<p>This thesis describes results from fundamental studies of liposomes intended for drug delivery and pH or temperature triggered release. In addition, the effect of lipid composition on bilayer disc formation and a potential application of the bilayer discs were investigated.</p><p>The lower pH encountered by endocytosed liposomes can be utilized to trigger drug release. The mechanisms behind cytosolic drug delivery were investigated using two different kinds of pH-sensitive liposomes. The results indicate that incorporation of non-lamellar forming lipids into the endosome membrane may allow for drug escape into the cytosol.</p><p>Temperature-sensitive liposomes containing lysolipid (LTSL) release their content almost instantly when heated to temperatures close to the gel to liquid crystalline phase transition temperature (T<i>C</i>). Morphological changes of the liposomes in response to temperature cycling were studied. Temperature cycling induced liposome openings and disintegration of the liposomes into bilayer discs. Incubation of LTSL in the presence of multilamellar liposomes (MLVs) resulted in relocalisation of lysolipid into the MLVs, which affected the rapid release from LTSL. We propose that the presence of micelle-forming components, such as lysolipids and PEG-lipids, facilitates the formation of defects and membrane openings during the initial phase of membrane melting, resulting in the observed rapid release. Similar to added lysolipids, also hydrolysis generated lysolipids induce disc-formation upon heating through T<i>C</i> of the lipid mixture.</p><p>Two fundamentally different micelles may form in PEG-lipid/lipid mixtures. We found that discoidal structures are preferred over cylindrical micelles when the mixture contains components that reduce the spontaneous curvature, increase the monolayer bending modulus, or reduce PEG-lipid/lipid miscibility. The large discoidal micelles found at low PEG-lipid content are better described as bilayer discs. We evaluated such discs as model membranes in drug partitioning studies, and suggest that they, in some cases, produce more accurate data than liposomes.</p>

The gastric mucosa is frequently exposed to endogenously secreted hydrochloric acid of high acidity. Gastric mucosal defense mechanisms are arranged at different levels of the gastric mucosa and must work in unison to maintain its integrity. In this thesis, several mechanisms underlying gastric mucosal resistance to strong acid were investigated in anesthetized rats and mice. The main findings were as follows: Only when acid secretion occurred did the pH gradient in the mucus gel withstand back-diffusion of luminal acid (100 mM or 155 mM HCl), and keep the juxtamucosal pH (pHjm) neutral. Thus, when no acid secretion occurred and the luminal pH was 0.8-1, the pH gradient was destroyed. Bicarbonate ions, produced concomitant with hydrogen ions in the parietal cells during acid secretion and blood-borne to the surface epithelium, were carried transepithelially through a DIDS-sensitive transport. Prostaglandin-dependent bicarbonate secretion seemed to be less important in maintaining a neutral pHjm. Removal of the loosely adherent mucus layer did not influence the maintenance of the pHjm. Hence, only the firmly adherent mucus gel layer, approximately 80µm thick, seemed to be important for the pHjm. Staining of the mucus gel with a pH-sensitive dye revealed that secreted acid penetrated the mucus gel from the crypt openings toward the gastric lumen only in restricted paths (channels). One crypt opening was attached to one channel, and the channel was irreversibly formed during acid secretion. Gastric mucosal blood flow increased on application of strong luminal acid (155 mM HCl). This acid-induced hyperemia involved the inducible but not the neural isoform of nitric oxide synthase. These results suggest a novel role for iNOS in gastric mucosal protection and indicate that iNOS is constitutively expressed in the gastric mucosa. It is concluded that a pH gradient in the gastric mucus gel can be maintained during ongoing acid secretion, since the acid penetrates the mucus only in restricted channels and bicarbonate is carried from the blood to the lumen via a DIDS-sensitive transporter.

Los biotensioactivos son compuestos producidos por microorganismos que poseen actividad superficial, baja toxicidad, carácter biodegradable y efectividad a temperaturas y valores de pH extremos. Los ramnolípidos, son un tipo de biotensioactivos con interesantes actividades biológicas, que parecen estar relacionadas con su interacción con las membranas. En esta tesis doctoral se estudia la interacción de la fracción dirramnolipídica purificada de cultivos de Pseudomonas aeruginosa con membranas modelo y membranas biológicas, la interacción con albúmina de suero bovino, utilizada como proteína modelo, y por último, se evalúa el papel de este biotensioactivo en la estabilización de liposomas sensibles a pH y su aplicación en la introducción de compuestos en células animales. Para el desarrollo de este trabajo de investigación se han utilizado técnicas como calorimetría, FT-IR, difracción de rayos X, dispersión de luz dinámica y microscopía electrónica. / Rhamnolipids are bacterial biosurfactants produced by Pseudomonas spp. These compounds have been shown to present several interesting biological activities. It has been suggested that the interaction with the membrane could be the ultimate responsible for these actions. This thesis presents a detailed molecular level study of the dirhamnolipid interactions with artificial phospholipids membranes and biological membranas, and the dirhamnolipid interactions with bovine serum albumin, used as a general globular protein model. Furthemore, this work includes the characterization of phosphatidylethanolamine/diRL pH-sensitive liposomes described as efficient systems for cytoplasmic delivery of foreign compounds into living cells. It has been employed a number of physical techniques such as calorimetry, FTIR, small angle X-ray (SAX) diffraction and dynamic light scattering.

This thesis describes results from fundamental studies of liposomes intended for drug delivery and pH or temperature triggered release. In addition, the effect of lipid composition on bilayer disc formation and a potential application of the bilayer discs were investigated. The lower pH encountered by endocytosed liposomes can be utilized to trigger drug release. The mechanisms behind cytosolic drug delivery were investigated using two different kinds of pH-sensitive liposomes. The results indicate that incorporation of non-lamellar forming lipids into the endosome membrane may allow for drug escape into the cytosol. Temperature-sensitive liposomes containing lysolipid (LTSL) release their content almost instantly when heated to temperatures close to the gel to liquid crystalline phase transition temperature (TC). Morphological changes of the liposomes in response to temperature cycling were studied. Temperature cycling induced liposome openings and disintegration of the liposomes into bilayer discs. Incubation of LTSL in the presence of multilamellar liposomes (MLVs) resulted in relocalisation of lysolipid into the MLVs, which affected the rapid release from LTSL. We propose that the presence of micelle-forming components, such as lysolipids and PEG-lipids, facilitates the formation of defects and membrane openings during the initial phase of membrane melting, resulting in the observed rapid release. Similar to added lysolipids, also hydrolysis generated lysolipids induce disc-formation upon heating through TC of the lipid mixture. Two fundamentally different micelles may form in PEG-lipid/lipid mixtures. We found that discoidal structures are preferred over cylindrical micelles when the mixture contains components that reduce the spontaneous curvature, increase the monolayer bending modulus, or reduce PEG-lipid/lipid miscibility. The large discoidal micelles found at low PEG-lipid content are better described as bilayer discs. We evaluated such discs as model membranes in drug partitioning studies, and suggest that they, in some cases, produce more accurate data than liposomes.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal

Cell-surface engineering has been attracting increased interest in the field of biotechnology, tissue engineering, cell therapy, or biosensors/bioelectronics. Thin nanocoatings or sometimes referred as nanoshells allow for modifying and controlling variety of cell properties, specifically retardation of cell division or growth, masking immunological properties, providing chemical and mechanical resistance to external stressors, and ability to further functionalize shells in order to guide cells attachment, their proliferation and function in artificial environment. Bottom-up approach, utilizing layer-by-layer (LbL) assembly of wide variety of different components (synthetic and natural polyelectrolytes, nanoparticles, and other nano-structures) has been introduced and elaborated to modify cell surfaces. Despite successful examples of the LbL-based cell encapsulation with polyelectrolytes, cytotoxicity of their polycation components possesses severe limitations for this approach. Additionally, by constructing rigid non-permeable shells can suppress the essential properties of cells. In this view, the goal of this research is to explore the formation of cyto-compatible ultrathin coatings from synthetic and natural polymers through utilization of non-cationic counterparts, with possibility to actively control cell division, provide protection from external environment, and temper shell properties in order to elicit or change specific cell response.

<div>Wounds are created in soft and hard tissue through surgery or disease. As the wound heals, the tissue is held in place using sutures or staples for soft tissue or plates, pins, or screws for hard tissues. These fixation methods inherently damage the surrounding healthy tissue. Surgical adhesives are a non-damaging alternative to these methods. In order to be effective, surgical adhesives must be biocompatible,</div><div>adhere strongly in a moist environment, and have mechanical properties similar to those of the native tissue.</div><div><br></div><div><div>To address the design criteria for surgical adhesives, we look to nature to find inspiration from compounds that provide these properties. Mussels use catechol-based</div><div>molecules to adhere to surfaces in wet and turbulent environments. Incorporating catechols into polymer systems can provide adhesion even in moist biological environments.</div><div>Mimics of elastomeric proteins from soft tissue can be used as backbones for soft and flexible adhesive systems. In particular, elastin-inspired proteins have a well-defined modular sequence that allows for a range of design choices. In this work, we explored the behavior of elastin- and mussel-inspired natural and synthetic polymers in biologically relevant environments.</div></div><div><br></div><div><div>First, the cytocompatibility of a catechol-containing poly(lactic acid) (cPLA) hard tissue adhesive was studied. The cPLA polymer was reacted with iron- or periodatebased</div><div>crosslinkers and compared to PLA. Fibroblasts grown directly on cPLA or cultured with leachate from cPLA had high viability but slower growth than cells on PLA. The periodate crosslinker was significantly cytotoxic, and cells grown on cPLA crosslinked with periodate had reduced metabolism and slowed growth. Cells grown on or in leachate from iron-crosslinked cPLA had similar viability, metabolism, and growth to cells on or in leachate from cPLA. The iron-crosslinked cPLA is a promising</div><div>cytocompatible adhesive for hard tissue applications.</div></div><div><br></div><div><div>Second, two elastin-like proteins (ELP) were developed that had pH-sensitive properties in solution and when crosslinked into hydrogels. Both ELPs had a large number of ionizable tyrosine and lysine residues, and one design also had a large number of ionizable histidine and aspartic acid residues. The stiffness of the hydrogels was maximized at pH values near the isoelectric point of the protein. The stoichometric ratio of crosslinker used affected hydrogel stiffness but did not significantly alter the pH-sensitivity of the gel. The crosslinked gel shrank when swelled at physiological pH. The pH-sensitive mechanical properties of hydrogels made from the two ELPs did not vary significantly. The tyrosine and lysine residues in one ELP were also</div><div>chemically blocked through acetylation to lower the isolectric point of the protein. The acetylated hydrogels had maximum stiffness at a pH near the isoelectric point of the acetylated ELP. The stiffness of both the native and acetylated gels were within the range of soft tissue. Through a combination of crosslinker ratio and chemical modification, the pH-responsive properties of the elastin-inspired hydrogels could be tuned.</div></div><div><br></div><div><div>Finally, adhesive proteins were created that were inspired by both elastin and mussels. An ELP was modified to include catechol groups (mELP). The ELP and mELP were optimized for adhesive use in a soft tissue system. A warm and humid environment was used to study the adhesion of these proteins on pig skin. Iron and (hydroxymethyl) phosphine crosslinkers increased the adhesive strength of both proteins, and periodate increased the adhesive strength of mELP. The adhesive strengths of the proteins were maximized when mELP was mixed with iron or when either protein were mixed with (hydroxymethyl)phosphine crosslinkers. These maximized adhesives were 12-17 times stronger than a commercially available sealant. In addition,</div><div>the iron and mELP adhesive formulation achieved high adhesive strengths even when cured for only ten minutes. This adhesive formula shows promise for adhesive</div><div>applications on soft tissue.</div></div>

Beaucoup d’applications en ingénierie demandent l’utilisation de matériaux intelligents qui peuvent se déformer en réponse à un stimulus extérieur. C’est dans ce contexte, que s’est posé ce projet de recherche. Bénéficiant d’un environnement pluridisciplinaire, grâce à l’association de deux axes de l'Institut Pascal : l’axe MMS (Mécanique, Matériaux et Structures) et l’axe PHOTON (Axe Photonique, Ondes, Nanomatériaux), cette thèse s’intègre parfaitement dans l’action transversale "Matériaux et Modélisations multi-échelles" du laboratoire. La première partie de ce travail s'appuie sur un système expérimental mis au point par une équipe américaine [Chang_10] qui permet la mesure sans contact du pH d'une solution en exploitant les caractéristiques photoniques du système. Ce système est composé d'un réseau d'hydrogel fixé sur un substrat rigide. Un modèle numérique est développé dans le but de simuler le fonctionnement de l'ensemble et d'optimiser le réseau d'hydrogel en vue d'applications dans le domaine médical. La seconde partie de ce travail concerne le développement d'une théorie sur le comportement mécanique de polymères sensibles à la lumière. L'objectif est d'établir une relation liant la déformation du matériau à l’intensité lumineuse. Les résultats obtenus sont comparés avec les résultats expérimentaux issus de la littérature. L'influence des interactions entre les molécules d'azobenzènes sur la déformation du matériau est étudiée. / Many engineering applications involve stimuli-responsive materials that can change their shape under the action of an external stimulus. It is in this context that this project takes place. Thanks to a multidisciplinary environment with the association of two lines of research of the Institut Pascal: the Mechanical area (Mechanic, Materials and structure) and the Photonic area (Nanostructures and Nanophotonics), this PhD perfectly fits with the “Materials and multi-scale Modeling” transversal action of the laboratory. The first part of this work relies on an experimental system developed by an American team [Chang_10] which allows to measure the pH of a solution without contact, making use of its photonic characteristics. This system is composed of a hydrogel network fixed on a rigid substract. A numerical model is developed in order to simulate its behavior and optimize the hydrogel network with a view to applications in the medical domain. The second part of this PhD is related to the development of a theory on the mechanical behavior of photo-sensitive polymers. The aim is to establish a link between the material deformation and the light intensity. The obtained results are compared to experimental ones from literature. The interaction influence of the azobenzenes molecules on the material strain is studied.

Il a été démontré que l’acidité de l’environnement tumoral jouait un rôle dans la résistance aux chimiothérapies. L’utilisation de nanovecteurs, tels que les nanocapsules lipidiques (LNC), permet non seulement d’améliorer le temps de biodistribution de substances actives, mais aussi de cibler l’environnement tumoral tout en protégeant les actifs de cet environnement acide. L’objectif de cette thèse porte ainsi sur l’optimisation et l’évaluation de LNC furtives et pH-sensibles dans le contexte du mélanome.Dans un premier temps, ces travaux ont consisté à caractériser la vascularisation de mélanomes humain et murin. Ces études ont permis de comparer différentes tumeurs (densité, taille et structure) et de déterminer si l’usage de nanomédecines est approprié dans ce contexte.La seconde partie s’est orientée sur l’élaboration de polymères combinant furtivité et pH-sensibilité. Ces copolymères composés de N-vinylpyrrolidone (NVP)et de vinylimidazole ont été synthétisés par polymérisation RAFT et post-insérés à la surface des LNC. Ces modifications ont donné lieu à des LNC présentant des charges de surface pH-dépendantes,entrainant une augmentation de leur internalisation à pH acide dans des cellules de mélanome. Finalement, des études de biodistribution ont mis en évidence l’intérêt de la NVP dans le développement de nanovecteurs furtifs. En conclusion, les copolymères développés ont permis de prolonger le temps de circulation, mais aussi d’apporter des propriétés pH-sensibles qui pourraient améliorer l’internalisation tumorale des LNC in vivo et donc de potentialiser l’effet d’une thérapie anticancéreuse. / Tumor acidity has been shown to play a major role in resistance to chemotherapy. The use of nanomedicines, as lipid nanocapsules (LNC), allows to protect drugs from this acidic environment. They can also improve the biodistribution of therapeutics and to target the tumor environment. The aim of this thesis concerns the evaluation and characterization of stealth and pH-sensitive LNC in the context of melanoma. Firstly, these works consisted in characterizing the vascularization of human and mice melanoma. These studies allowed to compare different tumors (density, size and structure), and determine if the used of nanocarrier is suitable in the context of melanoma.The second part of this thesis described the development and the characterization of new copolymers, combining stealth and pH-sensitive properties. These copolymers, composed of Nvinylpyrrolidone(NVP) and vinylimidazole, were synthesized by RAFT polymerization and were post in sertedonto LNC surface. These modifications allowed to obtain charge reversal nanocarriers, leading to increase their melanoma cell uptake underacid pH. Finally, biodistribution of these modified nanoparticles was studied in vivo and highlighted the interest of NVP in the development of stealth nanocarriers. To conclude, the developed copolymers able to extend nanocarrier circulation time and to provide pH-responsive properties which should increase the tumor internalization of LNC invivo and potentiate the effect of anticancer drugs.

<p>Physico-chemical characterisation of structure and stability of liposomes intended for drug delivery is the central issue in this thesis. In addition, targeted liposomes to be used in boron neutron capture therapy (BNCT) were developed.</p><p>Lysolipids and fatty acids are products formed upon hydrolysis of PC-lipids. The aggregate structure formed upon mixing lysolipids, fatty acids and EPC were characterised by means of cryo-TEM. A relatively monodisperse population of unilamellar liposomes was detected in mixtures containing equimolar concentration of the three components. </p><p>The interactions between alternative steric stabilisers (PEO-PPO-PEO copolymers) and conventional PC-and pH-sensitive PE-liposomes were investigated. Whereas the PE-liposomes could be stabilised by the PEO-PPO-PEO copolymers, the PC-liposomes showed an enhanced permeability concomitant with the PEO-PPO-PEO adsorption.</p><p>Permeability effects induced by different PEG-stabilisers on EPC liposomes were shown to be dependent on the length of the PEG chain but also on the linkage used to connect the PEG polymer with the hydrophobic membrane anchor.</p><p>An efficient drug delivery requires, in most cases, an accumulation of the drug in the cell cytoplasm. The mechanism behind cytosolic drug delivery from pH-sensitive liposomes was investigated. The results suggest that a destabilisation of the endosome membrane, due to an incorporation of non-lamellar forming lipids, may allow the drug to be released. </p><p>Furthermore, sterically stabilised liposomes intended for targeted BNCT have been characterised and optimised concerning loading and retention of boronated drugs. </p>