En raison de leur haute spécificité d’interaction, les protéines thérapeutiques sont de plus en plus utilisées et représentent une part majoritaire du marché pharmaceutique. Néanmoins, ces molécules sont fragiles et leur stabilité est une problématique majeure pour l'industrie pharmaceutique. La dégradation des protéines thérapeutiques peut survenir à chaque étape de leur cycle de vie : production, stockage, transport et administration au patient. Les modifications chimiques, l'exposition à des forces de cisaillement (fort débit fluidique), la température, le pH et les interactions avec les matériaux et/ou les interfaces gazeuses sont autant de facteurs qui peuvent nuire à la stabilité de ces protéines. De plus, l'utilisation croissante de dispositifs médicaux automatisés pour la manipulation et l'injection de protéines thérapeutiques augmente drastiquement le risque de dégradation. Dans cette thèse, nous étudions l’effet et le rôle de la triple interface solide/liquide/air sur l'agrégation des protéines. Ce phénomène se produit fréquemment dans les procédés de manipulation d’une solution de protéines thérapeutiques (cavitation, agitation…). Lors d’un mouillage intermittent, les interfaces air/liquide et liquide/solide se confondent en une seule et même interface appelée triple interface ou ligne triple. La ligne triple est une zone favorisant fortement l'agrégation des protéines. Notre étude, basée sur l’insuline, montre que la ligne triple cause une accumulation progressive de protéines qui déclenche, après une période de nucléation, leur agrégation, précisément à l’endroit de cette ligne triple. Nos résultats démontrent aussi que les forces de cisaillement, seules, n’entrainent pas l’agrégation de l’insuline. De plus, nous observons que la diminution de la tension superficielle (induite par l’ajout de polysorbates) d'une solution de protéines réduit le risque de formation d’agrégats. En conclusion de ce travail, nous proposons des recommandations pour la conception des dispositifs médicaux de préparation et d’administration de protéines thérapeutiques. / Due to the high specificity of their interactions, proteins are increasingly used in therapy and represent a vast majority of the global pharmaceutical market. Nevertheless, these molecules are fragile and therapeutic protein stability is a major concern in pharmaceutical industry. Protein degradation and aggregation can occur at every step during production, storage, transport and delivery. In this thesis, we interrogate the possible role of intermittent wetting in protein aggregation. Intermittent wetting frequently occurs in protocols involving pumping (cavitation), agitation, and liquid handling. During intermittent wetting, the air/liquid and liquid/solid interfaces meet at a triple line or triple interface, which is a local trigger for protein aggregation because it concentrates the mechanical action of the recessing fluid on the surface adsorbed proteins. We study the effect of surface intermittent wetting on insulin aggregation. Our results demonstrate that the triple interface line, where an air/water interface meets a hydrophobic surface, allows progressive protein accumulation, and finally triggers local insulin aggregation. We also show that shear stress, alone, is not detrimental for protein stability. Additionally, Additives such as polysorbates were tested, showing that the modification of the surface tension of a protein solution impacts its ability to form aggregates. Based on this work, we propose recommendations for the design of drug delivery and preparation devices in order to limit the risk of protein aggregation at the triple interface.

<p>The acceptance of blood contacting implants creating favorable conditions <i>in vivo</i> is decisively determined by their interaction with proteins that mediate inter cellular interactions with synthetic substrates. Adsorbed proteins can activate blood cascade systems like coagulation and complement that may result in serious blood clots, and/or immunological reactions. Poly (ethylene glycol) (PEG), heparin, and phosphoryl choline (PC) functional poly (methacrylates) are previously used polymers with known non-adhesive properties in blood contacting events.</p><p>This thesis contributes to this extensive research by introducing a novel type of biomaterial that equips biodegradable polymers with biomimetic functionalities. The phospholipid mimetic material is synthesized by combining biodegradable polymers with various functional polar end-groups consisting of zwitterionic phosphoryl choline (PC), anionic succinates, and cationic quaternary ammonium. The polymer backbone provides mechanical stability and biodegradability whilst the various head groups provide a variety of functions. The careful evaluation of the synthesis has allowed reaction conditions to be optimized leading to complete conversion at each step and subsequently high yields. Initially, poly (e-caprolactone) (PCL) was used since it provided a suitable synthetic starting point. However, the synthesis has also included poly (trimethylene carbonate) (PTMC) to provide a material that allows spontaneous surface enrichment of the polar PC group. This was achieved with an added hydrophilic environment. </p><p>Through the synthesis of multi PC functional PTMC, additional bulk organisation by the formation of zwitterionomers (PC ionomer) was achieved. Low modulus elasticity and water uptake were some of the properties of the formed material. As a result it was shown that the PC ionomer could be used for protein/drug loading and subsequent release. Furthermore, the material possessed non-adhesive properties in different biological environments.</p><p>Importantly, the result suggests that a versatile synthetic platform has been established that may provide a smorgasbord of different functional polymers, or combinations of such. This is indeed important since it was shown that the polymer in many ways dictates how the material may take advantage of an added functionality. </p><p>Such materials should be interesting for a variety of biomedical applications including the production of soft hemocompatible tissue.</p>

<p>The interactions between polymers and components of biological systems are an important area of interest within the fields of tissue engineering, polymer chemistry, medicine and biomaterials. In order to create such a biomimetic material, it must show the inherent ability to reproduce or elicit a biological function. How do we design synthetic materials in order to direct their interactions with biological systems?</p><p>This thesis contributes to this research with aspects of how polymers interact with biological materials with the help of ionic interactions. Polyesters, biodegradable or not, may after a hydrolytic cleavage interact ionically with protonated amines by the liberated carboxylate functions. Amines are found in proteins and this fact will help us to anchor proteins to polyester surfaces. Another type of interaction is to culture cells in polymeric materials, i.e. scaffolds. We have been working on compliant substrates, knitted structures, to allow cell culture in three dimensions. A problem that arises here is how to get a high cell seeding efficiency? By working on the interactions between polymers, proteins and finally cells, it is possible to create a polarized protein membrane that allows for very efficient cell seeding, and subsequent three dimensional cell cultures. Finally a synthetic route to taylor interaction was developed. Here a group of polymers known as ionomers were synthesized. In our case ionic end groups have been placed onto biodegradable polycarbonates, we have created amphiphilic telechelic ionomers. Functionalization, anionic or cationic, changes the properties of the material in many ways due to aggregation and surface enrichment of ionic groups. It is possible to add functional groups for a variety of different interactions, for example introducing ionic groups that interact and bind to the complementary charge of proteins or on the other hand one can chose groups to prevent protein interactions, like the phosphorylcholine zwitterionomers. Such interactions can be utilized to modulate the release of proteins from these materials when used in protein delivery applications. The swelling properties, Tg, degradation rate and mechanical properties are among other things that will easily be altered with the choice of functional groups or backbone polymer.</p>

The acceptance of blood contacting implants creating favorable conditions in vivo is decisively determined by their interaction with proteins that mediate inter cellular interactions with synthetic substrates. Adsorbed proteins can activate blood cascade systems like coagulation and complement that may result in serious blood clots, and/or immunological reactions. Poly (ethylene glycol) (PEG), heparin, and phosphoryl choline (PC) functional poly (methacrylates) are previously used polymers with known non-adhesive properties in blood contacting events. This thesis contributes to this extensive research by introducing a novel type of biomaterial that equips biodegradable polymers with biomimetic functionalities. The phospholipid mimetic material is synthesized by combining biodegradable polymers with various functional polar end-groups consisting of zwitterionic phosphoryl choline (PC), anionic succinates, and cationic quaternary ammonium. The polymer backbone provides mechanical stability and biodegradability whilst the various head groups provide a variety of functions. The careful evaluation of the synthesis has allowed reaction conditions to be optimized leading to complete conversion at each step and subsequently high yields. Initially, poly (e-caprolactone) (PCL) was used since it provided a suitable synthetic starting point. However, the synthesis has also included poly (trimethylene carbonate) (PTMC) to provide a material that allows spontaneous surface enrichment of the polar PC group. This was achieved with an added hydrophilic environment. Through the synthesis of multi PC functional PTMC, additional bulk organisation by the formation of zwitterionomers (PC ionomer) was achieved. Low modulus elasticity and water uptake were some of the properties of the formed material. As a result it was shown that the PC ionomer could be used for protein/drug loading and subsequent release. Furthermore, the material possessed non-adhesive properties in different biological environments. Importantly, the result suggests that a versatile synthetic platform has been established that may provide a smorgasbord of different functional polymers, or combinations of such. This is indeed important since it was shown that the polymer in many ways dictates how the material may take advantage of an added functionality. Such materials should be interesting for a variety of biomedical applications including the production of soft hemocompatible tissue.

The interactions between polymers and components of biological systems are an important area of interest within the fields of tissue engineering, polymer chemistry, medicine and biomaterials. In order to create such a biomimetic material, it must show the inherent ability to reproduce or elicit a biological function. How do we design synthetic materials in order to direct their interactions with biological systems? This thesis contributes to this research with aspects of how polymers interact with biological materials with the help of ionic interactions. Polyesters, biodegradable or not, may after a hydrolytic cleavage interact ionically with protonated amines by the liberated carboxylate functions. Amines are found in proteins and this fact will help us to anchor proteins to polyester surfaces. Another type of interaction is to culture cells in polymeric materials, i.e. scaffolds. We have been working on compliant substrates, knitted structures, to allow cell culture in three dimensions. A problem that arises here is how to get a high cell seeding efficiency? By working on the interactions between polymers, proteins and finally cells, it is possible to create a polarized protein membrane that allows for very efficient cell seeding, and subsequent three dimensional cell cultures. Finally a synthetic route to taylor interaction was developed. Here a group of polymers known as ionomers were synthesized. In our case ionic end groups have been placed onto biodegradable polycarbonates, we have created amphiphilic telechelic ionomers. Functionalization, anionic or cationic, changes the properties of the material in many ways due to aggregation and surface enrichment of ionic groups. It is possible to add functional groups for a variety of different interactions, for example introducing ionic groups that interact and bind to the complementary charge of proteins or on the other hand one can chose groups to prevent protein interactions, like the phosphorylcholine zwitterionomers. Such interactions can be utilized to modulate the release of proteins from these materials when used in protein delivery applications. The swelling properties, Tg, degradation rate and mechanical properties are among other things that will easily be altered with the choice of functional groups or backbone polymer.

La génération de cellules souches pluripotentes induites (iPSC) est très prometteuse en médecine régénérative, pour la modélisation physiopathologique et le criblage de nouveaux médicaments. A l’origine, des cellules somatiques ont été reprogrammées en iPSC par l'expression forcée de facteurs de transcription (FT) impliqués dans les cellules souches embryonnaires. Depuis, de nombreuses lignées d’iPSC ont été générées mais les vecteurs actuels les plus représentés et efficaces pour exprimer les FT sont les virus intégratifs. Ils comportent du matériel génétique. Des stratégies alternatives ont été développées dans un contexte de sécurisation et de transfert clinique mais sont ont encore besoin d’être acceptées par les comités d’éthique. La méthode la plus sûre et rationnelle serait alors d’apporter ces FT directement sous forme protéique mais le défi est de traverser les membranes. Dans ce contexte, notre laboratoire a développé un peptide de pénétration cellulaire (CPP) basé sur le FT ZEBRA du virus d’Epstein-Barr. La séquence impliquée dans la prise en charge cellulaire a été caractérisée au laboratoire et se nomme MD (Minimal Domain). Elle est capable de vectoriser des protéines et des biomolécules de haut poids moléculaire via un mécanisme indépendant de l'endocytose, permettant leur internalisation sous une forme biologiquement active. Dans ce projet, nous avons produit et purifié les protéines Oct4, Sox2, Nanog, Lin28, Klf4 et c-Myc chacune fusionnée au CPP MD. Ce domaine n'interfère pas avec la capacité d'Oct4 à lier sa séquence cible d’ADN. Le traitement in vitro de cellules primaires conduit à l’internalisation des protéines MD en 30 minutes à 1 heure. MD-Oct4 et MD-Nanog peuvent être localisés au noyau en 3 heures. Dans un contexte de reprogrammation, la combinaison de MD-Oct4, MD-Sox2, MD-Nanog et MD-Lin28 lors de traitements répétés conduit à l'activation transcriptionnelle de gènes cibles composant le réseau de pluripotence. / The generation of induced Pluripotent Stem Cell (iPSC) holds great promise for regenerative medicine, disease modelling and drug screening. Leading the original cell to an iPSC has been originally made by the forced expression of Transcription Factors (TF) involved in embryonic stem cells. Since the discovery of those mechanisms, many teams have engineered iPSC by well-defined cell culture tools such as the use of retroviruses in order to express TF. Those techniques use genetic material. Delivery techniques have evolved but most of reprogramming experiments still need TF. Development of alternative strategies has been conducted in a context of clinical application but still needs to be accepted by ethics comities. Thus, the use of recombinant proteins instead of genetic material is safe and rational but the challenge is to access the intracellular medium. In this context, our laboratory has developed a cell-penetrating peptide (CPP) based on the Epstein-Barr virus ZEBRA TF. The sequence implicated in cellular uptake has been characterized and is named MD (Minimal Domain). It is able to translocate high molecular weight proteins in an endocytosis-independent mechanism, allowing the internalization of cargos in fully biologically active form. Here we develop 6 MD fusions at the N-terminus of the following TF: Oct4, Sox2, Klf4, cMyc, Nanog & Lin28. This domain does not interfere with Oct4 capacity to associate with its own DNA sequence. Moreover, MD fused proteins transduce in vitro treated cells in 30 minutes to 1 hour ; MD-Oct4 & MD-Nanog can be localized in the nucleus after 3 hours only. In a context of reprogramming experiences, the combination of MD-Oct4, MD-Sox2, MD-Nanog and MD-Lin28 in repeated treatment leads to the activation of target genes transcription such as those constituting the pluripotency network.

Proteins are a diverse category of biomolecules with great therapeutic potential. Intracellular delivery of proteins can augment the deficient activities of dysfunctional or poorly expressed innate proteins and therefore represents a promising strategy to treat the associated diseases. One major barrier to intracellular protein delivery is the translocation of the protein across the cellular membrane. Endocytosis provides an important pathway for protein nanocarriers to enter cells across the plasma membrane. However, the cargo protein must then promptly escape from the endosomes to avoid degradation in the lysosome and to exert its cellular function. Previously, we reported a cationic lipid-coated magnesium phosphate nanoparticle (LPP) system for intracellular protein delivery. The intracellular delivery of catalase, an antioxidant enzyme, by LPP protected MCF-7 cells from a lethal level of exogenous H2O2 and lowered the reactive oxygen species (ROS) levels in EA.hy926 cells. These findings prompted us to further develop LPP to evaluate its protein delivery in animals. Two categories of LPP formulations, catalase-encapsulated (CE) LPP and catalase-complexed (CC) LPP, were successfully prepared by a modular approach. Catalase-encapsulated liposomes (CE LP) were prepared by hydrating a thin-film of lipids with catalase solution followed by extrusion. However, extrusion of CE LP resulted in substantial loss of catalase activity. Catalase-complexed liposomes (CC LP) were prepared by first extruding cationic liposomes with a LIPEX extruder and then mixing with catalase solution. The resultant CC LP was much smaller than CE LP and preserved all the catalase activity. Magnesium phosphate nanoparticles (MgP NP) were prepared by the microemulsion precipitation technique. CE LP or CC LP were mixed with MgP NP to yield LPP formulations (CE LPP or CC LPP, respectively). The formulations were then rendered isotonic with glucose (5% w/v). Transmission electron microscopy (TEM) confirmed the proposed structure of LPP comprising a shell of lipid bilayers with a core of MgP NP. Furthermore, TEM showed drastic morphological changes of LPP formulations at acidic pH, consistent with an osmotic explosion. The LPP formulations were administered by intravenous or intranasal routes to CD-1 mice. LPP formulations of fluorescently labeled catalase distributed substantially into the lung following intranasal administration, whereas intravenous administration of the same formulations caused catalase distribution mainly into the liver. In addition, intranasal administration of both the LPP formulations yielded higher pulmonary catalase activity and lowered the ROS levels in the healthy lung compared to free catalase solution. Based on these results, LPP’s antioxidant effects were further evaluated in mice with lipopolysaccharide-induced acute lung injury (ALI). Lack of LPP distribution into the lung following intranasal administration indicated that intranasal dosing did not deliver catalase substantially into inflamed lungs. In corroboration, the inflammatory biomarker tumor necrosis factor-alpha (TNF-α) remained unchanged after intranasal dosing of LPP formulations. Intratracheal dosing of LPP formulations delivered the fluorescently labeled catalase deep into the lung and significantly reduced TNF-α production in the inflamed lungs compared to free catalase solution. CC LPP, which was smaller and which better preserved catalase activity than CE LPP, showed greater intrapulmonary catalase activity compared to CE LPP in both healthy and inflamed lungs. Taken together, LPP represents a promising nanocarrier for intracellular protein delivery.

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Introduction Oral delivery of proteins faces numerous challenges due to their enzymatic susceptibility and instability in the gastrointestinal tract. In recent years, the polyelectrolyte complexes have been explored for their ability to complex protein and protect them against chemical and enzymatic degradation. However, most of the conventional binary polyelectrolyte complexes (PECs) are formed by polycations which are associated with toxicity and non-specific bio-interactions. The aim of this thesis was to prepare a series of ternary polyelectrolyte complexes (APECs) by introduction of a polyanion in the binary complexes to alleviate the aforementioned limitations. Method Eight non-insulin loaded ternary complexes (NIL APECs) were spontaneously formed upon mixing a polycation [polyallylamine (PAH), palmitoyl grafted-PAH (Pa2.5), dimethylamino-1-naphthalenesulfonyl grafted-PAH (Da10) or quaternised palmitoyl-PAH (QPa2.5)] with a polyanion [dextran sulphate (DS) or polyacrylic acid (PAA)] at 2:1 ratio, in the presence of ZnSO4 (4μM). A model protein i.e., insulin was added to a polycation, prior to addition of a polyanion and ZnSO4 to form eight insulin loaded (IL) APECs. PECs were used as a control to compare APECs. The complexes were characterised by dynamic light scattering (DLS) and transmission electron microscope (TEM). In vitro stability of the complexes was investigated at pH (1.2-7.4), temperature (25˚C, 37˚C and 45˚C) and ionic strength (NaCl-68mM, 103mM and 145mM). Insulin complexation efficiency was assessed by using bovine insulin ELISA assay kit. The in vitro cytotoxicity was investigated on CaCo2 and J774 cells by MTT (3-4,5 dimethyl thialzol2,5 diphenyl tetrazolium bromide) assay. All complexes were evaluated for their haemocompatibility by using haemolysis assay, oxidative stress by reactive oxygen species (ROS) assay and immunotoxicity by in vitro and in vivo cytokine generation assay. The potential of the uptake of complexes across CaCo2 cells was determined by flow cytometry and fluorescent microscopy. The underlying mechanism of transport of complexes was determined by TEER measurement, assessment of FITC-Dextran and insulin transport across CaCo2 cells. 15 Results NIL QPa2.5 APECs (except IL QPa2.5-DS) exhibited larger hydrodynamic sizes (228-468nm) than all other APECs, due to the presence of bulky quaternary ammonium moieties. QPa2.5 APECs exhibited lower insulin association efficiency (≤40%) than other APECs (≥55%) due to a competition between the polyanion and insulin for QPa2.5 leading to reduced association of insulin in the complexes. DS based APECs generally offered higher insulin association efficiency (≥75%) than PAA based APECs (≤55%) due to higher molecular weight (6-10kDa) of DS. In comparison to other complexes, Pa2.5 PECs and APECs were more stable at varying temperature, ionic strength and pH due to the presence of long palmitoyl alkyl chain (C16) which reduced the chain flexibility and provided stronger hydrophobic association. The cytotoxicity of polycations on CaCo2 and J774 cells is rated as PAH>Da10=Pa2.5>QPa2.5. The introduction of PAA in Pa2.5 and Da10 brought most significant improvement in IC50 i.e., 14 fold and 16 fold respectively on CaCo2 cells; 9.3 fold and 3.73 fold respectively on J774 cells. In comparison to other complexes, Da10 (8mgml-1) induced higher haemolytic activity (~37%) due to a higher hydrophobic load of 10 percent mole grafting of dansyl pendants. The entire range of APECs displayed ≤12% ROS generation by the CaCo2 cells. The degree of in vitro TNFα production (QPa2.5≥Da10≥Pa2.5=PAH) and in vitro IL-6 generation (QPa2.5≥Pa2.5=PAH≥Da10) by J774 cells established an inverse relationship of cytotoxicity with the cytokine generation. Similar to MTT data, the introduction of PAA in APECs brought more significant reduction in in vitro cytokine secretion than DS based APECs. Pa2.5-PAA brought the most significant reduction in both in vitro and in vivo cytokine generation. All the formulations were able to significantly reduce original TEER, however did not demonstrate appreciable paracellular permeation of a hydrophilic macromolecular tracer of paracellular transport i.e., FITC Dextran. The uptake study revealed internalisation of APECs predominantly by a transcellular route. Transcellular uptake of IL QPa2.5 (≤73%), IL QPa2.5-DS (67%) was higher than their NIL counterparts, whereas the uptake of NIL Pa2.5 (≤89%), NIL Pa2.5-PAA (42%) was higher than their IL counterparts. Conclusion In essence, amphiphilic APECs have shown polyanion dependent ability to reduce polycation associated toxicity and they are able to facilitate transcellular uptake of insulin across CaCo2 cells.