the permeate mass was noted down every minute until 3 mL are left in the retentate, so that the permeate flux could then be calculated.

The flux evolution during HSA filtration on every modified membrane has been plotted versus time and is compared to that obtained on the pristine membrane (Figure 4. 7). From these results

we can first notice that for a same volume (≈ 7 mL) that has been passed through the membrane,

the filtration lasted less time on the PM than on the modified membranes and that on the latter it lasted longer when the copolymer was larger. Indeed, it lasted 13 min on the pristine membrane, while it lasted 23 min, 33 min and 47 min on membranes coated with PS30-b-PAA5, PS26-b-PAA76 and PS100-b-PAA107 respectively. This makes sense since we showed earlier in this chapter (section 4.2) that the permeability of the membrane decreased with the size of the copolymer. fluctuating between 84 and 87 L•m 2 •h -1 but it was almost stable. This suggests that the adsorption of HSA on the pristine membrane does not affect its transport properties. In order to assess the effect of HSA adsorption on the membrane transport properties when it is coated with the different copolymers, the flux reduction was calculated using equation (4.5). The flux of water (Jw) was taken each time at 1 bar before HSA filtration was started, while J is the final flux at 1 bar after the filtration of 7 mL of protein solution. The obtained results plotted in Subsequently, the concentration of HSA in the permeate and in the retentate collected at the end of filtration was estimated using UV-visible spectroscopy. Thus, the membrane retention of HSA was evaluated using equation (4.3), to deduce the amount of protein adsorbed on the membrane surface by mass balance using equation (4.6). The outcomes of these calculations are summarized in Table 4. 3. One observes a decreasing HSA concentration in the permeate, while it increased in the retentate with the increase in copolymer size, which was then logically reflected by an increased retention rate. This is probably arising out of the fact that larger copolymers block more the pores and make them narrower. This has been shown earlier to decrease the membrane permeability and may also contribute to preventing the HSA passing through. Besides, the adsorption density of HSA onto the membrane surface was found to be around 0.11 mg•cm -2 , which is comparable to the protein adsorption density of the commercial membrane reported by the supplier to be 0.15 mg•cm -2 as shown in Table 3. 2. Then, the HSA adsorption density has been revealed to increase to 0.39 mg•cm -2 on membrane coated with PS30-b-PAA5, to reach 0.48 mg•cm -2 and 0.52 mg•cm -2 on that coated with PS26-b-PAA76 and PS100-b-PAA107 respectively. This is consistent with the FTIR and ATR results. In fact, the HSA adsorption has been shown to be higher on the membrane coated with PS30-b-PAA5 than on the pristine membrane, and even higher on membranes coated with PS26-b-PAA76 and PS100-b-PAA107 with not much difference between these two copolymers. As explained earlier, this The procedure detailed previously was followed for the membranes coated with PS-b-PEO or with both PS-b-PAA and PS-b-PEO at the same time. Thus, coating of the compacted pristine membrane was performed using a 5 mg•mL -1 solution of PS28-b-PEO13 copolymer. As for the coating of both copolymers at the same time, a mixture of 5 mg•mL -1 of PS26-b-PAA76 + 5 mg•mL -1 of PS28-b-PEO13 was used. The permeability of the membrane was evaluated before and after coating to assess the effect of coating on the membrane transport properties. After that, dead-end filtration of 10 mL HSA solution at 1 mg•mL -1 and pH ≈ 7 through the modified membranes was carried out until 3 mL are left in the retentate (VRF = 3.3). The flux was also noted down from the beginning to the end of the protein filtration to rate the reduction that could be due to fouling of the membrane with HSA.

Samples were characterized using FTIR mapping and maps were generated for the peaks area at 700 cm -1 and 1660 cm -1 to check the presence of copolymers and HSA respectively.

Thereby, the amount of HSA found after dead-end filtration on membranes coated with both PS26-b-PAA76 and PS28-b-PEO13 is compared to the amount on the PM and on membrane coated with each copolymer alone. in solution and during static adsorption. This is probably due to the increase of electrostatic attraction between the negatively charged PAA and the positive patches carried on HSA surface after the reduction of its negative overall charge when reducing pH to 5.

In order to validate that, the concentration of HSA in the permeate and in the retentate was estimated by UV-visible spectroscopy and the amount of HSA deposited onto the membrane surface was determined by mass balance. The results reported in Table 4. 5 show a retention rate of 63 % when the filtration was performed at pH 5, which is close (slightly lower) to that obtained previously at pH 7 that yields 68 %. However, these calculations support what has been noticed from FTIR maps, where HSA deposition onto the membrane surface at pH 5 was revealed slightly higher. Indeed, the adsorption density of HSA was found to be around 0.59 mg•cm -2 at pH 5, and 0.52 mg•cm -2 at pH 7. On the other hand, this increase (12 %) is not that sufficient to conclude definitively on the increase in protein adsorption at pH 5, especially as the method used (UV-visible) These results evince effectively that the HSA adsorption at pH 5 is greater than that at pH 7, where the mean intensity has been evaluated to be around 0.93 and 0.59 at pH 5 and 7 The membrane transport properties were also investigated before and after dead-end filtration. In fact, permeability measurements were performed after coating and it was found to be around 36 L•m 2 •h -1 •bar -1 (Figure 4. 15), which is higher than that obtained earlier for membrane coated with the same copolymer and that yields around 30 L•m 2 •h -1 •bar -1 . This was also noticed previously in the case of PM and it confirms that two different pieces of membrane prepared in exactly the same conditions could have different permeability, which would be due to the heterogeneity of pores distribution over the membrane surface.

After that, the flux evolution during HSA filtration was followed as shown in Figure 4. 15, and it was found to decrease from almost 36 L•m 2 •h -1 to 29 L•m 2 •h -1 after that a volume of 7 mL was passed through the membrane (VRF = 3.3). This makes a flux reduction J/Jw of 81 %, which is almost the same (79 %) as that obtained when the filtration was performed at pH 7.

This suggests that the flux reduction was not affected more by the additional deposition of HSA

Conclusions

Dead-end filtration is the process that is close to real conditions in which the suggested system in this project can work in long terms. Thus, it was interesting to run experiments in a dynamic mode via dead-end filtration of protein solutions. In fact, it allowed us to investigate the reproducibility of interactions proved to take place in the previous chapters whether in solution or at the interface of a modified membrane in static mode.

Dead-end filtration experiments provide interesting results on the stability of the coating layer deposited by physical adsorption on the membrane. The presence of the copolymer over the membrane surface has been every time confirmed by FTIR, after the filtration, although water and HSA solution were passed through the membrane under pressure after the coating was performed.

The presence of HSA was also investigated using FTIR and as in static mode, it was revealed to be deposited more on PS-b-PAA coated membrane than on pristine membranes. However, it was found to be slightly related to the copolymer size unlike in static mode where it was shown to be almost similar over different copolymer sizes. This suggests that the applied pressure during filtration helps HSA molecules interacting more with larger PAA, as it lead to the accumulation of HSA onto the surface.

In the case of membrane coated with PS-b-PEO, results showed a different trend than that shown in static mode in the same case, where HSA adsorption was found to be mitigated compared to that on PM. In fact, it seems that the pressure applied on HSA solution during filtration helped molecules overcoming the hydration layer created by PEO to be adsorbed onto the membrane surface. While in the case of membrane coated with both PS-b-PAA+PS-b-PEO copolymers, HSA adsorption was reduced compared to that on PS-b-PAA coated membrane.

The deposition of HSA onto membrane coated with PS-b-PAA copolymer increased at pH 5 during dead-end filtration, which is the same thing proved to happen during static adsorption or for complexation in solution.

The membrane transport properties were found to be slightly affected by the deposition of HSA during dead-end filtration where the flux was reduced around 20%. However, they seem considerably affected by the coating and highly related to the copolymer size. Indeed, the membrane permeability was found to be reduced of around 30 % with the shortest copolymer, and the decline reached 65 % with the largest copolymer.

IgG

Not investigated. Not investigated.

Chapter V

At the end, these results summarized in the Table 5. 1 regarding the proteins adsorption on coated membrane with copolymers (PS-b-PAA, PS-b-PEO) sustain those evidenced in solution by SAXS and chromatography concerning the interactions between the same proteins and functional groups such as PAA and PEG.

Overall, going from the understanding of local interactions, functionalization of the membrane and the study of its performance for filtration process, which are the objectives initially outlined for this thesis, one can consider that a good part has been covered in this study.

Perspectives

First, it would be interesting to complete some parts already covered in this thesis by bringing more elements to the discussion, and to go also further in experiments by considering other approaches. This will allow to improve even more the contribution of this study to the MOSAIC 3D project aiming at designing a suitable membrane technology for blood cells screening and in particular for platelets capture and separation.

Referring to Table 5. 1 for instance, it would be important to compare the behaviour of IgG to that displayed by HSA on PS-b-PAA coated membrane during dead-end filtration process. As stated previously, going further in experiments by considering new approaches could also improve significantly this study. For example, the use of other techniques can provide more information and reliability to the types of interactions that can be had in solution and at the interface of modified membrane. For instance, Isothermal Titration Calorimetry (ITC) has been

shown to be very sensitive to investigate the interactions between blood proteins and polymers in solutions [1], [2]. While Confocal Microscopy (CM) has been disclosed to be an effective technique for the study of blood proteins adsorptions onto membrane surfaces. Indeed, it was successfully used to evaluate the adsorption of white blood cells (WBC), red blood cells (RBC) and platelets on PVDF membrane modified with PS-r-PSBMA [3], or with PS-r-PAA [4].

It would also be possible to use microscopic methods such as fluorescence microscopy to explore the competitive adsorption of different proteins at the interface of a modified membrane using labelled proteins [5]. This could be tested either in static mode or during dead-end Another approach to explore the adsorption of proteins to polymeric surfaces is to use colloidal core-shell particles with a well-defined polymer shell [6]. In fact, functionalized nanoparticles have been proved to be a good alternative to study these interactions, using SAXS, ITC, FTIR, etc [7]. Thus, it would be interesting to investigate blood proteins (HSA, IgG and fibrinogen) interactions with colloidal core-shell particles with the core being PS and the shell formed of PAA, PEO or NIPAAM, or it could be the three of them.

One of the approaches that also can permit to investigate the interaction in solution is the measurement of the osmotic pressure. In fact, the complexation should drastically decrease the osmotic pressure of the mixture as the number of molecules of the solution changes from 5 molecules (for example 1 PAA and 4 HSA), to 1 molecule of complex (1PAA-4HSA).

Perspectives 196

Chapter V

A tentative of experimental measurement has been realised in dialysis bag with the help of a master student (Ema Louati), but aggregates of HSA and moisture have been observed on these experiments that need long time to reach the equilibrium. Experiments should be carried out with microfluidic devices using the membranes that are currently developed in the laboratory. Thus, for the same PAA + HSA mixture the measurement of the osmotic pressure should be performed by changing pH in the permeate. It should allow to confirm the stoichiometry (through the change of osmotic pressure) but also to have a quantitative measurement of the complexation reversibility degree.

Finally, it would be more worthwhile to run some experiments using PRP solutions (platelet rich plasma) to assess the interactions of platelets with the polymers stated previously either in solution and at the interface of a modified membrane. Besides, it could be interesting to explore the optimization of the PDGF transmission through the membrane by changing the coated functional groups size evidenced in this study to have a major role on the membrane retention.

After that, it will be substantial to consider the use of the real human blood as a feed during dead-end filtration process, and to sought to understand what could happen at the interface of the modified membrane.

Blood proteins interactions with coated polymer interfaces

The selective separation of blood proteins and cells is of a major importance to address some target diseases and for several medical applications such as wound healing. The challenge is to provide the most efficient and cost-effective technology that can ensure that. Among many other technologies, membranes processes have been revealed as promising techniques. However, their nonspecific interactions with proteins make them prone to biofouling, which in turn limits their application in biomedical fields. Therefore, the aim of this work is to design tailored membrane for blood proteins screening via surface modification. This latter is performed by coating onto the membrane surface some functional groups such as polystyrene-poly acrylic acid (PS-PAA) and polystyrene-poly ethylene glycol (PS-PEG) copolymers, either to promote a specific interaction with target proteins or to reduce their non-specific interaction. First, the potential interactions that could take place between PAA, PEG brushes and some abundant proteins in plasma such as human serum albumin (HSA) and ɣ-globulin (IgG) have been investigated in solution using Small Angle X-ray Scattering (SAXS) combined to chromatography. Thus, PAA has been found to form complexes with HSA in some specific physicochemical conditions of pH and ionic strength. This HSA-PAA binding has then been revealed to be reversible depending on the pH and the ionic strength of the medium and its stoichiometry was shown to be related to the PAA size. Whereas IgG did not exhibit the same behaviour as HSA towards PAA, it was found to be partially aggregated in our conditions. PEG did not display any interaction with both proteins. After that, Fourier Transform Infrared spectroscopy (FTIR) mapping showed that surface modification of the membranes by copolymers coating was successful, although the distribution of copolymer was heterogeneous over the membrane surface. The static adsorption of HSA onto membranes coated with PS-PAA was found to be also correlated to the pH and ionic strength as it was in solution, while that of IgG was not. Then, the adsorption of HSA onto a membrane coated with PS-PEG has been shown to be mitigated. Eventually, filtration experiments disclosed the same trend of HSA adsorption in dynamic mode even though the membrane permeability was reduced by the increase of the size of the copolymers brushes deposited onto the surface. Overall, this study enabled us to understand the behaviour of some abundant blood proteins in contact of some functional groups either in solution or at the interface of a modified membrane.

Keywords: interactions, blood proteins, polymers, membrane surface modification, SAXS, FTIR. n'a montré aucune interaction avec les deux protéines. Ensuite, la cartographie par spectroscopie infrarouge à transformée de Fourier (FTIR) a confirmé le succès de la modification de surface par dépôt de copolymères sur la surface de la membrane, bien que la distribution du copolymère y soit hétérogène. L'adsorption statique de l'HSA sur les membranes revêtues de PS-PAA s'est avérée également corrélée au pH et à la force ionique comme elle l'était en solution, alors que celle de l'IgG ne l'était pas. Ensuite, il a été démontré que l'adsorption de l'HSA sur la membrane recouverte de PS-PEG était réduite. Enfin, les expériences de filtration ont révélé la même tendance à l'adsorption de l'HSA en mode dynamique, même si la perméabilité de la membrane était réduite par l'augmentation de la taille des brosses de copolymères déposées sur la surface.

Interactions des protéines sanguines avec interfaces polymères modifiées

Globalement, cette étude nous a permis de comprendre le comportement de certaines protéines sanguines abondantes au contact de certains groupes fonctionnels que ce soit en solution ou à l'interface d'une membrane modifiée.

Mots clés : interactions, protéines sanguines, polymères, modification de surface des membranes, SAXS, FTIR.

Figure 1 . 1 . 4 Figure 1 . 2 . 7 Figure 1 . 3 . 8 Figure 1 . 4 . 9 Figure 1 . 5 . 16 Figure 1 . 6 . 26 Figure 1 . 9 . 29 Figure 2 . 1 . 42 Figure 2 . 4 . 44 Figure 2 . 5 . 45 Figure 2 . 6 . 46 Figure 2 . 7 .

 114127138149151616261929214224442545264627 Figure 1. 1. Membrane Separation Technology Market by Application -Global Forecast 2022 | MarketsandMarkets .................................................................................................................. 4 Figure 1. 2. Illustration of a/ improvement of protein adsorption on modified membrane with polymer brushes and b/ minimization of undesired interactions depending on the polymer brushes nature. Adopted from [16]. ........................................................................................... 7 Figure 1. 3. Scheme depicting the two processes "grafting from" or "grafting to" used for surface modification [18]. .......................................................................................................... 8 Figure 1. 4. Illustration of possible configuration of molecules attachment onto a surface during the chemisorption and physisorption. ......................................................................................... 9 Figure 1. 5. Illustration of contact angle over a flat surface [31]. ............................................ 16 Figure 1. 6. Architecture of a smart membrane for blood separation composed of a polymeric supporting layer surface modified with antifouling brushes and smart functional groups for selective trapping [5]. ............................................................................................................... 22 Figure 1. 7. Structure of the PVDF molecule ........................................................................... 23 Figure 1. 8. Design of different membrane systems to purify platelets from whole blood (separation of blood components) and enable the activation of platelets and purification of growth factor. ........................................................................................................................... 26 Figure 1. 9. Schematic representation of a membrane for selection and release of the platelets. .................................................................................................................................................. 27 Figure 1. 10. Confocal microscope images of blood platelets adhering onto the PS well plates surfaces modified with triblock PS-PAA and with random PS-PEG. ...................................... 28 Figure 1. 11. Illustration of the different steps and studies conducted to achieve the objectives of PhD thesis. ........................................................................................................................... 29

Figure 2 . 10 .

 210 Figure 2. 10. The theoretical SAXS scattering patterns of some simple geometrical bodies. Courtesy of Dr. Pierre Roblin. ................................................................................................. 51 Figure 2. 11. Principle of rigid body modelling showing A/ translational movement along r axis and rotational movement along 3 axes α, β and γ that can undergo the subunits and B/ how rigid domains can be connected with flexible linkers [41]. .............................................................. 53 Figure2. 12. Experimental SAXS scattering intensity I(q) of HSA 75 µM (blue line) compared to the SAXS intensity calculated with CRYSOL from HSA crystal structure 6EZQ (red curve). The inset represents the Guinier plot (Ln I(q) vs q 2 in red line) with the residual (green curve) that allow to determine the radius gyration using Primus. The HSA 3D structure shown in cartoon representation using PyMOL software. ....................................................................... 54 Figure 2. 13. Scattering intensities of HSA-PAA mixture and pH evolution for [HSA]=75 µM as a function of PAA concentration ([PAA]=0 to 50 µM). Evolution of the slope of the power law I(q) = q -P corresponding to small and intermediate angles. ............................................... 55 Figure 2. 14. A) Scattering intensities of [HSA]=75 µM (blue curve), [PAA]= 60 µM (green curve), the mixture of HSA and PAA at same concentrations (yellow curve) and the sum of both contributions without interaction (red curve). Table summarizing the radius of gyration, the maximal distance and the Porod volume of HSA, PAA and the complex. B) Autocorrelation function determined with GNOM of the complex and table summarizing the biophysical parameters extracted from the complexes' SAXS data. ........................................................... 57 Figure 2. 15. Comparison of chromatographic patterns for HSA 75 µM in PBS 1X mobile phase (red peak), for (75 µM-50 µM) HSA-PAA mixture in PBS 1X (black line) at pH 7, and in 50 µM PAA solution mobile phase at pH 5 (orange peak). .......................................................... 59 Figure 2. 16. Illustration of the protein-polyelectrolyte electrostatic interaction driven by the counterion release [46]. ............................................................................................................ 60 Figure 2. 17. SAXS scattering intensities I(q) of the HSA-PAA mixture as a function of HSA and PAA concentrations ([HSA]=75-600 µM and [PAA]=10-50 µM). .................................. 61 Figure 2. 18. Radius of gyration evolution as a function of both HSA and PAA concentrations showing the threshold PAA concentration needed for complexation phenomenon to occur ([HSA]=0-600 µM and [PAA]=0-50 µM). .............................................................................. 63 Figure 2. 19. Scattering intensities of HSA-PAA mixture for [HSA]=75 µM and [PAA]=50 µM, (A) as a function of ionic strength (I=0.137 to 1 M) for pH≈5, and (B) as a function of pH (pH=4.6 to 8) for I=0.137 M. ................................................................................................... 65 Figure 2. 20. A/ Scattering intensities of 75 µM HSA at different pH (5, 6, 7 and 8). B/ Scattering pattern of 75µM HSA at pH 5 compared to theoretical pattern calculated from the crystal structure. ....................................................................................................................... 66 Figure 2. 21. Radius of gyration evolution as a function of both pH and ionic strength showing the threshold needed for complexation phenomenon to occur ([HSA]=75 µM ; [PAA]=50 µM ; pH=5 to 7 and I=0.05 to 0.75 M). ............................................................................................ 67 Figure 2. 22. Scattering profiles of HSA-PAA mixture as a function of HSA amount ([HSA]=75 to 600 µM ; [PAA]=50 µM ; pH=5 and I=0.15 M).................................................................. 68 Figure 2. 23. The relative volume fraction of HSA, PAA and the complex as a function of [HSA]/[PAA] molar ratio calculated with the complexation model for a stoichiometry coefficient n=4 .......................................................................................................................... 71

Figure 2 . 24 .Figure 3 . 1 .Figure 3 . 21 .

 22431321 Figure 2. 10. The theoretical SAXS scattering patterns of some simple geometrical bodies. Courtesy of Dr. Pierre Roblin. ................................................................................................. 51 Figure 2. 11. Principle of rigid body modelling showing A/ translational movement along r axis and rotational movement along 3 axes α, β and γ that can undergo the subunits and B/ how rigid domains can be connected with flexible linkers [41]. .............................................................. 53 Figure2. 12. Experimental SAXS scattering intensity I(q) of HSA 75 µM (blue line) compared to the SAXS intensity calculated with CRYSOL from HSA crystal structure 6EZQ (red curve). The inset represents the Guinier plot (Ln I(q) vs q 2 in red line) with the residual (green curve) that allow to determine the radius gyration using Primus. The HSA 3D structure shown in cartoon representation using PyMOL software. ....................................................................... 54 Figure 2. 13. Scattering intensities of HSA-PAA mixture and pH evolution for [HSA]=75 µM as a function of PAA concentration ([PAA]=0 to 50 µM). Evolution of the slope of the power law I(q) = q -P corresponding to small and intermediate angles. ............................................... 55 Figure 2. 14. A) Scattering intensities of [HSA]=75 µM (blue curve), [PAA]= 60 µM (green curve), the mixture of HSA and PAA at same concentrations (yellow curve) and the sum of both contributions without interaction (red curve). Table summarizing the radius of gyration, the maximal distance and the Porod volume of HSA, PAA and the complex. B) Autocorrelation function determined with GNOM of the complex and table summarizing the biophysical parameters extracted from the complexes' SAXS data. ........................................................... 57 Figure 2. 15. Comparison of chromatographic patterns for HSA 75 µM in PBS 1X mobile phase (red peak), for (75 µM-50 µM) HSA-PAA mixture in PBS 1X (black line) at pH 7, and in 50 µM PAA solution mobile phase at pH 5 (orange peak). .......................................................... 59 Figure 2. 16. Illustration of the protein-polyelectrolyte electrostatic interaction driven by the counterion release [46]. ............................................................................................................ 60 Figure 2. 17. SAXS scattering intensities I(q) of the HSA-PAA mixture as a function of HSA and PAA concentrations ([HSA]=75-600 µM and [PAA]=10-50 µM). .................................. 61 Figure 2. 18. Radius of gyration evolution as a function of both HSA and PAA concentrations showing the threshold PAA concentration needed for complexation phenomenon to occur ([HSA]=0-600 µM and [PAA]=0-50 µM). .............................................................................. 63 Figure 2. 19. Scattering intensities of HSA-PAA mixture for [HSA]=75 µM and [PAA]=50 µM, (A) as a function of ionic strength (I=0.137 to 1 M) for pH≈5, and (B) as a function of pH (pH=4.6 to 8) for I=0.137 M. ................................................................................................... 65 Figure 2. 20. A/ Scattering intensities of 75 µM HSA at different pH (5, 6, 7 and 8). B/ Scattering pattern of 75µM HSA at pH 5 compared to theoretical pattern calculated from the crystal structure. ....................................................................................................................... 66 Figure 2. 21. Radius of gyration evolution as a function of both pH and ionic strength showing the threshold needed for complexation phenomenon to occur ([HSA]=75 µM ; [PAA]=50 µM ; pH=5 to 7 and I=0.05 to 0.75 M). ............................................................................................ 67 Figure 2. 22. Scattering profiles of HSA-PAA mixture as a function of HSA amount ([HSA]=75 to 600 µM ; [PAA]=50 µM ; pH=5 and I=0.15 M).................................................................. 68 Figure 2. 23. The relative volume fraction of HSA, PAA and the complex as a function of [HSA]/[PAA] molar ratio calculated with the complexation model for a stoichiometry coefficient n=4 .......................................................................................................................... 71

Figure 4 . 1 .Figure 4 . 2 .

 4142 Figure 3. 21. The intensity average of the peak area at 700 cm -1 as shown A/ for the coating of different PS-b-PAA and PS-b-PEO using ID process, 2 hours of immersion time and a copolymer concentration of 5 mg•mL -1 and B/ the intensity as a function of the hydrophilic/hydrophobic ratio. ............................................................................................... 132 Figure 3. 22. FTIR maps for the coating of PVDF membranes with various PS-b-PAA copolymers using ID process, 2 hours of immersion and different copolymer concentrations, and the adsorption of HSA on these same modified membranes; [HSA] = 1 mg•mL -1 ; pH ≈ 7. ................................................................................................................................................ 134 Figure 3. 23. The intensity average of the peaks area at 700 cm -1 and 1715 cm -1 for the coating of different PS-b-PAA copolymers as function of copolymer concentration using ID process, 2 hours of immersion time. ....................................................................................................... 136 Figure 3. 24. The intensity average of the peak area at 1660 cm -1 for the HSA adsorption on PVDF membranes coated with different PS-b-PAA copolymers, [HSA]= 1 mg•mL -1 ; pH=7. ................................................................................................................................................ 137 Figure 3. 25. FTIR maps for the adsorption of HSA protein on PVDF membranes coated with different concentrations of PS30-b-PEO13 copolymer, as well as the evaluated mean intensities for the peaks area at 700 cm -1 and 1660 cm -1 ; [HSA]= 1 mg•mL -1 . ...................................... 139 Figure 3. 26. FTIR maps for the adsorption of HSA protein on PVDF membranes coated with PS26-b-PAA76 and PS28-b-PEO13 as well as the evaluated mean intensities for the peaks area at 700 cm -1 and 1660 cm -1 ; [HSA]= 1 mg•mL -1 . ........................................................................ 140 Figure 3. 27. FTIR maps for the adsorption of HSA at different pH (5, 7 and 8) on PVDF membranes coated with PS30-b-PAA5 and PS100-b-PAA107 copolymers using ID process, 2 hours of immersion and a copolymer concentration of 5 mg•mL -1 ; [HSA]= 1 mg•mL -1 . ..... 142 Figure 3. 28. Evaluated mean intensities of the peaks area at 700 cm -1 and 1660 cm -1 showing respectively the uniformity of coating on different samples and the adsorption of HSA onto these samples at different pH; [HSA]= 1 mg•mL -1 . ............................................................... 142 Figure 3. 29. Illustration of the HSA adsorption onto PS-PAA coated membrane due its hydrophobic interactions with the visible PS and electrostatic interactions with PAA brushes. ................................................................................................................................................ 143 Figure 3. 30. FTIR maps for the adsorption of HSA at different ionic strength on PVDF membranes coated with PS30-b-PAA5 and PS100-b-PAA107 using ID process, 2 hours of immersion and a copolymer concentration of 5 mg•mL -1 ; [HSA]= 1 mg•mL -1 . .................... 145 Figure 3. 31. The evaluated mean intensities of the peaks area at 700 cm -1 and 1660 cm -1 showing respectively the coating on different samples and the adsorption of HSA onto these same samples at different ionic strength; [HSA]= 1 mg•mL -1 . .............................................. 146 Figure 3. 32. FTIR maps for the adsorption of IgG at different pH (5, 7 and 8) on PVDF membranes coated with PS30-b-PAA5 and PS100-b-PAA107 using ID process, 2 hours of immersion and a copolymer concentration of 5 mg•mL -1 ; [IgG]= 1 mg•mL -1 . ..................... 147 Figure 3. 33. The evaluated mean intensities of the peaks area at 700 cm -1 and 1660 cm -1 showing respectively the uniformity of coating on different samples and the adsorption of IgG onto these samples at different pH; [IgG]= 1 mg•mL -1 . ......................................................... 148

I

  ................................................................................................................................................ 106 Table 3. 2. Specific properties of the commercial PVDF membrane used in this study as provided by Millipore. ............................................................................................................ 107 Table 3. 3. List of copolymers used for surface modification with their properties. ........... 109 Table 3. 4. Absorption bands of functional groups in PS-b-PAA copolymers and those in proteins (HSA and IgG). ........................................................................................................ 119 Table 3. 5. The calculated coating density and the measured mean intensity of the peak area (700 cm -1 ) with their standard deviations for different coating times. .................................. 129 Table 3. 6. The amounts of HSA adsorbed onto PVDF membranes coated with PS30-b-PAA5 and PS100-b-PAA107 estimated using UV-visible spectroscopy. ............................................ 144 Table 4. 1: contact angles of PVDF membranes coated with different PS-b-PAA copolymers compared to that of the pristine membrane. ........................................................................... 166 Table 4. 2. Permeability of membranes coated with different PS-b-PAA copolymers compared to that of the pristine membrane, as well as the decline percentage caused by coating. ........ 167 List of tables XVIII Table 4. 3. Calculations of protein retention by the PM and membranes coated with different PS-b-PAA copolymers as well as the evaluation of the amount of HSA adsorbed on the membrane surface and/or structure by mass balance. ............................................................ 176 Table 4. 4. Calculations of protein retention as well as the amount of HSA adsorbed on the membrane surface in the case of PM and of membranes coated with PS28-b-PEO13 and with both PS28-b-PEO13 + PS26-b-PAA76. ...................................................................................... 181 Table 4. 5. Comparison of HSA retention and adsorption on PVDF membrane coated with PS100-b-PAA107 during dead-end filtration at different pH (5 and 7). .................................... 183 Table 5. 1 : Summary of the blood proteins (HSA and IgG) interactions with polymers (PAA and PEG) in solution, and at the interface of a functionalized PVDF membrane in static mode and during dead-end filtration. ............................................................................................... 193 SI-Table 1. Quantitative analysis of the confocal microscope images performed using Image J software for the adhesion of the platelets on polystyrene (PS) well plate modified with triblock PS-PAA and with random PS-PEG copolymers. ..................................................................-PAA Polystyrene-block-poly(acrylic acid) PS-b-PEG Polystyrene-block-poly(ethylene glycol) This chapter constitutes a state of the art on the use of membrane technology in separation processes in different fields for versatile applications, especially medical ones. Besides, in this chapter are reported the different membrane materials, how they can be tailored and functionalized depending on the application, as well as how to characterise them. It also introduces the concept of smart biomedical membranes for blood separation and the application behind. Eventually, the positioning of the PhD project with regards to the application, and the different parts constituting this work and structuring the manuscript are presented.

Figure 1 . 1 .

 11 Figure 1. 1. Membrane Separation Technology Market by Application -Global Forecast 2022 | MarketsandMarkets.

Figure 1 . 2 .

 12 Figure 1. 2. Illustration of a/ improvement of protein adsorption on modified membrane with polymer brushes and b/ minimization of undesired interactions depending on the polymer brushes nature. Adopted from [16].

Figure 1 . 4 . 1 .

 141 Figure 1. 4. Illustration of possible configuration of molecules attachment onto a surface during the chemisorption and physisorption.

4 .

 4 Membrane surface functionalization by polymers adsorption 4.2. Interactions of functionalized membrane with proteins Chapter I

4 .

 4 Membrane surface functionalization by polymers adsorption 4.3. Methods for characterization of functionalized membranes and protein adsorption 13

4 .

 4 Membrane surface functionalization by polymers adsorption 4.3. Methods for characterization of functionalized membranes and protein adsorption 14 Chapter I Table

4 .

 4 Membrane surface functionalization by polymers adsorption 4.3. Methods for characterization of functionalized membranes and protein adsorption Chapter I

  For samples that have either extremely faint or bright, nonspecific fluorescence, they can be attached with fluorescent stains or fluorescent-tagged antibodies. This technique is capable of detecting a single molecule. Using multiple fluorescence labelling and different probes, it can also simultaneously identify several target molecules. Thus, fluorescence microscopy could be advantageous to observe the competitive adsorption of proteins on the modified membrane surface. 4. Membrane surface functionalization by polymers adsorption 4.3. Methods for characterization of functionalized membranes and protein adsorption

Figure 1 . 5 . 4 . 3 . 3 . Assessment of the modification layer density 4 . 3 . 3 . 1 .

 154334331 Figure 1. 5. Illustration of contact angle over a flat surface [31].

4 .

 4 Membrane surface functionalization by polymers adsorption 4.3. Methods for characterization of functionalized membranes and protein adsorption Chapter I

5 .

 5 Smart biomedical membranes for blood separation 5.2. Concept of smart membranes 20 Chapter I fractions: erythrocytes in the bottom of the centrifugation tube, plasma as the supernatant, and

5 .

 5 Smart biomedical membranes for blood separation 5.2. Concept of smart membranes 21 Chapter I

5 .IFigure 1 . 6 .

 516 Figure 1. 6. Architecture of a smart membrane for blood separation composed of a polymeric supporting layer surface modified with antifouling brushes and smart functional groups for selective trapping [5].

5 .Figure 1 . 7 .

 517 Figure 1. 7. Structure of the PVDF molecule Nowadays, PVDF (Figure 1. 7) is one of the most popular material for ultrafiltration and microfiltration membrane manufacturing. This material provides robust membranes due to its structural and chemical stability. Further, reasonable cost compared to other membrane materials, add to the popularity of this polymer [54].

5 . 5 . 4 .

 554 Smart biomedical membranes for blood separation 5.4. Surface-Modifying functional groups Chapter I Surface-Modifying functional groups

  Toulouse, CNRS, INPT, UPS, Toulouse, France) and the Research and Development Center for Membrane Technology (Chung Yuan Christian University, ChungLi, Taiwan). The funding for this project was provided by the Agence Nationale de la Recherche (ANR 17-CE08-0041) and by the Ministry of Science and Technology in Taiwan (MOST 107-2923-E-033-001) for a budget of 250 k€ each, which makes a total budget of 500k. Within this framework, a 6-weeks stay was made in the Research and Development Center for Membrane Technology (Chung Yuan Christian University, ChungLi, Taiwan), where training on copolymer synthesis and membrane surface modification was initiated. The visit was concluded with a workshop between the two groups and a participation to the International Membrane Conference in Taipei (2019).

  membranes can be functionalized through surface modification allowing it to specifically interact with the targeted components resulting in selective separation.

  ) enable the activation of platelets and release of PDGF (Figure1. 8). Thus, the systems schematized below allow to consider several routes for the production of PDGF. The first one to select platelets, release the platelets and activate them in solution to induce the formation of growth factor (Membrane I). The second option is to select the platelets and purify the growth factors at the interface (Membrane II). Finally, for a comparative study, the third route is to select erythrocytes and leukocytes, let the platelets permeate through the membrane and activate them in solution similarly as in (1) (Membrane III).

Figure 1 . 8 .

 18 Figure 1. 8. Design of different membrane systems to purify platelets from whole blood (separation of blood components) and enable the activation of platelets and purification of growth factor.

Figure 1 . 9 .I

 19 Figure 1. 9. Schematic representation of a membrane for selection and release of the platelets.For that reason, different copolymers were used in this study to modify hydrophobic PVDF membrane surface. The polymers used for the surface modification are chosen from a range of polymers known to have limited or specific interaction with other target solutes. Polystyreneblock-poly(acrylic acid) (PS-b-PAA) copolymer was used to investigate its interaction with blood proteins. PAA is considered as bioactive polymer that can be conjugated with blood proteins which could induce platelet capture and activation. Then the polystyrene-blockpoly(ethylene glycol) (PS-b-PEG) should avoid unwanted interactions with other blood proteins. Finally, the polystyrene-block-poly(N-isopropylacrylamide) (PS-b-PNIPAAM) role is to release platelets by changing the temperature.

Figure 1 . 10 .

 110 Figure 1. 10. Confocal microscope images of blood platelets adhering onto the PS well plates surfaces modified with triblock PS-PAA and with random PS-PEG.

6 .

 6 PhD strategy and positioning 6.3. Contribution of PhD thesis 29 Chapter I

Figure 1 . 11 .

 111 Figure 1. 11. Illustration of the different steps and studies conducted to achieve the objectives of PhD thesis.

Chapter II 2 .

 2 Materials and samples preparationHuman serum albumin (HSA, lyophilized powder, ≥96% agarose gel electrophoresis), and human γ-globulin (IgG, ≥99% electrophoresis), with a molecular weight of 66.5 kDa and of around 155 kDa respectively, were purchased from Sigma Aldrich and used as received. The poly acrylic acid (PAA) of molecular weight around 100 kDa (35 wt. % in H2O), the poly ethylene glycol (PEG) with a molecular weight of 8 kDa (40wt. % in H2O), and the PEG methyl ether methacrylate of 2 kDa (50wt. % in H2O) were bought from Sigma Aldrich.The other sizes of PAA (5, 30 and 50 kDa), and the PEG with a molecular weight of 20 kDa, were purchased from Biovalley.Protein powder was dissolved in PBS 1X (0.012M phosphate buffer solution at pH 7.4, ionic strength 0.137 M) to obtain a stock solution of 50 g•L -1 (750 µM) for HSA and 30 g•L -1 (200µM) for IgG, from which samples of lower concentration were prepared. The same way, the polymer solutions were prepared by dilution in PBS 1X buffer from a stock PBS solution of 100 g•L -1 PAA (1 mM).

  The chromatography unit used consisted of an Agilent 1260 infinity system connected to a UV-visible detector set at a wavelength of 280 nm. Analyses were performed using an Agilent Bio Sec-3 (3µm, 4.6x300 mm, 300 Å) size exclusion column at room temperature. In a typical analytical run, 50 µL of the protein solution were injected, and PBS 1X was used as mobile phase for protein structure investigation at a flow rate of 0.4 ml•min -1 . The mobile phase was changed depending on the studied parameter (pH, ionic strength …), but also on the composition of the sample. For instance, depending on the desired pH, PBS was replaced by another buffer from those listed in the materials section. For the study of protein-polymer complexation, solutions containing both solutes were injected and the mobile phase was a buffer solution (chosen depending on the desired pH) containing the polymer. These experiments can help to know if the complexation is reversible or if, at opposite, it leads to stable (irreversible) complex (Figure2. 1). Indeed, if complexes have been formed initially between the protein and the polymer, they would not be then dissociated due to the absence of polymer in the mobile phase. In the latter case, the equilibrium may move in the opposite direction of complexation, which is dissociation as illustrated in Figure2. 1.

Figure 2 . 1 .

 21 Figure 2. 1. Illustration of the chromatography response according to the stability of complexes and to the composition of the mobile phase. Courtesy of Dr. Pierre Roblin.

Figure 2 . 2 .

 22 Figure 2. 2. Calibration curve of the molecular weight as a function of the retention time.

Figure 2 . 3 .

 23 Figure 2. 3. Schematic representation of a SAXS experiment, and illustration of the final scattering intensity calculation by subtracting the solvent contribution from the scattering pattern of the solution. Adopted from [26] and modified.

Figure 2 .

 2 4 displays what information could be obtained from a SAXS scattering pattern.

Figure 2 . 4 .

 24 Figure 2. 4 . Information extracted from different regions of the experimental scattering intensity.

Figure 2 .

 2 Figure 2. 5 presents typical scattering patterns and distance distribution functions of geometrical bodies with the same maximum size. Globular particles (red curve) display bellshaped p(r) functions with a maximum at about Dmax/2. While, flattened particles display a

Figure 2 . 5 .

 25 Figure 2. 5. Scattering intensities and distance distribution functions of geometrical bodies. Adopted from Svergun & Koch [29].

Figure 2 . 6 .II 3 . 2 . 2 .

 26322 Figure 2. 6. Example of calculation of the Porod invariant Q using the constant K with the homemade Python code.

Figure 2 . 7 .

 27 Figure 2. 7. Illustration of A/ the XEUSS 2.0 SAXS bench of Laboratoire de Genie Chimique, Toulouse, and B/ the chromatographic equipment connected upstream to the SAXS cell.

Figure 2 .

 2 8 illustrates a merge performed on experimental SAXS scattering data of HSA obtained through chromatography and in direct injection mode.

Figure 2 . 8 .

 28 Figure 2. 8. Illustration of the merge of experimental scattering data of HSA.

3 . Methods 3 . 2 .

 332 Small angle X-ray scattering (SAXS) measurement 50 Chapter II considered a valid description of the structure under the solution conditions used for the measurement.

  transform, will allow to calculate the scattered intensity. The theoretical scattering curve is obtained by spherical averaging of the intensities.The experimental scattering curves are obtained by subtracting the scattering of the solvent from that of the solution containing the molecule as illustrated in Figure2. 9 . However, solvated molecules are characterized by a boundary where the scattering density differs from that of disordered solvent[28]. Therefore, to calculate the diffusion curve from a structure, two additional contributions to the diffusion must be taken into account. The first one is linked to the volume excluded by the particle, and that by considering this volume filled with solvent with a constant diffusion density. The second contribution is related to the hydration layer, i.e. a layer of solvent molecules interacting with the surface of the molecule immobilized on its surface. In this very particular layer the diffusion density is considered constant but with a different value.

Figure 2 . 9 .

 29 Figure 2. 9. Illustration of the different contributions taken into account in the calculation of the theoretical scattering intensity with GRYSOL program.

3 . Methods 3 . 2 .

 332 Small angle X-ray scattering (SAXS) measurement 51 Chapter II already defined (Figure2. 10). These constitute then models to which data obtained for particles with unknown geometry can be compared. Thus, a wide variety of models have been implemented in various software packages that allow to fit the experimental data to existing theoretical models. The diversity of the available models, and the possibility that multiple models having very different physical bases can fit the data equally well, can make the process of data analysis challenging.

Figure 2 . 10 .

 210 Figure 2. 10. The theoretical SAXS scattering patterns of some simple geometrical bodies. Courtesy of Dr. Pierre Roblin.

  modeling. It uses dummy residues to model missing regions in both protein complexes and multi-domain proteins connected by flexible linkers (Figure2. 11.B). Besides a simulated annealing (SA) minimization process is used to locate a best fitting arrangement of the rigid bodies and also the optimal local conformation of dummy residues. The SA process consist on 3. Methods 3.2. Small angle X-ray scattering (SAXS) measurement 53 Chapter II decreasing the energy of the system by searching for the most suitable conformation but occasionally allows an increase in energy to explore a larger conformational space.Initially the missing parts are materialized as chains structured in 'zig-zag' linking the domains together. Then, the domains are moved relative to each other with minimal modifications at the hinge regions, and as the domains stabilize in certain orientations, the loops are progressively rearranged to best mimic the experimental data. BUNCH can also handle multiple diffusion curves corresponding to partial structures of the protein simultaneously.

Figure 2 . 11 . 1 .

 2111 Figure 2. 11. Principle of rigid body modelling showing A/ translational movement along r axis and rotational movement along 3 axes α, β and γ that can undergo the subunits and B/ how rigid domains can be connected with flexible linkers [41].

Figure 2 .

 2 Figure 2. 12. Experimental SAXS scattering intensity I(q) of HSA 75 µM (blue line) compared to the SAXS intensity calculated with CRYSOL from HSA crystal structure 6EZQ

Figure 2 .

 2 Figure 2. 13. Scattering intensities of HSA-PAA mixture and pH evolution for [HSA]=75 µM as a function of PAA concentration ([PAA]=0 to 50 µM).Evolution of the slope of the power law I(q) = q -P corresponding to small and intermediate angles.

4 .

 4 Results and discussion 4.2. Study of HSA-PAA interaction 56 Chapter II As shown in Figure 2. 13, starting from 40 µM of PAA with 75 µM HSA, a strong change

3. 8

 8 for single HSA and the mixture of HSA+10, 20 and 30 µM of PAA, to 4 when increasing the PAA concentration to 40 and 50 µM (Figure2. 13). These results indicate a same type of surface despite the big change in the general shape shown by an increase in the slope at small angles (0.005 to 0.05 Å -1 ). In fact, the difference is more obvious, where the slope is going from almost a plateau (P=0.3) for single HSA, to a slope of 1.2 when adding 50 µM PAA, which corresponds to the slope of an elongated object[28].

Figure 2 .

 2 Figure 2. 13 also reveals that despite being in PBS buffer, PAA strongly influences the pH of the solution. Thus, we can suppose that the change in the SAXS curve could be correlated to a pH variation rather than to an effect of the concentration of PAA (explicitly discussed later in this chapter).

Figure 2 .

 2 14.A. Although the curves are calibrated in cm -1 , it is impossible to directly determine the mass concentration of the heterocomplex from the I(0) because we do neither know the association stoichiometry nor precisely the experimental specific volume and contrast of the PAA.

Figure 2 .

 2 Figure 2. 14. A) Scattering intensities of [HSA]=75 µM (blue curve), [PAA]= 60 µM (green curve), the mixture of HSA and PAA at same concentrations (yellow curve) and the sum of both contributions without interaction (red curve). Table summarizing the radius of gyration, the maximal distance and the Porod volume of HSA, PAA and the complex. B)

Figure 2 .

 2 Figure 2. 14.A) in the same conditions of concentration reflecting the case without interactions between both partners, discloses clearly that the SAXS curve observed corresponds to the creation a new object resulting from interactions between HSA and PAA.

4 .IIFigure 2 . 15 .

 4215 Figure 2. 15. Comparison of chromatographic patterns for HSA 75 µM in PBS 1X mobile phase (red peak), for (75 µM-50 µM) HSA-PAA mixture in PBS 1X (black line) at pH 7, and in 50 µM PAA solution mobile phase at pH 5 (orange peak).

4 .IIFigure 2 . 16 . 4 .

 42164 Figure 2. 16. Illustration of the protein-polyelectrolyte electrostatic interaction driven by the counterion release [46].

Figure 2 .

 2 Figure 2. 17. SAXS scattering intensities I(q) of the HSA-PAA mixture as a function of HSA and PAA concentrations ([HSA]=75-600 µM and [PAA]=10-50 µM).

  ) corroborate also the fact that when increasing the amount of added polymer to HSA solution, the measured intensity starts to raise suddenly at small angles from a PAA concentration of 40 µM. Indeed, it can be noticed that from 10 to 30 µM of PAA and for 75 µM of HSA, the curves almost overlap with the one representing single protein. Then, SAXS pattern are translated when protein concentration increases without any change in the form factor. That confirms what has been noticed previously, the complexation phenomenon takes place only from a certain critical polymer concentration happened to be around 40 µM of PAA.In order to support these observations, the scattering profiles were drawn as diagram representing the radius of gyration evolution as function of both HSA and PAA concentrations (Figure2. 18). Rg values were obtained by fits of the scattering intensities at small angles using Foxtrot software, and they are quoted in Table2. 1.

4. Results and discussion 4 . 2 .

 42 Study of HSA-PAA interaction 63 Chapter IIThe same was observed for single proteins, where greater Rg was obtained for smaller HSA concentrations. In fact, at high HSA concentration, molecules are closer to each other leading to intermolecular interactions and electrostatic repulsions. That distorts the I(q) at small angles and so the structure factor which results in a decrease in the radius of gyration.Then, for each polymer concentration from 10 to 30 µM, Rg decreases when the protein concentration increases from 75 to 600 µM until reaching a value around 30 Å, which is almost the size of single HSA in solution. This finding is reflected by the diagram in Figure2. 18.

Figure 2 . 18 . 4 .

 2184 Figure 2. 18. Radius of gyration evolution as a function of both HSA and PAA concentrations showing the threshold PAA concentration needed for complexation phenomenon to occur ([HSA]=0-600 µM and [PAA]=0-50 µM).

4 .

 4 [HSA] was set at 75 µM and the [PAA] at 50 µM (pH ~ 5 remains controlled by the polymer concentration), where the complexation was observed, while the ionic strength was varied from 0.137 M to 1 M by adding a needed amount of a 4 M NaCl solution.Data obtained and reported below (Figure2. 19.A) reveal a decrease in scattering intensity with the increase in ionic strength. Obviously curves are moving down especially at medium and small angles (0.005 to 0.1 Å -1 ), changing the slope, to collapse for an ionic strength of 0.75 M and 1 M with the theoretical sum (plotted as red line) of two independent signals without interaction (signal of HSA 75 µM+ signal of PAA 50 µM) obtained at I=0.137 M. This finding confirms the previous assumption and bears out the fact that the binding of HSA-PAA is due to electrostatic interactions. Results and discussion 4.3. Ionic strength and pH effect on HSA/PAA complexation 65 Chapter II Afterwards, salts concentration was set at 0.15 M and pH was changed from 8 to 4.6 using the different buffer solutions listed in section 39. As shown in Figure2. 19.B, the decrease in pH down to 5 promotes the formation of the complex whereas at pH > 5 the complex is dissociated giving the same SAXS signal as a mix of two independent contributions.

Figure 2 . 19 .

 219 Figure 2. 19. Scattering intensities of HSA-PAA mixture for [HSA]=75 µM and [PAA]=50 µM, (A) as a function of ionic strength (I=0.137 to 1 M) for pH≈5, and (B) as a function of pH (pH=4.6 to 8) for I=0.137 M.

Figure 2 .

 2 Figure 2. 20. A/ Scattering intensities of 75 µM HSA at different pH (5, 6, 7 and 8). B/ Scattering pattern of 75µM HSA at pH 5 compared to theoretical pattern calculated from the crystal structure.

4. Results and discussion 4 . 3 .

 43 Ionic strength and pH effect on HSA/PAA complexation 67 Chapter II particles begin to dissociate into free HSA molecules and free PAA chains. This dissociation is not complete at 0.75 M and some complex particles are still in there since the obtained Rg (69.8 Å) is still higher than the average value (50 Å) found when the HSA and PAA do not interact. This outcome sustains the previous results (Figure2. 19.A) suggesting that complete dissociation of the complex is taking place between 0.75 M and 1 M.

Figure 2 . 21 .

 221 Figure 2. 21. Radius of gyration evolution as a function of both pH and ionic strength showing the threshold needed for complexation phenomenon to occur ([HSA]=75 µM ; [PAA]=50 µM ; pH=5 to 7 and I=0.05 to 0.75 M).

4. Results and discussion 4 . 4 . 4 . 4 .

 4444 Determination of the complex stoichiometry (PAA-nHSA) 68 Chapter II investigated latter in chapter 3 for the adsorption of HSA on PAA deposited on membrane surface. In fact, we can imagine that to enhance the platelets capture on PAA, pH > 5 should be favoured to prevent the adsorption of HSA on PAA. Now that the conditions of complex formation are controlled, the stoichiometry of complexation can be investigated. Determination of the complex stoichiometry (PAA-nHSA) In order to determine the number of proteins that could bind to each polymer chain, SAXS experiments were performed by setting the concentration of PAA at 50 µM and varying the HSA concentration between 75 and 600 µM. In other words, the [HSA]/[PAA] molar ratio ranged from 1.5 to 12. The experiments were performed at pH 5 and an ionic strength I = 0.15 M to ensure complexation.

Figure 2 . 22 . 4 .

 2224 Figure 2. 22. Scattering profiles of HSA-PAA mixture as a function of HSA amount ([HSA]=75 to 600 µM; [PAA]=50 µM; pH=5 and I=0.15 M).

4 .

 4 10 3 Å 3 , which represents the complex Porod volume (Figure 2. 14). Then, we notice that increasing the HSA concentration reduces the volume to 239.10 3 Å 3 for a molar ratio of 12. That makes sense because after saturation of all PAA chains, the excess of HSA contributes to the average of the Porod volume. Results and discussion 4.4. Determination of the complex stoichiometry (PAA-nHSA) Chapter II On the other hand, a complexation model has been developed to describe the evolution of the Porod volume for the same range of HSA/PAA molar ratio, making the assumption of the following complexation equilibrium: 𝑛 𝐻𝑆𝐴 + 1 𝑃𝐴𝐴 ⇄ 1 𝑐𝑜𝑚𝑝𝑙𝑒𝑥(𝑃𝐴𝐴 -𝑛𝐻𝑆𝐴)

4. Results and discussion 4 . 4 .IIFigure 2 . 23 .

 44223 Figure 2. 23. The relative volume fraction of HSA, PAA and the complex as a function of [HSA]/[PAA] molar ratio calculated with the complexation model for a stoichiometry coefficient n=4

4. Results and discussion 4 . 4 .IIFigure 2 .

 442 Figure 2. 24 displays the Porod volume calculation for 3, 4 and 5 HSA molecules bounded to each PAA chain, where the symbols represent the estimated Vp (reported in Table 2. 2) from the SAXS experimental data. The comparison of these experimental data to the theoretical model suggests a stoichiometry of saturation for an [HSA]/[PAA] molar ratio around 4. Indeed, the results show that a complex model containing 4 HSA with 1 PAA gives a better fit than those run assuming 3 or 5 HSA per PAA (Figure 2. 24).

Figure 2 . 24 .

 224 Figure 2. 24. The Porod volume evolution as function of the initial [HSA/[PAA] ratio, using experimental SAXS data and calculation from the complexation model for different stoichiometry coefficient n.

Figure 2 .

 2 Figure 2. 25 exposes also the HSA 3D structure as shown in cartoon using PyMOL software, and it illustrates how the protein can be represented by a disk model (diameter=74 Å , length=31 Å) from different angles.

Figure 2 .

 2 Figure 2. 25. Experimental SAXS scattering intensity I(q) of HSA 75µM (blue line) and the optimal fit obtained with a cylinder (disk) model (red line) using Sasview. (a) The HSA crystallographic 3D structure shown using PyMOL software and (b) its representation by disk model as seen from different angles.

Figure 2 .

 2 Figure 2. 26. The SAXS scattering intensity for HSA-PAA complex as dotted thick blue line, fitted by cylinder model (Radius=22 Å ; Length=380 Å) as yellow line, and by 4 stackeddisks model (Radius=22 Å ; Length=392 Å) as dashed red line, with the shape of the two patterns how they can be illustrated.

Figure 2 . 27 .

 227 Figure 2. 27. Representative computer simulation snapshot of the HSA-PAA complexation performed with a short PAA (2 kDa) by Ballauff and al [20].

Figure 2 . 28 . 4 . 4 . 4 . 6 .

 2284446 Figure 2. 28. Superimposition of the experimental SAXS data (dotted thick blue circle) of the HSA-PAA complex and those of the compatible model (red line) calculated with the program BUNCH.

Figure 2 .

 2 Figure 2. 29. SAXS scattering intensities I(q) of the HSA-PAA mixture as a function of HSA concentration and for different PAA sizes ([HSA]=50-300 µM and [PAA]= 50 µM). Table

Figure 2 . 30 .

 230 Figure 2. 30. Comparison of chromatographic pattern of HSA 225 µM and those of HSA-PAA mixtures using different PAA sizes.

4. Results and discussion 4 . 6 .

 46 Influence of PAA size on HSA-PAA complexation Chapter II Eventually, in order to make sure good information can be extracted at small and high angles, data obtained from these experiments were merged with those obtained from the SAXS sampling of the elution peaks as explained in the methods section. Results exposed an increase of I(q) at small angles, while it remains almost unchanged in the intermediate and high angles range (Figure2. 31). This indicates that all HSA-PAAs complexes exhibit the same type of surface with an increase in the global size.

Figure 2 . 31 .

 231 Figure 2. 31. Scattering intensities of HSA-PAA complexes obtained using different sizes of PAA, and the corresponding autocorrelation function calculated from each I(q) using Primus.

Figure 2 . 32 .

 232 Figure 2. 32. Comparison on the HSA-PAA complex molecular weight estimated from SAXS data and from chromatography data to the theoretical Mw calculate from the stoichiometry of complexation (PAA-nHSA) for each size of PAA.

4. Results and discussion 4 . 7 .

 47 Study of HSA-PAAs complexation reversibility 83 Chapter II

Figure 2 . 33 .

 233 Figure 2. 33. Chromatographic patterns of HSA-PAA mixture as studied in various mobile phases (with PAA in the mobile phase at pH 5 -orange line-and at pH 7 -green line-, and without PAA in the mobile phase at pH 5 -black line-) and compared to the pattern of single HSA in blue.

Figure 2 . 4 .

 24 Figure 2. 34 did not display changes on the size or the form of IgG with the pH change, and the evaluation of the radius of gyration found to be around 68 Å has confirmed that. The evaluated Rg for single IgG and for the IgG-PAA mixture at different pH are summarized in the table attached to Figure 2. 34.

Figure 2 .

 2 Figure 2. 34. SAXS scattering intensities I(q) of the IgG-PAA mixture with different PAA sizes (30 and 100 kDa) and at different pH (5, 7 and 8). ([IgG]=65µM and [PAA]= 50µM).

4. Results and discussion 4 . 8 .

 48 Study of interaction between PAA and IgGChapter II PAA as it was the case with HSA. It could be rather related to a bigger amount of aggregates which increases their contribution in the estimation of Rg. A previous study has showed that, in 0.1 M NaCl solutions, PAA could have a role on IgG unfolding, association, and aggregation via Coulomb and hydrophobic interaction[51]. While, a stabilization of oligomers with PAA chains leading to slow aggregation rate was suggested in solutions without NaCl.

Figure 2 . 35 . 4 .

 2354 Figure 2. 35. Chromatographic profiles of IgG-PAA30 kDA and IgG-PAA100 kDa compared to that of IgG at different pH (5, 7 and 8). [IgG]=65µM and [PAA]=50µM.

4. Results and discussion 4 . 9 .IIFigure 2 . 36 .

 49236 Figure 2. 36. SAXS scattering intensities I(q) of HSA-PEG mixture with different PEG sizes (2, 8 and 20 kDa) and at different pH (5, 7 and 8). ([HSA]=150µM and [PEG]= 500µM).

Figure 2 .

 2 Figure 2. 37. SAXS scattering intensities I(q) of IgG-PEG mixture with different PEG sizes (2, 8 and 20 kDa) and at different pH (5, 7 and 8). ([IgG]=65µM and [PEG]= 500µM).

2 .

 2 Literature review on parameters affecting coating 2.2. Effect of copolymer concentration 102ChapterIIIMany other studies have investigated the effect of copolymer concentration on the coating density. For instance, it has been assessed in the case of PS-r-PSBMA deposited on PVDF membrane surface[7]. Results revealed a decrease of the water contact angle and fibrinogen adsorption when increasing the copolymer concentration in solution until the protein adsorption reached a plateau implying the potential increase of coating on the surface until it reached saturation. As shown in Figure3. 1, similar trend was obtained when the coating density increased with the copolymer concentration and eventually reaches a plateau in the case of PVDF membranes coated with poly(propylene oxide)-block-poly(sulfobetaine methacrylate) PPO-b-PSBMA[11].

Figure 3 . 1 .

 31 Figure 3. 1. Effect of varying concentration of poly(propylene oxide)-block-poly (sulfobetaine methacrylate) copolymer in solution and PPO/PSBMA ratio on copolymer coating density [11].

2 .

 2 Literature review on parameters affecting coating 2.3. Effect of Hydrophobic/hydrophilic chain length 103 Chapter III Studies of PS-PEGMA coating on hydrophobic PVDF membranes have shown that increasing

Figure 3 . 2 .

 32 Figure 3. 2. PS-b-PEGMA coating density on PVDF membrane vs concentration of copolymer solution used as coating bath for different PS/PEGMA ratio [9].

Figure 3 . 3 .

 33 Figure 3. 3. Proposed assembly combinations of random, diblock and triblock PS-PEGMA copolymers on PS surfaces [8].

Figure 3 . 4 .

 34 Figure 3. 4. Block and random PS-PEGMA coating density on PVDF membrane vs concentration of copolymer solution for a) PS/PEGMA ratio = 0.8-0.9, b) PS/PEGMA ratio = 1.7-2.1, and c) PS/PEGMA ratio = 3.7-3.9 [12].

  copolymers set used in this study were purchased from Sigma Aldrich for PS30-b-PAA2, PS30-b-PAA5 and PS30-b-PAA8, and from Polymer Source (Montreal, Canada) for the other copolymers: PS13-b-PAA22, PS26-b-PAA76, PS100-b-PAA107, PS28-b-PEO13, PS16-b-PEO72 and PS12-b-PEO30. They all have a Polydispersity Index (PDI) ≤ 1.3, implying minor heterogeneities on the distribution of the molecular mass. The PDI of a polymer is calculated as follows:

3 . 6 *

 36 List of copolymers used for surface modification with their properties. Data obtained by DLS measurement performed in this study. ° Data provided by the furnishers of the copolymers.

Figure 3 . 5 .

 35 Figure 3. 5. Illustration of the studied coating procedures of PVDF membranes.

Figure 3 . 6 .

 36 Figure 3. 6. Illustration of the procedure of protein adsorption on the coated membrane.

3. Materials and methods 3 . 3 .

 33 Methods for characterization of coating and proteins adsorption Chapter III been removed are analysed with the help of a UV-visible spectrometer Lambda 365, PERKIN

Figure 3 . 7 . 3 .III 3 . 3 . 2 .

 373332 Figure 3. 7. Working principle of the Lambda 365, PERKIN ELMER UV visible spectrometer.

Figure 3 . 8 .

 38 Figure 3. 8. Working principle of Attenuated Total Reflectance Spectroscopy (ATR) [16]. Attenuated Total Reflectance (ATR) spectroscopy (Nicolet 6700, Thermo Scientific) with diamond crystal, 45ᵒ incident angle, 16 scans, and 4 cm -1 spectral resolution was used to obtain spectra and verify the presence of the coating and the proteins on the membranes surface.

3. Materials and methods 3 . 3 .

 33 Methods for characterization of coating and proteins adsorption 115 Chapter III

Figure 3 . 9 .

 39 Figure 3. 9. Illustration of operating steps of the FTIR spectrometer to get to the sample spectrum.

Figure 3 . 10 . 3 .

 3103 Figure 3. 10. Schematic representation of FTIR-ATR operation [15].

4 .III 4 .

 44 Methodology for the analysis of ATR-FTIR results 4.1. Determination of copolymers and proteins characteristic peaks 118 Chapter Methodology for the analysis of ATR-FTIR results

  maps. The obtained ATR spectra of PVDF pristine membrane and the pure PS-b-PAA copolymers are shown in Figure 3. 11.

Figure 3 .

 3 Figure 3. 11. ATR spectra of PVDF pristine membrane and the pure PS-b-PAA copolymers.

4 .

 4 Methodology for the analysis of ATR-FTIR results 4.1. Determination of copolymers and proteins characteristic peaks 119 Chapter III the small length of PAA (copolymer is only 10 wt.% of PAA). As for the O-H stretching, it starts being seen in the spectra when the PAA molecular weight is higher than 20 kDa. Table 3. 4. Absorption bands of functional groups in PS-b-PAA copolymers and those in proteins (HSA and IgG). C=C stretching of the aromatic in PS 700 C-H deformation of monosubstituted aromatic in PS Absorption (cm -1 ) Functional groups of proteins 3500-3070 N-H stretching in the amide 1680-1630 C=O stretching in the amide Table 3. 4 summarizes the peaks of interest representing the chemical structure of PS-b-PAA and that of proteins (HSA and IgG) with the corresponding functional groups of each one, as well as their wavenumbers of absorption.

Figure 3 . 12 .

 312 Figure 3. 12. Example of the conversion of a color-coded map generated for the peak at 700 cm -1 for the coating of PS30-b-PAA2 at different concentrations in solution as compared to a pristine membrane (PM), to a map coded with a grey scale.

Figure 3 . 5 )

 35 Figure 3. 5) on the coating of PS30-b-PAA5 and PS30-b-PAA8 is investigated. The best coating

13 andFigure 3 .Figure 3 . 13 .

 133313 Figure 3. 14 for the coating of PS30-b-PAA5 and PS30-b-PAA8 respectively.

Figure 3 .

 3 Figure 3. 14. FTIR maps obtained for three functional groups of the PS30-b-PAA8 coated on PVDF membranes using different processes (IWD, IDW and ID), 2 hours of coating time and a copolymer concentration of 5 mg•mL -1 .

Figure 3 .

 3 Figure 3. 15. FTIR maps generated for the peak area at 700 cm -1 and converted in the same grey scale for both copolymers.

Figure 3 . 16 .

 316 Figure 3. 16. Intensity average of the peak area (symbols) and the standard deviation (error bars) at 700 cm -1 as function of the operating process for the coating of PS30-b-PAA5 and PS30-b-PAA8 with a concentration of 5 mg•mL -1 and coating time of 2 hours.

Figure 3 .

 3 Figure 3. 17. FTIR maps for membranes modified with the PS30-b-PAA5 and PS30-b-PAA8 using different coating times (1, 2, 4 and 6 hours) while keeping the copolymer concentration at 5 mg•mL -1 .

  were already given enough time to adsorb on the surface until a plateau is reached indicating saturation of the surface. The same trend is obtained with both copolymers but with higher intensities of absorption for PS30-b-PAA5 than PS30-b-PAA8 suggesting again a better adhesion of the first copolymer than the second one. That confirms what has been noticed in the previous 5. Results and discussions 5.2. Variation of coating time Chapter III explained in section 3.3.1. The experiments have been repeated at least three times to calculate

Figure 3 . 19 .

 319 Figure 3. 19. Coating density calculated as function of the immersion time (1, 2, 4 and 6 hours) for the coating of PVDF membranes with a PS30-b-PAA5 and PS30-b-PAA8 solutions with a concentration of 5 mg•mL -1 .

  obtained in the previous sections show different abilities of adhesion to the surface by two copolymers having the same hydrophobic group and a different hydrophilic chain length. In order to corroborate this outcome, coating has been conducted this time with six different sizes of PS-b-PAA copolymer and three of PS-b-PEO using ID process, 2 hours of immersion and a copolymer concentration of 5 mg•mL -1 . Since we can put four samples at most for each mapping, one of which is the pristine membrane (PM) as reference, two mapping have been done to analyze all the coated samples with the six PS-b-PAA copolymers and one mapping for PS-b-PEO. The first mapping was to confirm what has been noticed on the effect of the hydrophilic chain length, as the copolymers used are PS30-b-PAA2, PS30-b-PAA5 and PS30-b-PAA8. As for the second and the third mapping, it was done to assess the effect of both hydrophobic/hydrophilic chain lengths on the attachment of the copolymer on the membrane surface. One of them was for the coating of PS13-b-PAA22, PS26-b-PAA76 and PS100-b-PAA107, while the second one assesses the coating of PS28-b-PEO13, PS12b-PEO30 and PS16-b-PEO72.

Figure 3 .

 3 Figure 3. 20. FTIR maps for the coating of different sizes of PS-b-PAA and PS-b-PEO using ID process, 2 hours of immersion and a copolymer concentration of 5 mg•mL -1 .

Figure 3 . 21 .

 321 Figure 3. 21. The intensity average of the peak area at 700 cm -1 as shown A/ for the coating of different PS-b-PAA and PS-b-PEO using ID process, 2 hours of immersion time and a copolymer concentration of 5 mg•mL -1 and B/ the intensity as a function of the hydrophilic/hydrophobic ratio.

5. Results and discussions 5 . 4 .

 54 Variation of the copolymer concentration 134 Chapter III HSA over the modified membrane. It has to be mentioned that the maps showing the presence of PS block, PAA segment and HSA protein, were all generated from the same membrane sample.

Figure 3 .

 3 Figure 3. 22. FTIR maps for the coating of PVDF membranes with various PS-b-PAA copolymers using ID process, 2 hours of immersion and different copolymer concentrations, and the adsorption of HSA on these same modified membranes; [HSA] = 1 mg•mL -1 ; pH ≈ 7.

  membrane. It has to be mentioned that the maps showing the presence of PS block, PAA segment and HSA protein, were all generated from the same sample. This excludes the theoretical scheme of vertical deposition of the copolymer brushes on the membrane proposed in the presentation of the project in the general introduction (section 6.3 of chapter I). It would be more logical to imagine a horizontal deposition of the hydrophobic part (PS block) due to the hydrophobic interactions with the PVDF membrane and maybe a, stretched orientation perpendicular to the membrane surface of the hydrophilic chains (PAA segment). This configuration of coating has been suggested in a study aimed at coating PS surfaces with different PS-b-PEGMA copolymers (Figure3. 3)[7].These results support also what has been noticed in the previous section. In fact, the copolymers do not exhibit all the same ability of adhesion onto the membrane surface by having different intensities of absorbance. But the scale of intensity has been found to be also correlated to each part (hydrophilic or hydrophobic) and its size. This means that larger hydrophilic chains either introduce higher steric hindrance preventing the access of PS moieties, or they hide certain parts of PS that we can no longer see with the FTIR. These are maybe the reasons of a weaker presence of the PS in the case of PS26-b-PAA76 and PS100-b-PAA107, despite of having almost the same size of PS block or higher than PS30-b-PAA5 and PS30-b-PAA8.The mean intensities (Figure3. 23) of the peaks area (at 700 cm -1 and 1715 cm -1 ) evaluated from the converted maps using the same grey scale, illustrate better what has been said above.Logically, the presence of PAA was found to be greater with PS100-b-PAA107, PS26-b-PAA76, and then PS30-b-PAA8 and PS30-b-PAA5 where no big differences are displayed. As for the PS presence, the biggest intensity was obtained for PS30-b-PAA5 followed by PS30-b-PAA8, and 5. Results and discussions 5.4. Variation of the copolymer concentration 136 Chapter III then PS100-b-PAA107 and PS26-b-PAA76, which confirms what was previously suggested regarding the effect of hydrophilic segment size on the PS presence or visibility.

Figure 3 . 23 .

 323 Figure 3. 23. The intensity average of the peaks area at 700 cm -1 and 1715 cm -1 for the coating of different PS-b-PAA copolymers as function of copolymer concentration using ID process, 2 hours of immersion time.

Figure 3 . 24 .

 324 Figure 3. 24. The intensity average of the peak area at 1660 cm -1 for the HSA adsorption on PVDF membranes coated with different PS-b-PAA copolymers, [HSA]= 1 mg•mL -1 ; pH=7.

5. Results and discussions 5 . 4 .

 54 Variation of the copolymer concentration 138 Chapter III

5 .IIIFigure 3 . 25 .

 5325 Figure 3. 25. FTIR maps for the adsorption of HSA protein on PVDF membranes coated with different concentrations of PS30-b-PEO13 copolymer, as well as the evaluated mean intensities

5 .IIIFigure 3 . 26 .

 5326 Figure 3. 26. FTIR maps for the adsorption of HSA protein on PVDF membranes coated with PS26-b-PAA76 and PS28-b-PEO13 as well as the evaluated mean intensities for the peaks area at 700 cm -1 and 1660 cm -1 ; [HSA]= 1 mg•mL -1 .

5. Results and discussions 5 . 6 .III 5 . 6 .

 5656 Influence of pH on HSA adsorption onto membrane coated with PS-b-PAA 141 Chapter Influence of pH on HSA adsorption onto membrane coated with PS-b-PAA

4 M

 4 -1 of HSA on PVDF membrane coated with PS-b-PAA was studied again in this section. However, the adsorption was carried out this time at different pH (5, 7 and 8) and for the coating of two copolymers (PS30-b-PAA5 and PS100-b-PAA107), with a short PAA segment and with a larger one. The coating was performed using ID process, 2 hours of coating time and a copolymers concentration of 5 mg•mL -1 . The pH of HSA solutions used for the immersion of the coated membranes was controlled using different buffer solutions at 0.1 M (TRIS base pH 8, HEPES pH 7, and a mixture of sodium citrate di-hydrate and citric acid at pH 5). While, the ionic strength of these protein solutions was fixed at 0.15 M by adding the required amounts of a NaCl solution.

5. Results and discussions 5 . 6 .IIIFigure 3 . 27 .

 56327 Figure 3. 27. FTIR maps for the adsorption of HSA at different pH (5, 7 and 8) on PVDF membranes coated with PS30-b-PAA5 and PS100-b-PAA107 copolymers using ID process, 2 hours of immersion and a copolymer concentration of 5 mg•mL -1 ; [HSA]= 1 mg•mL -1 .

Figure 3 . 28 . 5 .

 3285 Figure 3. 28. Evaluated mean intensities of the peaks area at 700 cm -1 and 1660 cm -1 showing respectively the uniformity of coating on different samples and the adsorption of HSA onto these samples at different pH; [HSA]= 1 mg•mL -1 .

Figure 3 . 29 .

 329 Figure 3. 29. Illustration of the HSA adsorption onto PS-PAA coated membrane due its hydrophobic interactions with the visible PS and electrostatic interactions with PAA brushes.

Table 3 . 6 .

 36 The amounts of HSA adsorbed onto PVDF membranes coated with PS30-b-PAA5 and PS100-b-PAA107 estimated using UV-visible spectroscopy.HSA adsorption on PVDF membranes coated with PS30-b

5. Results and discussions 5 . 7 .

 57 Ionic strength effect on HSA adsorption onto membrane coated with PS-b-PAA 145 Chapter III acid.

Figure 3 .

 3 30 or from the evaluated mean intensities shown in Figure3. 31, it can be confirmed that both copolymers have been coated all over the membrane surface. As for the adsorption of HSA, it has been found to decrease with the increase of the ionic strength. Indeed, for membranes coated with PS30-b-PAA5, the intensity yields 0.53 for an ionic strength (I) of 0.05 M and 0.15 M, against 0.3 for I=0.5 M. As for membranes modified with PS100-b-PAA107 the intensity yields 0.51 and 0.58 for a salts concentration of 0.05 M and 0.15 M respectively, against 0.28 for I=0.5 M.

Figure 3 .IIIFigure 3 . 31 .

 3331 Figure 3. 30. FTIR maps for the adsorption of HSA at different ionic strength on PVDF membranes coated with PS30-b-PAA5 and PS100-b-PAA107 using ID process, 2 hours of immersion and a copolymer concentration of 5 mg•mL -1 ; [HSA]= 1 mg•mL -1 .

5. Results and discussions 5 . 8 .III 5 . 8 .

 5858 IgG adsorption on membranes coated with PS30-b-PAA5 and PS100-b-PAA107 147 Chapter IgG adsorption on membranes coated with PS30-b-PAA5 and PS100-b-PAA107 Previous results revealed the adsorption of HSA protein on PS-b-PAA coated membranes.This adsorption was found to be correlated to the pH and the ionic strength of the protein solution as it was also proved in the previous chapter. Indeed, it took place more at low ionic strength (≤ 0.15 M) and a pH of 5, while at higher pH and salts concentration the adsorption was much lower. In this section, the adsorption of IgG protein on membranes coated with two copolymers (PS30-b-PAA5 and PS100-b-PAA107) was studied at different pH and I=0.15 M.

Figure 3 .

 3 Figure 3. 32. FTIR maps for the adsorption of IgG at different pH (5, 7 and 8) on PVDF membranes coated with PS30-b-PAA5 and PS100-b-PAA107 using ID process, 2 hours of immersion and a copolymer concentration of 5 mg•mL -1 ; [IgG]= 1 mg•mL -1 .

Figure 3 . 33 .

 333 Figure 3. 33. The evaluated mean intensities of the peaks area at 700 cm -1 and 1660 cm -1 showing respectively the uniformity of coating on different samples and the adsorption of IgG onto these samples at different pH; [IgG]= 1 mg•mL -1 .

  -1 were enough to allow copolymers (PS-b-PAA and PS-b-PEO) to adsorb on the membrane surface and to cover it almost completely.

  showed a clear response to blood proteins whether adsorption or rejection. Hence, we used PS30-b-PAA5, PS30-b-PAA8, PS26-b-PAA76, PS100-b-PAA107 and PS28-b-PEO13.

  permeate was measured every 5 minutes during 20 min to observe the instantaneous flux change and verify its stability. At 1 bar, the flux was noted until it reached almost a steady state.

4. Results and discussions 4 . 1 . 4 . Results and discussions 4 . 1 .

 41441 Membrane conditioning and permeability measurementChapter IV Membrane conditioning and permeability measurementAs explained in section 3.1, the permeability of the PVDF membrane was determined only once the compaction had been achieved. Results shown in Figure4. 1 show a hysteresis evolution of the flux when changing the pressure. In fact, the flux logically increased with the increase in pressure until 1 bar, before starting decreasing during the compaction of the membrane at this same pressure. Indeed, it decreased from around 280 L•m -2 •h -1 before compaction to around 86 L•m -2 •h -1 after compaction. This evinces the importance of the compaction step to avoid unsteady transport properties during filtration experiments. However, this significant decrease in flux is probably not only due to membrane compaction but also to the solvent exchange because the membrane had been soaked in absolute ethanol before water was passed through. Once the flux had reached almost a steady state at 1 bar, its evolution showed a linear correlation with the pressure. Thus, the permeability was obtained from the slope of this linear evolution of the flux versus the pressure according to Darcy's law given by equation 4.1, and it was found to be around 86 L•m -2 •h -1 •bar -1 .

Figure 4 . 1 .

 41 Figure 4. 1. Compaction of pristine membrane and solvent exchange, and the plot of the water flux versus pressure given the permeability of the pristine membrane.

4. Results and discussions 4 . 2 .

 42 Effect of the copolymer size on the permeability of coated membranes 165 Chapter IV

Figure 4 . 2 .

 42 Figure 4. 2. FTIR maps generated at 700 cm -1 and 1715 cm -1 for the coating of PVDF membranes with different PS-b-PAA copolymers with a concentration of 5 mg•mL -1 and during 2 hours of coating time.

IVFigure 4 . 3 .

 43 Figure 4. 3. Plots of the water flux vs pressure after coating with each copolymer from which the permeability was calculated.

Table 4 . 2 . 3 .IV 4 . 3 . 4 . 3 . 1 .

 42343431 Permeability of membranes coated with different PS-b-PAA copolymers compared to that of the pristine membrane, as well as the decline percentage caused by coating. Dead-end filtration of HSA through membrane coated with PS-b-PAA 168 Chapter Dead-end filtration of HSA through membrane coated with PS-b-PAA Assessment of HSA adsorption onto the membrane during dead-end filtration Once the coating of compacted membrane had been successfully performed, the dead-end filtration of HSA was carried out in the Amicon cell. For that, the cell was filled with 10 mL of HSA solution and the filtration was performed at 1 bar until around 3 mL are left in the retentate (VRF = 3.3). The presence of HSA over the membrane surface was assessed using FTIR as shown in Figure 4. 4.

Figure 4 . 4 .

 44 Figure 4. 4. FTIR maps generated at 1660 cm -1 and 3300 cm -1 for the adsorption of HSA during dead-end filtration on PVDF membranes coated with different PS-b-PAA copolymers. pH ≈ 7 ; [HSA]= 1 mg•mL -1 ; I ≈ 0.15M.

4. Results and discussions 4 . 3 .

 43 Dead-end filtration of HSA through membrane coated with PS-b-PAA 169 Chapter IV the negatively charged PAA brushes. This is what has been suggested in the previous chapter (section 5.7

  The obtained averages representing the chemical composition of the membrane surface after the adsorption of HSA on membrane coated with different PS-b-PAA copolymers are compared to that of HSA adsorption on PM as shown Figure 4. 5. 4. Results and discussions 4.3. Dead-end filtration of HSA through membrane coated with PS-b-PAA Chapter IV

4 .

 4 Results and discussions 4.3. Dead-end filtration of HSA through membrane coated with PS-b-PAA Chapter IV

Figure 4 . 7 .

 47 Figure 4. 7. Flux evolution versus time during HSA filtration at 1 bar on pristine and modified membranes with different PS-b-PAA copolymers.

Figure 4 .

 4 Figure 4. 8 confirm first that the permeability of the PM was almost not affected by HSAadsorption, where J/Jw was found to be 0.98. However, deviations of the flux in the case of coated membranes have been displayed. Indeed, the J/Jw ratio has been found to be 0.82, 0.87 and 0.79, after the filtration of HSA on membranes coated with PS30-b-PAA5, PS26-b-PAA76 and PS100-b-PAA107 respectively. Meanwhile, the flux reduction does not exhibit much difference between the three copolymers and it does not seem to be related to the copolymer size. Thus, one can consider that the deviations observed could be due to deviations on the measurement during the experiments.

Figure 4 . 8 .

 48 Figure 4. 8. Flux reduction after HSA filtration at 1 bar through pristine and modified membranes with different PS-b-PAA copolymers.

4 .Figure 4 . 9 .

 449 Figure 4. 9. The evolution of water flux Jw after surface modification by different PS-b-PAA copolymers, and the flux of HSA solution J in the end of filtration through these modified membranes.

4. Results and discussions 4 . 3 . 4 . 4 . 4 .

 43444 Dead-end filtration of HSA through membrane coated with PS-b-PAA 176 Chapter IV could be due to the fact that with the applied pressure, larger part of PAA is accessible to HSA molecules to interact with, which means that larger PAA would adsorb more HSA. Table 4. 3. Calculations of protein retention by the PM and membranes coated with different PS-b-PAA copolymers as well as the evaluation of the amount of HSA adsorbed on the membrane surface and/or structure by mass balance. Dead-end filtration of HSA through membrane coated with PS-b-PEO and with both PSb-PAA and PS-b-PEO in the mixture Chapter IV Dead-end filtration of HSA through membrane coated with PS-b-PEO and with both PS-b-PAA and PS-b-PEO in the mixture

Figure 4 .Table 4 . 4 . 5 .IV 4 . 5 .

 444545 Figure 4. 10. FTIR maps generated at 700 cm -1 and 1660 cm -1 showing the presence of the coating layer and that of the protein after HSA filtration through modified membrane (pH ≈ 7;[HSA]= 1 mg•mL -1 ).

Figure 4 .

 4 Figure 4. 13. FTIR maps generated at 700 cm -1 , 1720 cm -1 and 1660 cm -1 showing the presence of the coating layer and that of the protein after HSA dead-end filtration through membrane coated with PS100-b-PAA107 (pH = 5 and 7; [HSA]= 1 mg•mL -1 ).

Table 4 . 5 .

 45 Comparison of HSA retention and deposition on PVDF membrane coated with PS100-b-PAA107 during dead-end filtration at different pH (5 and 7).

  previously showed standard deviations of around 10% or more. The standard deviations are not calculated here because the experiment was performed once as stated earlier. Thus, to confirm HSA adsorption increase, we resorted to calculating the mean intensities from the maps converted into grey scales as explained in section 4.2 of chapter III. The obtained results exposed in Figure4. 14 were then compared to the mean intensities reported in section 5.6 of chapter III regarding the static adsorption of HSA onto PS100-b-PAA107 coated membrane at different pH.

4. Results and discussions 4 . 5 .

 45 Dead-end filtration of HSA at pH 5 through membrane coated with PS-b-PAA 184 Chapter IVrespectively. It can also be noticed in Figure4. 14 that the HSA deposition during filtration is clearly greater than its static adsorption. Indeed, the evaluated intensities in static adsorption of HSA yields 0.64 and 0.41 at pH 5 and 7. This outcome sustains the hypothesis we established earlier assuming that HSA deposition during filtration is enhanced by the flow through the membrane. Thus, the HSA observed on the membrane surface would be due to the adsorption and the accumulation of HSA once all the adsorption sites were occupied.

Figure 4 . 14 .

 414 Figure 4. 14. Evaluated mean intensities of the peak area at 1660 cm -1 showing the adsorption of HSA onto PS100-b-PAA107 coated membrane at pH 5 and 7 during filtration as compared to those obtained for static adsorption of HSA in the same conditions; [HSA]= 1 mg•mL -1 .

4 .

 4 Results and discussions 4.5. Dead-end filtration of HSA at pH 5 through membrane coated with PS-b-PAA 185 Chapter IV shown at pH 5. Thus, one can consider once again that the small differences in flux reduction could be due to deviations on the measurement during the experiments.

Figure 4 . 15 .

 415 Figure 4. 15. Plot of the water flux versus pressure from which the permeability was calculated for membranes coated with PS100-b-PAA107 and evolution of HSA solution flux versus time during HSA filtration (pH=5) through this same membrane.

Table 5 . 1 :

 51 Summary of the blood proteins (HSA and IgG) interactions with polymers (PAA and PEG) in solution, and at the interface of a functionalized PVDF membrane in static mode and during dead-end filtration. -PAA complexes only at pH 5 and ionic strength ≤ 0.15 M, which is attributed to electrostatic interactions. No specific interaction has been shown IgG Partial aggregation of IgG No specific interaction has been shown Medium Protein At the interface of a modified membrane in static mode PS-PAA PS-PEO HSA -HSA adsorption onto PS-b-PAA PVDF coated membrane at pH 7 due to electrostatic interactions of HSA with PAA, and due to hydrophobic interactions between HSA and the visible PS. -Increase of HSA adsorption on the same membrane at pH 5 due to increased electrostatic interaction of HSA with PAA. Reduction of HSA adsorption on PS-PEO PVDF coated membrane compared to its adsorption on PVDF pristine membrane, which maybe owing to the hydration layer created by the PEO preventing the adsorption of proteins. IgG Adsorption of IgG similarly on PVDF pristine membrane as on PSb-PAA PVDF coated membrane Not investigated. Medium Protein At the interface of a modified membrane during dead-end filtration PS-PAA PS-PEO HSA The same trend of HSA adsorption obtained in static mode has been observed during dead-end filtration but with greater amounts due to the pressure applied on the HSA solution. The role of the pH and ionic strength leading to the complexation is observable but is screened by the role of the filtration rate on the deposit formation. Slight increase of HSA adsorption on PS-PEO PVDF coated membrane due to the pressure applied on the HSA solution which probably overcome the hydration layer created by the PEO.

  Besides, it could be interesting to study the adsorption of IgG on PS-b-PEO coated membrane to confirm the effect of PEO on preventing the non-specific adsorption of proteins either in static mode and during dead-end filtration. The study of HSA deposition at pH 5 on PVDF pristine membrane may provide more clarifications and new elements that would maybe sustain the hypotheses we have already assumed about the difference of HSA adsorption on coated membrane at pH 5 and 7.Moreover, the study of interactions could be extended to other blood proteins such as the Fibrinogen from human blood. In fact, some experiments conducted by our partners in the Research and Development Center for Membrane Technology (Chung Yuan Christian University, ChungLi, Taiwan) showed that the Fibrinogen conjugated to the PAA could even enhance the platelets capture. Thus, it would be substantial to explore the interaction of Fibrinogen with PAA at different levels as done for the other proteins. Otherwise, it would be perhaps advisable to think to graft the fibrinogen onto the PS-b-PAA modified membrane, and then test the adsorption of other blood proteins.It would be also of interest to investigate the behaviour of these proteins (HSA, IgG and fibrinogen) towards the thermosensitive NIPAAM suggested to release the platelets after being 2the temperature. Thereby, it will be significant to probe the proteins-NIPAAM interactions at different temperatures in solution. Then, as it has been done for the other copolymers, the adsorption of blood proteins onto PS-b-NIPAAM coated membrane either in static mode and during dead-end filtration should be examined using FTIR.

  filtration operations. This study should be carried out either on PS-b-PAA, PS-b-PEO and on PS-b-NIPAAM coated membranes. Subsequently, it would be even more significant to perform the adsorption of the blood proteins mixture on PVDF membrane coated with the three copolymers mentioned previously. For that, the functionalization of the membrane surface by the three copolymers should be first performed as it has been done in this study using two of them (PS-b-PAA and PS-b-PEO).

  La séparation sélective des protéines et des cellules sanguines est d'une importance capitale pour traiter certaines maladies cibles et pour plusieurs applications médicales telles que la cicatrisation des plaies. Le défi consiste à fournir la technologie la plus efficace et la plus rentable qui puisse garantir cette séparation. Parmi de nombreuses autres technologies de séparation, les technologies membranaires se sont révélées être prometteuses. Cependant, leurs interactions non spécifiques avec les protéines les rendent vulnérables au bio-colmatage, ce qui limite leur application dans le domaine biomédical. Par conséquent, l'objectif de ce travail est de concevoir une technologie membranaire adaptée pour le dépistage des protéines sanguines en utilisant la modification de surface. Cette dernière est réalisée en déposant certains groupements fonctionnels sur la surface de la membrane, tels que des copolymères polystyrène-poly acrylique acide (PS-PAA) et polystyrène-polyéthylène glycol (PS-PEG), soit pour favoriser une interaction spécifique avec des protéines cibles ou pour réduire leur interaction non spécifique. Dans un premier temps, les interactions potentielles qui pourraient avoir lieu entre les brosses de PAA, PEG et certaines protéines abondantes dans le plasma telles que la sérum-albumine humaine (HSA) et la ɣ-globuline (IgG) ont été étudiées en solution en utilisant la Diffusion des rayons-X aux petits angles (Acronyme anglais : SAXS) combinée à la chromatographie. Ainsi, nous avons constaté que le PAA forme des complexes avec l'HSA dans certaines conditions physico-chimiques spécifiques de pH et de force ionique. Cette liaison HSA-PAA s'est ensuite révélée réversible en fonction du pH et de la force ionique du milieu, et il a été démontré que sa stoechiométrie était liée à la taille du PAA. En revanche, l'IgG n'a pas présenté le même comportement que l'HSA vis-à-vis du PAA, mais elle s'est avérée être partiellement agrégée. Quant au PEG, il

Table 1 .

 1 1. Main forces governing the interfacial interactions between nanomaterials and biological systems. Adopted from Nel et al. [27]. ................................................................... 12

Table 1 .

 1 2. Spectroscopic methods used in membrane surface characterization[30]. ............. 14

Table 2 .

 2 1. Rg values (in Å) as a function of both HSA and PAA concentrations obtained by fits of the scattering intensities at small angles using Foxtrot software ([HSA]=0-600µM and [PAA]=0-50µM). ..................................................................................................................... 62

Table 2 .

 2 2. The evolution of radius of gyration as well as the Porod volume for the HSA-PAA mixture as a function of the molar ratio ([PAA]=50 µM; [HSA]=75-600 µM)....................... 69

Table 2 .

 2 3 : Various physical parameters of the HSA-PAA complex that can be formed with every PAA size. ........................................................................................................................ 81

Table 3 .

 3 1. Effects of the different parameters reported in this section on the coating density evolution, as well as the deviations from Langmuir adsorption model related to each parameter.

Table 1 .

 1 1. Main forces governing the interfacial interactions between nanomaterials and biological systems. Adopted from Nel et al.[27].

	. 1.

Force Origin and nature Possible impact on the interface

	Hydrodynamic and	Convective drag, shear, lift and	Increase the frequency of collisions
	Brownian interactions	Brownian diffusion are often	between nanoparticles and other
		hindered or enhanced at nanoscale	surfaces responsible for transport.
		separations between interacting
		interfaces.
	Electrodynamic	VDW interactions arising from	Universally attractive in aqueous
	interactions	each of the interacting materials	media; substantially smaller for
		and the intervening media.	biological media and cells owing to
					high water content.
	Electrostatic	Charged	interfaces	attract	Overlapping double layers are
	interactions	counter-ions and repel co-ions	generally repulsive as most
		through Coulombic forces, giving	materials acquire negative charge
		rise to the formation of an	in aqueous media, but can be
		electrostatic double layer.		attractive for oppositely charged
					materials.
	Solvent interactions	Lyophilic	materials	interact	Lyophilic	materials	are
		favourably	with	solvent	thermodynamically stable in the
		molecules.			solvent and do not aggregate.
	Steric interactions	Polymeric species adsorbed to	Generally increase stability of
		inorganic particles or biopolymers	individual particles but can
		expressed at the surfaces of cells	interfere in cellular uptake,
		give rise to spring-like repulsive	especially when surface polymers
		interactions with other interfaces.	are highly water-soluble.
	Polymer bridging	Polymeric species adsorbed to	Generally, promote aggregation or
	interactions	inorganic particles or biopolymers	deposition, particularly when
		expressed at the surfaces of cells	charge functionality is carboxylic
		containing charged functional	acid and dispersed in aqueous
		groups can be attracted by	media containing calcium ions.
		oppositely charged moieties on a
		substrate surface.

  (Figure 2. 12), as it gives the forward scattering intensity I(0) from the intercept. The result yields approximately 30 Å (30.4 ± 0.2 Å), which is almost what was found with Rg (30.2 ± 0.4

	4. Results and discussion
	4.2. Study of HSA-PAA interaction
	vs q 2
	II
	Chapter
	55

Table 2 .

 2

	[PAA] µM
		0	10	20	30	40	50
	[HSA] µM
	0	0	116	114.3	105.4	103.2	106.1
	75	30.4	35.7	41.4	44.3	102.8	101.7
	150	29.6	34.6	37	40.8	101.6	102.3
	300	28.8	31.4	32.6	37.6	79.7	100
	450	27.9	30	29.5	34.1	84	96.4
	600	25.2	27.2	30	30.2	77.5	90.2

1. Rg values (in Å) as a function of both HSA and PAA concentrations obtained by fits of the scattering intensities at small angles using Foxtrot software ([HSA]=0-600µM and [PAA]=0-50µM).

Table 2 .

 2 2. The evolution of radius of gyration as well as the Porod volume for the HSA-PAA mixture as a function of the molar ratio ([PAA]=50 µM; [HSA]=75-600 µM).

	[HSA] µM	75	75	150	180	225	300	450	600
	nHSA/PAA	Free HSA	1.5	3	3.6	4.5	6	9	12
	Rg (± 3 Å)	30.4	108.3	107	110.1	108.6	90.6	81	74.1
	Vp .10 3 (Å 3 )	99.1	428	502	500	491	400	305	239

Table summarizing

 summarizing Rg values as evaluated by Primus program.

	4. Results and discussion
	4.6. Influence of PAA size on HSA-PAA complexation
	These primary results gave us an idea about the potential stoichiometry of HSA-PAA
	complexation with every size of PAA.
	II
	Chapter

Table 2 . 3

 23

: Various physical parameters of the HSA-PAA complex that can be formed with every PAA size.

Parameter Complex Rg (Å) Vp × 10 3 (Å 3 ) Mw (kDa) SAXS Retention time (Rt) Mw (kDa) HPLC Rc (Å) Dmax

								(Å)
	HSA	29.2 ± 0.3	100	65.7	7.8	64.5	36	89
	HSA+PAA	35.2 ± 0.6	130	85.2	7.7	71.5	36	105
	5kDa
	HSA+PAA	62.5 ± 1.6	210	125.3	7.24	115	38	138
	30kDa
	HSA+PAA	81.7 ± 2.4	380	304.9	6.25	320	41	227
	50kDa
	HSA+PAA	97.7 ± 3.4	520	433.6	6.02	401.6	42	390
	100kDa

Table summarizing

 summarizing Rg values as evaluated by Primus program.

Literature review on parameters affecting coating

	2. Literature review on parameters affecting coating
	2.1. Effect of coating time
	2.
	III
	Chapter
	101

Table 3 .

 3 2. Specific properties of the commercial PVDF membrane used in this study as provided by Millipore.

HSA adsorption on PVDF membranes coated with PS100-b-PAA107

	PM (pH=7)	1.15	0.96	0.19	0.06 (32%)
	5	1.15	0.63	0.52	0.07 (13%)
	7	1.15	0.82	0.33	0.05 (15%)
	8	1.15	0.8	0.35	0.05 (14%)
	PM (pH=7)	1.15	0.99	0.16	0.05 (31%)
	5	1.15	0.57	0.58	0.06 (10%)
	7	1.15	0.76	0.39	0.04 (10%)
	8	1.15	0.79	0.36	0.06 (17%)

An instrument funded by the FERMAT federation (https://www.federation-fermat.fr/) and operated on a shared time basis.

AppendicesD. Scientific communications and publication

Acknowledgements

My special thanks also go to all the trainees who worked with me (Abigail Rosales, Sharath Jayakumar and Ema Louati), for their contribution to the progress of this project.

Acknowledgements I

Summary

Chapter

II

The formation of the complex seems to be specific to the HSA and PAA solutes. Whether SAXS or chromatography measurements, they do not reveal any specific interaction between IgG and PAA or between neither HSA or IgG and PEG. In fact, IgG has been revealed to be partially unfolded and aggregated via Coulomb and hydrophobic interaction, while both proteins (HSA and IgG) did no display any interaction with PEG.

First of all, I would like to acknowledge the Agence Nationale de la Recherche (MOST-ANR International Program: ANR 17-CE08-0041) for the financial support, and FERMaT Federation FR3089 for providing the Small Angle X-ray Scattering laboratory facility.

Chapter II.

Study of blood proteins-polymers interactions in solution

Chapter III.

Membrane surface modification and investigation of blood proteins adsorption onto

Chapter III Most of the experiments were conducted using the HSA because it was the one that showed specific affinity with PAA as discussed in the previous chapter. IgG was used to compare its affinity to that of HSA regarding PAA.

Methods for characterization of coating and proteins adsorption

UV Visible spectroscopy

As stated in section 4.3.3.2 of chapter I, UV-visible spectroscopy could be used to evaluate the coating density and the amount of the proteins adsorbed onto the membrane surface by analysing the solute concentration before and after the adsorption. Since the absorbance is proportional to the concentration according to Beer-Lambert's Law (equation 3.3), the final concentration of the copolymer inside the coating bath can be calculated by measuring its absorbance.

𝐴 = 𝜀 • 𝑐 • 𝑙 (3.3)

Where ε is absorptivity (M -1 cm -1 ), c is concentration (M), and l is optical path length (cm).

For that, the absorbance of different standard solutions with known concentration of copolymer should be measured first to establish a calibration curve of the absorbance versus the concentration of the copolymer. That was done for all the copolymers used for surface modification as well as the proteins (HSA and IgG) used for the proteins adsorption study.

Whether for the copolymers or the proteins, the calibration curves were obtained using concentrations ranging between 0 and 2 mg•mL -1 , and results are given in section 1 of Appendix C. After that, since the initial concentrations are already known, the quantity of the coated copolymer or the adsorbed protein can be calculated by difference of mass using the equation below:

Where, mads is the adsorbed mass of the copolymer or the protein, Ci and Ceq are the initial concentration and that at the equilibrium respectively of the concerned solutes (copolymer or protein), and V is the volume.

Samples for coating are prepared the same way as mentioned previously depending on the studied parameter. 1 mL of the initial copolymer solution is prepared in addition to check the exactitude of the concentration and to avoid possible fluctuation in the UV apparatus response.

Once the modification has been performed, the final solutions from which the membranes have In order to compare quantitatively the maps, we proceeded to apply a similar images analysis as previously to determine the mean intensity of the peak area. Wherefore, the maps were converted using the same grey scale, and the calculated mean intensity was drawn versus the coating time for the two copolymers (Figure 3. 18). The obtained results confirm what has been observed on the FTIR maps, and that 2 hours of immersion was enough to achieve almost the maximum of intensity. Indeed, the mean intensity of the peak area was found to be 0.55 after 2 hours of immersion in PS30-b-PAA5 solution, against 0.57 and 0.58 after 4 and 6 hours respectively, which makes a variation of 5% in the mean intensity. As for the PS30-b-PAA8 copolymer, the mean intensity was 0.28 for 2 hours against 0.29 for 4 and 6 hours, which makes a variation of 3 %. This also sustains the fact that a greater amount of PS30-b-PAA5 copolymer has been attached to the PVDF membrane surface than that of PS30-b-PAA8 using the same operating conditions. The mean intensities for the peak area are given in Table 3. 5 with the standard deviation to the average value.

To validate these outcomes, we proceeded to the evaluation of the coating density as a function of the coating time. That was done by measuring the difference of the copolymer mass inside the coating bath before and after immersion using the UV-visible spectroscopy as [HSA]= 1 mg•mL -1 ; I ≈ 0.15M.

The bar graph in Figure 4. 6 represents the contact angle versus different conditions previously discussed. First, it is obvious that the contact angle decreased after the filtration of HSA through the membrane whether modified or not. This indicates that the adsorption of the protein onto the membrane surface increased its hydrophilicity. This makes sense since the HSA is adsorbed on the membrane due to interactions between its hydrophobic pockets and the membrane, which leave the hydrophilic amino acids of the protein more exposed on the surface.

However, the contact angle is still higher on the pristine membrane than on coated one even after the adsorption of HSA. This could be indicative that the PM is not completely covered by the protein which would be consistent with what has been noticed from FTIR maps. Because if the coverage of the membrane surface by HSA were the same on PM as on coated membrane, the hydrophilicity would be the same, so we would expect the same contact angle on both membranes.

However, if FTIR and ATR results suggest a relatively higher adsorption on membranes coated with PS26-b-PAA76 and PS100-b-PAA107, than on membrane coated with PS30-b-PAA5, the contact angle results do not exhibit exactly the same trend. Indeed, contact angles of around 76°, 79° and 78° have been obtained on membranes coated with PS30-b-PAA5, PS26-b-PAA76 and PS100-b-PAA107 respectively after the deposition of HSA. Thus, the hydrophilicity doesn't again that the copolymers distribution over the membrane surface is heterogeneous. Besides, these maps display a higher intensity with the coating of both copolymers than the coating of each one alone, which indicates clearly the presence of both of them on the membrane surface in the first case.

A visual analysis of these maps evinces the presence of HSA on the membrane surface, and confirms once again that this amount is greater on the membrane coated with PS26-b-PAA76

than on the PM. In addition, it has been shown to be also greater on membrane coated with copolymers in mixture than on a PM, but lower than that on a membrane coated with PS26-b-PAA76, which is consistent with what has been shown in the previous chapter (section 5.5). This is necessarily due to the presence of PS28-b-PEO13, which reduces the contact of HSA with PS26-b-PAA76. In fact, the PEO is able to create a hydration layer [10], which makes the PAA chains less accessible to the HSA. In addition, this could reduce the hydrophobic interactions of the protein with the visible blocks of PS on the membrane. However, the adsorption of HSA on membrane coated with PS28-b-PEO13 seems to be equivalent to that on the PM or even slightly greater. This is not exactly what is reported in the previous chapter (section 5.4.2),

where the adsorption of HSA is found to be reduced on membranes modified with this copolymer. This resistance to the protein adsorption was mainly attributed to PEO ability to form a surface hydration layer, its large excluded volume, and strong coordination capability to water molecules [11]. Apparently, this is not valid in this case, and one can assume that the flow through the membrane during filtration overcomes this hydration layer leading to the adsorption of protein molecules on the PS blocks due to hydrophobic interactions.

As explained earlier in this section, the permeability was evaluated after coating and it was logically found to be reduced as shown in Thereby, the reduction is higher when the membrane is coated with both copolymers because more copolymers are deposited onto the surface. We noticed here that the permeability of the pristine membrane found to be around 91 L•m 2 •h -1 •bar -1 is slightly higher than that obtained in the previous section that yields 86 L•m 2 •h -1 •bar -1 . This indicates that two pieces of the same membrane could have different permeability, which is probably due to the heterogeneity of the pores distributions over the same membrane coupon. Subsequently, the retention of HSA was evaluated by measuring the protein concentration in the permeate and in the retentate using UV-visible spectroscopy and by using equation (4.3).

Thereby, the amount of protein deposited on the membrane surface was calculated by mass balance using equation (4.6). The results of these calculations are summarized in Table 4. 4.

From these results, it can be first noticed that the membrane coated with PS28-b-PEO13 displayed an HSA adsorption density of 0.16 mg•cm -2 , which is close (slightly higher) to that obtained on the PM that yields 0. Overall, the different elements brought by this thesis constitute a fairly important knowledge base on the interactions between some abundant blood proteins and certain polymers used for membrane surface modification. This study allows not only to understand these interactions but also to control them by tuning the operating physicochemical conditions at different level i.e.

in solution and at the interface of a modified membrane either in static mode or during deadend filtration operations.

For instance, the bioactive PAA suggested to capture blood platelets has been found to form complexes in solution with the most abundant protein in plasma namely HSA, whereas another blood protein namely IgG tended to aggregate in the presence of the same polyelectrolyte. Then, neither HSA or IgG exhibited any specific interactions with the neutral PEG assumed to prevent the nonspecific adsorption of proteins.

Deeper investigations using SAXS and chromatography proved that the HSA-PAA complexation takes place at some specific physicochemical conditions of pH and ionic strength disclosing that this phenomenon was driven by electrostatic attractive interactions. In fact, the HSA-PAA binding was found to take place only at pH 5 and an ionic strength ≤ 0.15 M, and it was reversible at higher pH or ionic strength where the complex was shown to be dissociated.

The dissociation has been also revealed to occur when the complex is put in a medium not containing PAA. The evaluation of the complexation stoichiometry suggested that 4 HSA molecules could be bound to each PAA100kDa chains as assumed in the equilibrium given below:

Besides, the occurrence and the reversibility of this binding has been shown for different PAA sizes. Moreover, the evaluation of the HSA-PAA complexes size has evinced that the number of HSA bound to each PAA chain was related to the size of the latter.

After that, functionalized PVDF membrane were prepared by coating PS-b-PAA copolymers onto the membrane surface to investigate the interactions stated above. For that, the modification process has been first optimized (procedure of coating, coating time, etc.). Thus, the physical adsorption of copolymers due to hydrophobic interactions of the anchoring PS block with the hydrophobic PVDF membrane has been an effective technique for the membrane surface modification. In fact, the presence of the copolymer layer was confirmed every time using FTIR before the study of proteins adsorption onto the modified membrane.

General conclusions

Chapter V

Subsequently, the adsorption of HSA on PS-b-PAA coated membrane was shown using FTIR to be higher than its deposition onto PVDF pristine membrane (PM). This interaction was disclosed to be once again specific to HSA because IgG exhibited a different behaviour, which adsorbs similarly on PVDF PM as on PS-b-PAA coated membrane. In fact, besides the electrostatic interactions assumed to be the driving force of the HSA-PAA complexation in solution, the hydrophobic interactions were revealed to play also a role on HSA adsorption onto PS-b-PAA coated PVDF membrane. As a matter of fact, the HSA adsorption was shown to take place at pH 7 mostly due to hydrophobic interactions between hydrophobic pockets of HSA and the PS, but also due the electrostatic interactions of HSA with PAA. Then, the adsorption has been evinced to be enhanced at pH 5 owing to increased electrostatic interactions after the reduction of the HSA overall negative charge (isoelectric point ≈ 4.7) to come closer to the negatively charged PAA chains (pKa = 4.5). The ionic strength effect assessment confirmed then the major role of the attractive electrostatic interactions on the HSA adsorption onto the PS-b-PAA coated membrane. In fact, the adsorption was greater at an ionic strength ≤ 0.15 M, and it was mitigated at higher ionic strength. Furthermore, the HSA adsorption was not related to the PAA size as it was the case for HSA-PAA complexation in solution.

As for the adsorption of HSA onto PS-b-PEO coated membrane in static mode, it has been mitigated compared to its adsorption onto PVDF pristine membrane. In fact, the hydration layer that could be formed by PEO has been suggested to be the barrier that prevents the adsorption of proteins. However, this barrier was not sufficient to preclude the HSA deposition onto the same membrane during dead-end filtration operations. Indeed, the HSA deposition was found to increase slightly on PS-b-PEO coated membrane compared to that on PM suggesting that the pressure applied on HSA solution helped overcoming the hydration layer created by PEO.

Otherwise, the same trend of HSA adsorption on PS-b-PAA coated membrane obtained in static mode has been observed during dead-end filtration operations. However, the deposition was greater due to the flow of HSA solution which maybe helped the HSA getting more in contact with the membrane surface, and so increase its interactions with the copolymer and raise the deposited amount due to the accumulation of HSA. Eventually, the assessment of the membrane transport properties unveiled that the membrane permeability and its selectivity can be tuned with the size of the copolymer used for coating.

General conclusions

Appendices

A. Supporting information on analysis of confocal microscope images

Appendices

A. Supporting information on analysis of confocal microscope images

The quantitative analysis of the confocal microscope images is exposed in SI-Table 1 given below. The images are shown in section 6.3 of chapter I, and they were performed to explore the adhesion of platelets on polystyrene well plates modified with triblock PS-PAA and with random PS-PEG copolymers.

SI-Table 1. Quantitative analysis of the confocal microscope images performed using Image J software for the adhesion of the platelets on polystyrene (PS) well plate modified with triblock PS-PAA and with random PS-PEG copolymers.

Parameter

Appendices

B. Supporting information on SAXS data analysis

The SAXS data (I(q) being the scattering intensity obtained from SAXS measurement) have been analysed to obtain the main properties of the HSA and the HSA-PAA complex. The Python home code used for different calculations presented in Chapter II of the manuscript is found in the following link: https://github.com/CharafMerzougui/Supporting-information/blob/master/Porod%20volume %20estimation %20-%20modif%20.ipynb

Determination of the radius of gyration and I(0)

As stated in section 3.2.1 of chapter II, the radius of gyration Rg is the average root-meansquare distance to the center of density in the molecule weighted by the scattering length density as illustrated in SI-Figure 1.

SI-Figure 1. Illustration of the radius of gyration meaning Thus, Rg is obtained by applying the Guinier law given by following equation at small q:

Close to q=0, the scattering intensity of a particle is described by a Gaussian curve. The Guinier law is equivalent of a linear variation of Ln I(q) vs q 2 (Guinier plot), providing Rg and I(0). The code given in the link above performs the curve fitting to obtain the values of Rg and I(0) for the 10 first values of q. Here q is then less than 0.012 and should then met the criteria q.Rg < 1.5 if Rg is less than 125 nm. It is important to note that in the different sections of Chapter II, Rg was evaluated using Foxtrot or Primus program that consider the Guinier approximation given above, and not the Python code.

B. Supporting information on SAXS data analysis 2. Determination of the Porod volume 203

It can be checked from Fig. 1 and Fig. 2 that the data fitting is correct: there is no deviation from the straight line indicating no intermolecular interaction or aggregation in this concentration range.

Determination of the Porod volume

The calculations of the Porod volume exposed in section 4.4 of chapter II were carried out for all the studied HSA/PAA molar ratio (1.5 to 12) and were performed using the Python code mentioned earlier. The table below summarize the figures that show the estimation of radius of gyration Rg, the constant K as well as the invariant Q, which allow then the evaluation of the Porod volume for the studied range of HSA/PAA molar ratio.

Calibration curves for UV-visible analysis

In order to evaluate the amount of the copolymer attached to the membrane during the modification process, some calibration curves of the absorbance versus the concentration of the copolymer have been established using UV-visible spectroscopy. For that, the absorbance of different standard solutions with known concentration of copolymer were measured. Hence, the concentration of the remain copolymer in the coating bath can be calculated after the membrane is removed. Thus, the mass of the copolymer attached to the membrane surface can be then evaluated as stated in section 3.3.1 of Chapter III by mass balance using the following equation:

The same procedure has been used to evaluate the amount of the protein adsorbed onto the membrane surface.

Therefore, below are exposed the calibration curves (SI-Figure 3 C. Supporting information on characterization of surface modification and protein adsorption 3. Python code used for FTIR mapping analysis 209 Appendices

Python code used for FTIR mapping analysis

The Python code that allowed to convert the color-coded maps with different rainbow scales into maps coded with same grey scale and permitted then to evaluate the mean intensities for the chosen peaks is given below.