Dissertationen zum Thema „Interaction of Dendrimers and Liposomes“

In considering the concept of surface-immobilised liposomes as a drug release system, two factors need to addressed, the interfacial surface density of the liposomes for maximum drug loading and the stability of these liposomes to allow for controlled drug release. This thesis investigates a multilayer system for the affinity immobilisation of liposomes and their stability to various applied stresses. In the work presented here an allylamine monomer was used to create plasma coatings that were stable, thin and amine-rich. The aging studies using AFM showed these films to rapidly oxidise on exposure to water. The freshly deposited films were used for further surface modifications, by the covalent grafting of PEG layers of different interfacial densities under the conditions of varying polymer solvation. The AFM was used to measure the interaction forces between the grafted PEG layers and modified silica interfaces. It was found that the polydispersity of the PEG species resulted in bridging interactions of ???brush???-like PEG layers with the silica surface. These interactions were screened minimised by increasing the ionic strength of the solution. Although the densely grafted PEG layers were found to be highly protein-resistant by the XPS and QCM-D some minor protein-polymer adhesions were observed by the AFM. The densely anchored biotinylated PEG chains served as an optimum affinity platform for affinity-docking of NeutrAvidinTM molecules, which assembled in a rigid, 2-D layer as confirmed by the QCM-D. The submonolayer surface density of NeutrAvidin, as determined by Europium-labelling, was attributed to steric hindrance of the immobilised molecules. The final protein layer enabled specific binding of biotin-PEG-liposomes as a highly dissipative, dense and stable layer verified by tapping mode AFM and QCM-D. We found that these liposomes were also stable under a range of stresses induced by the shearing effects of water, silica probe and HSA layer at increased loads and velocities. The frictional response of the liposome layer also demonstrated the viscoelasticity and stability of these surface immobilised liposomes. Finally, the minimal adhesive interaction forces, as measured by the AFM, demonstrated the repellency of these liposomes to commonly found proteins, such as HSA.

La créatine kinase mitochondriale (CKmt) est une protéine octamérique qui, in vivo, est associée à la face externe de la membrane mitochondriale interne. La liaison de l'enzyme, via des lysines C-terminales, à des liposomes contenant des phospholipides acides affecte la fluidité membranaire et entraîne une modification conformationnelle de la CKmt. Nous avons étudié par la technique de Langmuir couplée à la microscopie à l'angle de Brewster le comportement interfacial de la protéine en l'absence et en présence de phospholipides. En l'absence de monocouche, elle s'adsorbe spontanément à l'interface air/eau et doit donc posséder des zones hydrophobes accessibles. En présence de monocouches constituées de phospholipides acides ou zwitterioniques, à chaînes saturées ou insaturées, les isothermes ont un profil différent selon la présence ou non de charges négatives et la pression de surface au moment de l'injection. La présence de phospholipides acides facilite l'adsorption de la protéine

Les protéines à ancre glycosylphosphatidyl inositol (GPI) sont ancrées dans le feuillet externe des membranes plasmiques. L'objectif du travail a été de caractériser le comportement de la phosphatase alcaline (AP) à ancre GPI dans des monocouches, en présence ou en absence de lipides, à l'interface air/eau et dans des liposomes. Nous avons étudié le comportement de la phosphatase alcaline seule à l'interface air/eau en utilisant la technique de Langmuir et la spectroscopie IR de modulation (PM-IRRAS). L'AP avec l'ancre GPI présente une plus forte affinité pour l'interface air/eau que la forme dépourvue de ce motif. Dans les deux cas, l'activité enzymatique est conservée au niveau de la surface. Les résultats obtenus à partir de l'analyse des isothermes de compression de Langmuir ou des spectres IR suggèrent une orientation particulière de l'AP/GPI à l'interface air/eau telle que le site actif est dirigé vers le milieu aqueux. On a également mis en évidence des mouvements réversibles de domaines intramoléculaires qui pourraient rendre compte des propriétés allostériques de ces enzymes. La microscopie à angle de Brewster a permis de mettre en évidence la formation réversible d'agrégats résultant d'interactions protéine-protéine très fortes. L'insertion de la protéine dans des monocouches de phospholipides se fait en plusieurs étapes et dépend de la pression de surface initiale de la monocouche. De plus, l'interfaction de la protéine avec les phospholipides diffère selon la nature de la tête polaire. Par PM-IRRAS, des mouvements de domaines sont également observés montrant que le comportement de la protéine est sans doute identique en présence ou en l'absence de phospholipides. La phosphatase alcaline insérée dans des liposomes montre une hétérogénéité de distribution à la surface des liposomes et la protéine peut être mesurée, ce qui correspond à la longueur de l'ancre.

Le ciblage par glycosylation, que nous avons appliqué à un médicament antitumoral : la daunorubicine (DNR) dont la cardiotoxicité limite l'usage thérapeutique, consiste à greffer sur le principe actif des structures glucidiques susceptibles d'être spécifiquement reconnues par les lectines membranaires des cellules-cibles. Ayant retenu comme modèles d'étude des cellules possédant les récepteurs des asialoglycoprotéines : les hépatocytes de rat et les cellules d'hépatome humain HepG2, nous avons choisi, comme vecteurs, l'orosomucoïde désialylé et ses glycopeptides. Ces glycoconjugués ont été greffés sur la DNR par l'intermédiaire du bras de couplage Glc-S-Et-Arg-Leu. Nous avons tiré parti des propriétés de fluorescence de la DNR pour analyser les produits de couplage synthétisés et étudier l'interaction entre le médicament libre ou glycosylé et les cellules. L'utilisation des techniques de microspectrofluorimétrie LASER (Microsonde Moléculaire, microfluorimètre à détection multicanale) a permis de mettre en évidence l'internalisation, par les cellules HepG2, de la DNR couplée aux glycoconjugués ainsi que la modification du mode de pénétration de la DNR couplée au glycopeptides d'asialoorosomucoïde par rapport à la DNR libre. Ces résultats sont en faveur d'une endocytose de la DNR "glycosylée" par l'intermédiaire du récepteur spécifique du galactose des cellules HepG2. Des expériences préliminaires ont été effectuées pour mettre au point la méthodologie d'étude de la destinée in vivo de la DNR libre ou couplée et pour étudier certains des mécanismes liés à l'activité ou à la toxicité de l'anthracycline (interaction avec l'ADN, interactions avec certains organes). Nos résultats démontrent donc que le ciblage cellulaire de substances actives est parfaitement réalisable par une glycosylation spécifique de ces dernières.

PONTIFÍCIA UNIVERSIDADE CATÓLICA DO RIO DE JANEIRO
COORDENAÇÃO DE APERFEIÇOAMENTO DO PESSOAL DE ENSINO SUPERIOR
CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO
PROGRAMA DE SUPORTE À PÓS-GRADUAÇÃO DE INSTS. DE ENSINO
PROGRAMA DE EXCELENCIA ACADEMICA
Norfloxacina (NFX) é um antibiótico fluorescente de amplo espectro bacteriológico, membro da família das fluorquinolonas (FQs). A interação das FQs com íons metálicos pode incrementar a ação bactericida e agir contra a resistência das bactérias frente aos antibióticos. Diversos exemplos de complexos mistos de norfloxacina e Cu(II) podem ser encontrados na literatura. A compreensão dos mecanismos moleculares de interação desses antibióticos com os diferentes componentes das células e com nanopartículas de ouro, como transportadores do fármaco, é extremamente importante. Para entender essas interações, neste trabalho utilizamos diferentes técnicas espectroscópicas, como espectroscopia de fluorescência estacionária e resolvida no tempo, absorção de luz UV-Visível e ressonância paramagnética eletrônica (RPE). A interação de NFX e do complexo ternário Cu(II):L:NFX, onde o ligante L é a 1,10-fenantrolina (Phen) ou 2,2-bipiridina (Bipy), com micelas e lipossomas unilamelares pequenos de fosfatidilcolina de ovo (PC) foi estudada usando espectroscopia de fluorescência em estado estacionário e resolvida no tempo. Foi estudada a estabilidade dos complexos ternários formados em micelas. Foram obtidas constantes de estabilidade no interior de micelas de SDS, as quais mostraram valores muito maiores do que em tampão. Já os espectros de RPE deram maiores detalhes sobre a estrutura dos complexos e confirmaram a formação do complexo ternário dentro das micelas. Foram estudadas as interações de FQs e seus complexos de cobre com lipossomas de PC preparados com diferentes densidades superficiais de carga elétrica negativa. No estudo da interação de FQs com nanopartículas de ouro sintetizadas por ablação a laser (nanocompósitos, AuNCs), NFX mostrou maiores mudanças, tanto na absorbância como na fluorescência, do que as FQs ciprofloxacina (CFX) e levofloxacina (LFX). Os resultados sugerem mudanças no índice de refração na superfície dos AuNCs, por associação com o fármaco e/ou formação de aglomerados como resultado da interação. Observou-se também uma supressão lenta, porém significativa, na fluorescência da NFX, sem mudança na posição do pico, indicando que NFX mantém o seu estado inicial de protonação ligando-se à superfície dos AuNCs. Também foi observada a liberação de FQs ligadas à superfície de AuNPs mediante substituição por tióis, que ocasiona recuperação parcial da fluorescência da fluorquinolona. Por último, como o surfactante aniônico SDS se mostrou promissor na interação com NFX, em comparação com surfactantes catiônicos e neutros, e como os AuNCs são estáveis em SDS, estudamos a interação de NFX com AuNCs sintetizados em presença de SDS e, para comparação, com AuNCs colocados em solução de SDS após a síntese.
Norfloxacin (NFX) is a fluorescent antibiotic of broad bacteriological spectrum, member of the fluoroquinolone (FQ) family. The interaction of FQs with metal ions can increase the bactericidal action and act against antibiotics bacterial resistance. Several examples of mixed-ligand norfloxacin Cu (II) complexes can be found in the literature. Understanding the molecular mechanisms of interaction of these antibiotics with different cell components and with gold nanoparticles as drug transporters is extremely important. To clarify these interactions, we used different spectroscopic techniques, such as steady-state and time-resolved fluorescence spectroscopy, UV-Visible light absorption, and electron paramagnetic resonance (EPR). The interaction of NFX and the ternary complex Cu(II):L:NFX, where the L is the ligand 1,10-phenanthroline (Phen) or 2,2-bipyridine (Bipy), with micelles and small unilamellar liposomes of egg phosphatidylcholine (PC) was studied using steady-state and time-resolved fluorescence spectroscopy. The stability of the ternary complexes formed in micelles was studied, and stability constants were obtained inside SDS micelles, which showed values much larger than in buffer. The EPR spectra gave further details on the structure of the complexes, and confirmed the formation of the ternary complex inside the micelles. The interactions of FQs and their copper complexes with PC liposomes prepared with different surface densities of negative electrical charge were studied. In the study of the interaction of FQs with gold nanoparticles synthesized by laser ablation (nanocomposites, AuNCs), NFX showed greater changes than FQs ciprofloxacin (CFX) and levofloxacin (LFX) in both absorbance and fluorescence. The results suggest changes in the surface refractive index of the AuNCs and/or cluster formation, as result of the interaction with the drug. A slow but significant quenching of NFX fluorescence was also observed, with no change in peak position, indicating that NFX maintains its initial state of protonation by binding to the surface of the AuNCs. Release of FQs attached to the surface of AuNCs by thiols has also been observed, which causes partial recovery of FQ fluorescence. Finally, as the anionic surfactant SDS showed to be promising in the interaction with NFX, compared to cationic and neutral surfactants, and because the AuNCs are stable in SDS, we studied the interaction of NFX with AuNCs synthesized in the presence of SDS and, for comparison, with AuNCs placed in SDS solution after synthesis.

Les analogues nucléosidiques peuvent être associés de manière covalente avec des lipides afin d’améliorer leurs propriétés pharmacocinétiques. C’est dans ce contexte qu’a été découvert le concept de « squalénisation »: le couplage covalent du squalène confère des propriétés particulières aux molécules auxquelles il est lié, leur permettant de s’auto-assembler en nanoparticules. Nous nous sommes intéressés à l’un de ces dérivés: la gemcitabine-squalène qui possède une activité antinéoplasique plus importante que celle de la gemcitabine libre. Ce travail a permis (i) de caractériser l’organisation supramoléculaire des nanoparticules de gemcitabine-squalène, (ii) d’identifier les interactions moléculaires du squalène, de la gemcitabine et de la gemcitabine-squalène avec une membrane modèle, (iii) de modéliser l’interaction des nanoparticules avec une membrane et (iv) d’encapsuler la prodrogue dans des liposomes, dont l’activité thérapeutique a été testée in vivo
Nucleoside analogues may be associated covalently with lipids to improve their pharmacokinetics. It’s in this context that the concept of "squalénisation" has been discovered: the covalent coupling of squalene, a natural lipid precursor of cholesterol, confers specific properties to the molecules bound with it, allowing them self-assemble into nanoparticles. We have been interested in one of these prodrugs: gemcitabine-squalene which has got a greater antineoplastic activity than that of free gemcitabine. These thesis work has allowed (i) to characterize supramolecular organization of nanoparticles of gemcitabine-squalene, (ii) to identify the molecular interactions of squalene, of gemcitabine and of gemcitabine-squalene with a membrane model, (iii) to model the interaction of nanoparticles with a membrane and (iv) to encapsulate the prodrug into liposomes, which therapeutic activity was tested in vivo on a solid tumor model

Les triterpènes sont un groupe important et structurellement diversifié de produits naturels issus du squalène. Les progestatifs et les glucocorticoïdes sont des triterpènes oxygénés ayant un squelette tétracyclique et reconnus pour leurs diverses propriétés thérapeutiques. Par ailleurs, l'érythrodiol et l'uvaol sont des triterpènes pentacycliques reconnus pour leurs effets bénéfiques dans l'alimentation humaine. Dans ce travail, l'interaction avec les membranes des vésicules lipidiques et la liaison à la sérum albumine humaine de ces molécules sont étudiées dans le but de mieux comprendre leurs propriétés pharmacologiques. Les vésicules lipidiques ont été caractérisées par DSC, spectroscopie Raman, FTIR et polarisation de fluorescence du DPH pour comprendre l'effet des molécules sélectionnées sur la fluidité membranaire.Également, nous avons étudié la liaison du cholestérol, d'une série de progestatifs et de glucocorticoïdes à l'albumine humaine par la spectroscopie de fluorescence.Les résultats ont démontré que les progestatifs, les glucocorticoïdes, l'érythrodiol et l'uvaol altèrent les propriétés physiques de la bicouche lipidique.Les progestatifs et les glucocorticoïdes démontrent un attachement modéré à l'albumine. Par ailleurs, la dydrogestérone présente la constante de liaison la plus importante. Enfin, notre étude a démontré que la constante de liaison du cholestérol à l'albumine est faible en comparaison avec les autres molécules étudiées. Notre étude a conduit à une connaissance approfondie des mécanismes moléculaires et des caractéristiques structurales impliqués dans l'interaction des triterpènes avec les protéines et les membranes synthétiques
The triterpenoids are a large and structurally diverse group of natural products derived from squalene. Progesterone derivatives and glucocorticoids are a group of oxygenated triterpenes having a tetracyclic skeleton and identified for their therapeutic properties. Whereas, erythrodiol and uvaol are pentacyclic triterpenes, known for their beneficial effects on human diet. In this thesis, we studied their interaction with the membranes of lipid vesicles and with human serum albumin to better understand their pharmacological properties. DSC, Raman spectroscopy, FTIR and fluorescence polarization of DPH were used to investigate the effect of triterpenes on the membrane fluidity. Besides, we used fluorescence spectroscopy to study the binding of cholesterol, a series of progesterone derivatives and another series of glucocorticoids to albumin. The results revealed that progesterone derivatives, glucocorticoids, erythrodiol and uvaol changed the physical properties of the bilayers. Progesterone derivatives and glucocorticoids have been proven to bind moderately to albumin. Dydrogesterone showed the highest binding constant. Finally, our study demonstrated that cholesterol exhibited a much weaker interaction with albumin compared to progesterone derivatives and glucocorticoids.Our work has led to a better understanding of triterpenes molecular mechanisms of their interaction with proteins and biological membranes and structural features controlling these interactions

La créatine kinase mitochondriale (CKmt) appartient à la famille des phosphagènes kinases, c'est un enzyme qui, contrairement aux isoformes cytoplasmiques exclusivement dimériques, existe également sous forme octamérique. In vivo, seule cette dernière est associée à la face externe de la membrane mitochondriale interne, probablement par des liaisons électrostatiques impliquant la cardiolipine. Les isoenzymes de la CK forment des complexes structuraux et fonctionnels impliqués dans le transfert d'énergie entre les sites producteurs et utilisateurs d'ATP. Afin d'étudier la protéine dans un environnement proche de son milieu naturel, nous avons tenté de caractériser les interactions entre la CKmt recombinante de cœur de lapin et des liposomes unilamellaires constitués de phospholipides naturels saturés ou insaturés renfermant un phospholipide anionique. Des mesures de taille et de potentiel zêta indiquent que la fixation de la protéine sur les liposomes peut induire la formation d'agrégats stables. L'utilisation de la spectroscopie de fluorescence et de la spectroscopie infrarouge à transformée de Fourier, a permis de mettre en évidence un changement réversible de structure tant au niveau des phospholipides (diminution de la fluidité des chaînes acyles) que la CKmt (réduction de mobilité d'un ou plusieurs tryptophanes, diminution du rapport hélice " sur feuillet β) à la suite de l'adsorption de l'enzyme sur les liposomes. Cette adsorption induit la formation de nouvelles structures β intramoléculaires, et tend à protéger la pp̂rotéine contre la dénaturation thermique. Parallèlement, des mesures de spectroscopie infrarouge différentielle associée à l'utilisation de substrats photo activables ont permis de détecter des modifications de la structure secondaire de la protéine soluble lors de la fixation de substrats adényliques. La fixation de ces derniers provoque la désorption de la CKmt adsorbée sur les liposomes. Cette technique a en outre permis de vérifier l'intégrité de la protéine dans la complexe CKmt-liposomes.

Les solvants eutectiques profonds (DES) sont récemment apparus comme une nouvelle classe de solvants verts présentant un potentiel élevé pour remplacer les solvants organiques usuels. Bien que découverts récemment, les DES ont fait l'objet de nombreuses recherches au cours des dernières années en raison de leurs propriétés intéressantes. Cependant, il reste encore beaucoup à découvrir étant donné le nombre quasiment illimité de DES potentiels et de leur polyvalence. Notre étude vise à examiner l'effet des DES sur les liposomes, adoptés comme modèles membranaires, et sur les membranes cellulaires. Elle a également cherché à évaluer la capacité de solubilisation des DES envers des composés bioactifs volatils. Ainsi, une sélection de DES ainsi que de nouveaux solvants ont été tout d'abord préparés et caractérisés. Des mesures de densité, de viscosité et de polarité ont été effectuées et ont montrées que les propriétés des DES pouvaient être ajustées en fonction de leur composition. L'organisation des phospholipides et des liposomes au sein des DES a ensuite été étudiée à l'aide de microscopies optique et à force atomique. Les phospholipides s'auto-assemblent en vésicules dans les DES à base de chlorure de choline tandis que les liposomes se convertissent en bicouches lipidiques avant leur reconstitution en vésicules. De plus, des études de cytotoxicité et des examens morphologiques ont été combinés afin d'évaluer l'impact de quelques DES sur MDA-MB-231, une lignée cellulaire de cancer du sein humain. Les résultats ont montrés que l'effet dépendait fortement de la composition du DES. D'autre part, la capacité de solubilisation des DES envers des composés bioactifs volatils a été testée par chromatographie en phase gazeuse couplée à un espace de tête. L'influence de la présence d'eau et de certains systèmes d'encapsulation tels que les liposomes et les cyclodextrines sur la capacité de solubilisation des DES ont été analysés. Enfin, la libération du trans-anéthole à partir des DES a été suivie par extraction multiple de l'espace de tête. Les DES ont été capables de mieux solubiliser les composés bioactifs volatils et de contrôler leur libération par rapport à l'eau. Dans l'ensemble, ces travaux mettent en évidence l'utilisation potentielle des systèmes à base de DES comme véhicules de solubilisation de composés bioactifs
Deep eutectic solvents (DES) recently emerged as a novel class of green solvents with a high potential to replace common organic solvents. Despite their novelty, DES were extensively explored in the past years owing to their remarkably interesting properties. Yet, a lot remains to be uncovered given the limitless number of possible DES and their versatility. The current sudy aimed to examine the effect of DES on liposomes, adopted as model membranes, and on cell membranes. It also sought to evaluate the solubilizing ability of DES toward bbioactive volatile compounds. Therefore, a group of selected DES along with new solvents were first prepared and characterized. Density, viscosity and polarity measurements were mainly carried out and showed that DES' properties can be tuned depending on their composition. The organization of phospholipids and liposomes within the DES was then investigated using optical- and atomical force microscopies. Phospholipids self-assembled into vesicles in choline chloride-based DES while liposomes converted to lipid bilayers before their reconstitution into vesicles. Moreover, cytotoxicity studies and morphological examinations were combined to evaluate the impact of some DES on MDA-MB-231, a human breast cancer cell line. Results showed that the effect is highly dependent on the DES' composition. On the other hand, the solubilizing ability of the DES toward bioactive volatile compounds was tested using static headspace-gas chromatography. The influence of the presence of water and some encapsulation systems such as liposomes and cyclodextrins on the overall DES' solubilization efficiency was further analyzed. At last, the release of trans-anethole from the DES was monitored via multiple headspace extraction. DES were able to greatly solubilize the bioactive volatile compounds and to control their release when compared with water. Altogether, this work highlights the potential use of the DES-based systems as solubilization vehicles for bioactive compounds

Les interactions protéines-carbohydrates sont à la base de nombreux processus biologiques physiologiques aussi bien que pathologiques. Ces interactions incluent la synthèse et la dégradation enzymatique des oligosaccharides, la cohésion des tissus, l'immunité, le cancer ou encore l'infection bactérienne et virale. L'inhibition de ce type d'interaction par des molécules multivalentes synthétiques telles les glycopolymères, glycodendrimères, glycoclusters, etc. fait l'objet d'études importantes depuis plusieurs décennies. L'obtention de telles molécules pourrait permettre de développer de nouvelles thérapies qui pourraient palier notamment la multi-résistance aux antibiotiques. De plus, la détection de telles interactions par des méthodes simples et faciles à mettre en oeuvre permettrait une amélioration de la compréhension de ces phénomènes, ainsi que le diagnostic rapide de la présence de microorganismes. C'est dans ce contexte, que nous avons développé une nouvelle classe de composés glycosylés multivalents à coeur triazine. Ces glycoclusters de basse valence, ont la particularité de présenter une double fonctionnalité : l'inhibition d'interactions lectine-sucre par des effets de multivalence ainsi que la détection de ces interactions. Nous présentons dans ce manuscrit, la synthèse d'une nouvelle famille de glycoclusters polysulfurés à coeur triazine portant des épitopes saccharidiques tels que D-glucose, D-galactose, D-mannose, L-fucose, ainsi que leurs évaluations biologiques réalisées sur des lectines de Pseudomonas aeuriginosa. Nous avons ainsi mis en évidence la possibilité de reconnaître et de détecter les interactions lectine-sucre dans un premier temps par association d'un cluster mixte portant un fluorophore, et de façon plus sophistiquée, grâce à un système à géométrie variable incorporant dans le scaffold même un switch photochimique variant l'arrangement des sucres dans l'espace
Protein-carbohydrate interactions mediate a wide range of biochemical processes. Amongst these is the process of bacterial infection, which often proceeds through carbohydrate-binding lectins involved in biofilm formation. Even if the individual associations result from weak interactions, the assembly of multiple carbohydrate-protein interactions, typically more than additive, confers to the system the required specificity and avidity for their biological functions. In order to study this « glycocluster effects », a number of scaffold systems presenting multivalent carbohydrate ligands have been prepared in the literature. Dendrimers, polymers, peptides, calixarenes, to name a few, have been used as core molecules for the synthesis of multivalent glycoconjugates. The purpose of this work is to design new glycoclusters which exhibit dual functionality: the inhibition of carbohydrate-protein interactions via a multivalency effect; and detection of the interactions via fluorescence spectroscopy. A first generation of polysulfurated glycoclusters, organized around a heteroaromatic core, was synthesized using click chemistry reactions, which provided a family of highly soluble and readily accessible clusters. The glycoclusters were evaluated for their ligand-lectin interactions, multivalency effects, thermodynamic parameters, and abilty to modulate biofilm formation by Pseudomonas aeruginosa, a major causative agent of lung infections in cystic fibrosis patients. We describe a new family of ‘switchable glycoclusters’ based on photochromic behavior. They are designed to generate a modulated fluorescence signal as well as a defined change in the three-dimensional arrangement of the sugar epitopes, and may eventually provide significantly improved probes for studying the distribution, dynamics, interactions, and activities of specific lectins

Au cours de cette thèse, nous avons étudié la possibilité d’utiliser des cibles membranaires pour le développement de nouveaux médicaments antipaludiques. Nous avons également proposé un protocole original pour l’étude du mécanisme d’action de certaines molécules à activité antipaludique. Nos travaux reposent sur la caractérisation à l’échelle moléculaire et nanométrique des interactions entre les molécules antipaludiques et des modèles membranaires mimant les membranes parasitaires. En utilisant diverses techniques biophysiques, nous avons montré que la sphingomyéline des membranes du Plasmodium pourrait être une cible intéressante pour les molécules potentiellement antipaludiques comme la Cyclosporine A. De plus, nous avons mis en évidence l’importance des membranes lipidiques dans le mécanisme de détoxification de l’hématine que le parasite met en œuvre en incorporant ces molécules dans un cristal inerte : l’hémozoïne. Ainsi, les observations AFM ont permis de visualiser pour la première fois et en temps réel la formation de ce cristal dans une bicouche lipidique. Enfin, nous avons montré que l’association des molécules antipaludiques avec l’hématine pourrait inhiber la formation de l’hémozoïne en empêchant l’insertion de l’hématine dans les membranes (cas de la chloroquine) ou en augmentant le pouvoir membranotrope de cette molécule (cas des dérivés pipéraziniques de l’acide ursolique)
In this thesis, we have studied the possibility to use membrane targets for the development of new antimalarial drugs. Furthermore, we have proposed an original protocol to study the mechanism of action of certain antimalarial drugs. Our work is based on the characterization at the molecular and nanoscale levels of the interactions between antimalarial drugs and membrane models mimicking parasite membrane. Indeed, using various biophysical techniques, we have shown that sphingomyelin membranes of Plasmodium could be an attractive target for many potential antimalarial compounds such as Cyclosporin A. In addition, we have demonstrated the importance of lipid membranes in the hematin detoxification that is implementing carried out by the parasite by incorporating these molecules in an inert crystal inert called hemozoin. Thus, the AFM observations have allowed us to visualize for the first time and in real time the formation of this crystal in a lipid bilayer. Finally, we have showed that the combination of antimalarial drugs with hematin could inhibit the formation of hemozoin by inhibiting of the insertion of hematin in the membranes (e. G. As in chloroquine) or by the increasing of the membranotrope effect of hematin (for e. G. Derivatives of piperazine ursolic acid derivatives)

This thesis focuses on the design, synthesis and development of dynamic multivalent nanostructures such as supramolecular dendrimers, liposomes and gold-functionalized nanostructures. These structures can be used for drug delivery and molecular sensing applications. This thesis is divided into three parts: In part one, a general introduction to self-assembly, dynamic systems, metalligand exchange, nanostructured dendritic scaffolds, liposomes and gold nanostructures is given. In part two, a microwave approach is presented as an efficient method for the regioselective deuteration of bipyridine scaffolds. Dynamic systems based on transition metal-bipyridine coordination complexes were investigated. The compositional self-adaptation and kinetics of these dynamic systems were successfully assessed by ESI-MS. Based on this amphiphilic dendrimers/metallodendrimers were also designed and synthesized via  a convergent strategy. Their ability to self-assemble into supramolecular assemblies and their controlled disassembly was effectively demonstrated. In part three, two types of drug delivery systems based on dynamic multivalent nanostructures of glycodendrimers/metalloglycodendrimers and drugpresenting liposomes were developed. The dynamic self-assembly of these architectures into supramolecular nanostructures with site-specific functionality through interacting carbohydrate or cholesterol moieties was assessed. The host-guest interaction/encapsulation and controlled release with external stimuli were studied using a fluorescent probe, as well as selected drug molecules. The antibacterial property of the drug delivery systems was also evaluated, demonstrating an enhanced bactericidal activity. A new, rapid and simple approach for the functionalization of plasmonic gold nanostructured surfaces was also developed. The optical performance and light-specific sensitivity of the fluorescent probe on the resulting nanostructures were also presented.

QC 20151119

Nesta tese serão discutidos alguns aspectos químicos, fotoquímicos e fotofísicos importantes no desenvolvimento de fotossensibilizadores para aplicação em terapia fotodinâmica (TFD). Os estudos abrangeram a investigação de 29 espécies, sendo que 20 delas continham um anel porfirínico convencional e 9 eram porfirinas duplamente N-confusas. As primeiras foram sintetizadas para apresentarem diferentes números e tipos de substituintes catiônicos coordenados à posição meso do anel porfirínico, mais especificamente as meso(N-4-piridil)fenilporfirinas e as meso(N-3-piridil)fenilporfirinas contendo um, dois (em trans), dois (em cis), três e quatro grupos [Ru(bipy)2Cl]+ ou CH3+ (figura 1). Em contraste com as porfirinas convencionais. as outras 9 estruturas consistiram das formas protonadas, neutras e desprotonadas de porfirinas que exibem dois anéis pirrólicos adjacentes voltados para fora do anel porfirínico, mais especificamente a base livre (H2N2CP) e os respectivos complexos de prata (AgHN2CP) e de cobre (CuHN2CP). Esta peculiaridade estrutural permite coordenar metais de transição por meio de dois átomos de carbono e dois de nitrogênio, estabilizando fortemente íons metálicos em estados de oxidação anormalmente elevados como Ag3+ e Cu3+. Os dois nitrogênios pirrólicos externos ao anel são susceptíveis tanto a protonação quanto a desprotonação, possibilitando a modulação das propriedades eletrônicas desses compostos por simples alteração no pH do meio. De um modo geral, a acidificação das amostras perturbou mais significativamente suas propriedades fotoquímicas que a desprotonação. Em especial, as espécies neutras metaladas apresentaram bandas bastante intensas na região fototerapêutica de 600 a 750 nm, além de um elevado rendimento quântico de formação de oxigênio singlete (ΦΔ > 0,90 para o complexo Ag(III)). Todavia, um processo de fotodecomposição atribuído ao ataque do 1O2 formado, também foi observado. Ambos os processos, ΦΔ e fotodecomposição, parecem ser modulados pelo metal coordenado no centro do anel. Tanto a coordenação de Ag(lII) como Cu(lII) aumentou o ΦΔ, provavelmente devido ao efeito do metal pesado sobre o cruzamento interssistema. Todavia, verificou-se que o derivado AgHN2CP é muito menos reativo que o CuHN2CP e a base livre. Os motivos deste comportamento ainda não são completamente compreendidos, mas é evidente que o complexo de Ag(lII) apresenta melhores propriedades fotodinâmicas que as demais. Em comparação com as porfirinas duplamente N-confusas, as demais 20 porfirinas catiônicas são muito mais solúveis em água. Neste caso, o enfoque dos experimentos foi direcionado às aplicações biológicas propriamente ditas. Assim, além das propriedades fotofísicas associadas à formação de oxigênio singlete (1O2), também foram exploradas a influência do número dos substituintes periféricos sobre a ligação e danos fotooxidativos provocados em eritrócitos, lipossomos, mitocôndrias e células cancerígenas. Os resultados de ΦΔ mostraram ser inversamente proporcionais ao número de cargas positivas, para as porfirínas contendo grupos N-4metilpiridíniom. Estas diferenças foram consistentes com os efeitos de agregação, causados pela formação de dímeros ou oligômeros, no caso das espécies mais hidrofóbicas. Os coeficientes de partição em n-octanol/água (logPOA) corroboraram estes resultados, apresentando uma dependência inversamente proporcional ao número de cargas positivas ou proporcional ao número de resíduos fenila. As eficiências de ligação à bicamada lipídica mitocondrial, lipossomal e celular e os processos de peroxidação lipídica fotoinduzidos mostraram uma dependência proporcional aos valores de logPOA, exceto para as porfirinas contendo complexos de rutênio. O comportamento não linear neste último caso pode estar associada a dissociação da ligação rutênio-porfirina, devido a elevada força iônica das soluções fisiológicas utilizadas. Neste caso, a descoordenação dos complexos periféricos deve promover uma drástica diminuição da solubilidade do composto. Por este motivo os experimentos foram conduzidos somente com as espécies metiladas. No caso, de mitocôndrias isoladas verificou-se que a interação também apresenta uma razoável participação do potencial de membrana. De fato, quando a mitocôodria estava energizada foi observado um acréscimo de 15% no acúmulo da 3P2cMe em comparação com à mitocôndria desacoplada. Para as demais porfirinas houve um menor acúmulo com o aumento do número de resíduos N-3-metilpirídínio. Em todos os experimentos, um comportamento diferenciado foi observado no caso dos isômeros dicatiônicos. A espécie com cargas dispostas nas posições cis (3P2cMe) apresentou praticamente o dobro de eficiência que o correspondente isômero trans (3P2tMe). Este comportamento parece consistente com uma maior penetração da primeira espécie na bicamada lipídica, proporcionado pelo caráter antipático de sua estrutura química. Os resultados apresentados neste estudo mostraram que estas séries de meso-porfirinas catiônicas constituem sistemas bastante interessantes para a compreensão dos efeitos estruturais envolvidos na ação fotodinâmica dos compostos sobre membranas lipídicas, devido à possibilidade de modulação da relação hidrofilicidade/lipofilicidade, sem alterar significativamente a eficiência na formação de 1O2. Além disso, a disposição espacial das cargas positivas na estrutura do fotossensibilizador demonstrou ser extremamente crítica no processo de ancoragem do mesmo na membrana celular ou mitocondrial e consequentemente em causar efeitos fotooxidativos mais eficientemente. As porfirinas contendo grupos 3-metilpiridínio apresentam vantagens sobre as 4-metilpiridínio, normalmente estudas, por serem mais hidrofílicas, terem menor tendência de agregação e possuírem similar atividade fotodinâmica. Em especial, a estrutura anfipática da 3P2cMe associada a sua elevada formação de 1O2 e estabilidade lhe conferem uma interessante potencialidade como agente fototerapêutico.
In this thesis the chemical, photochemical and photophysical relevant aspects for the development of new photosensitizers for photodynamic therapy applications are discussed. 20 conventional porphyrins species and 9 doubly Nconfused porphyrins species were investigated. The first series contains variable number (1 to 4) of cationic groups, [Ru(bipy)2CI]+ or CH3+ (figure 1), bound to the meta or para-pyridil N-atoms of meso-phenylpyridylporphyrins. On the other hand, the protonated, neutral and deprotonated doubly N-confused porphyrins possess two rotated adjacent pyrrol rings, such that they have two coordinating camon atoms. Consequently, transition metal ions in unusually high oxidation states such as Ag(lII) and Cu(lII) can be stabilized by this structure, while the outer pyrrol N-atoms are susceptible to protonation and deprotonation reactions, allowing the modulation of their electronic properties simply by controlling the pH of the solution. In general, the protonation perturbed more significantly the photochemical properties than the deprotonation. The neutral species exhibit intense absorption bands in the phototherapeutical range (600 to 750 nm), associated with a high singlet oxygen sensitization quantum yield (ΦΔ> 0,90 for the Ag(lII) complex). Furthermore, a photodecomposition process due to the reaction with 1O2 was also identified. Both ΦΔ and photodecomposition are influenced by the metal ion coordinated to the doubly N-confused porphyrin ring. The coordination of Ag(lIl) and Cu(lII) increased ΦΔ, probably due to the enhancement of intersystem crossing quantum yield associated with the heavy atom effects. However, AgHN2CP is much more stable than CuHN2CP or the free-base, but the reasons are not clear and should be further investigated. The above results clearly evidence the superior properties of the Ag(lIl) complex as photosensitizer for PDT applications. The 20 cationic pyridylporphyrin derivatives are much soluble in water and experiments directed to biological applications could be carried out. Accordingly, in addition to the quantum yield for singlet oxygen generation(1O2), the effect of the stereochemistry and number and position of the electrically charged substituents on the binding constants and photooxidative damage on erythrocytes, lipossomes, mitochondria and HeLa cells were evaluated. The ΦΔ values were constant in the case of the N-3-methylpyridinium derivatives but inversely proportional to the number of positive charges for the N-4-methylpyridinium derivatives. This was assigned to an increase of aggregation of the sensitizers as the number of meso-phenyl rings and the lipophylicity increase. The partition coefficients in n-octanol/water (logPOA) corroborate that assertion showing a linear correlation with increasing of the number of phenyl groups. The binding efficiency towards the mitochondrial, lipossomal and cellular membrane and the photo-induced lipidic peroxidation processes were directly proportional to the logPOA, except for the ruthenated porphyrins. This last behavior may be associated with the dissociation reactions of the rutheniumpyridylporphyrin bond in the physiologic solution. Accordingly, those experiments were carried out only with the methylpyridynium derivatives. For the first time, we showed inequivocally that the interaction of the methyllated porphyrins with mitochondria are significantly influenced by the membrane potential. In fact, when the mitochondria was energized a 15% increase was observed in the binding constants of 3P2cMe in comparison with teh results with decoupled mitochondria. Also, the amount of bond porphyrin sensitizer decreased as the number of methylpyridynium groups was increased, following the same tendency as above. The behavior of the cis (3P2cMe) and trans (3P2tMe) isomers didn\'t follow the general tendency for the other derivatives in a series, such that the binding constants of the cis isomers were always about twice higher than for the trans, which were much higher than predicted by the general behavior. This means that they can associate more strongly and penetrate deeper in the membrane than the more charged porphyrin derivatives. In particular, the cis species is amphiphilic, i.e. possess an adequate structure for that interaction. The results presented in this thesis showed conclusively that the two series of meso-phenylpyridylporphyrins are adequate for the investigation of the effect of the stereochemistry on the bonding ability and photodynamic properties of those photosensitizers. This is particularly true due to the possibility of modulating the ratio between hydrophobicitylhydrophylicity and the stereochemistry without influencing significantly the quantum yield for 1O2 generation. The meta- series showed an advantage over the conventionally used para- series of methylpyridynium porphyrins because it is more soluble and shows lesser tendency to aggregate. In condusion, the amphiphilic 3P2cMe is the species with the highest potentiallity as por sensitizer because of its high binding constants, low tendency to associate and high ΦΔ.

Sur le plan moléculaire, le transport de petites molécules à travers les bicouches lipidiques est un processus fondamental. L'objectif de ce travail consiste a étudier les différents mécanismes de transfert de molécules hydrophiles à travers la bicouche liposomale. La calcéine a été choisie comme molécule hydrophile modèle pour simuler la vectorisation de principes actifs. Dans une première étape, la formulation des liposomes a été optimisé en considérant ses propriétés physico-chimiques (taille, efficacité d'encapsulation, fluidité et etc.) par différentes méthodes (DSC, TEM, SAXS et etc). Les résultats montrent que la taille moyenne, le potentiel zêta, Tc, l'efficacité d'encapsulation et la fluidité, sont influencés par la composition lipidique des liposomes. Les interactions entre les molécules hydrophiles encapsulées et le vecteur ont été étudiés par spectroscopie Raman, balance de Langmuir et analyse thermique différentielle. Les résultats obtenus montrent que la calcéine est capable d'interagir avec le groupement polaire de la phosphatidylcholine, en s'intercalant entre les chaînes acyles et modifiant de ce fait l'organisation de la membrane. La perméabilité des membranes à la calcéine a d'abord été évaluée sur la base d'une cinétique du premier ordre par spectrofluorimètrie. L'effet de la composition en lipides sur la fluidité membranaire a été étudié en fonction des conditions environnementales. Un modèle simulant les conditions de la digestion a été élaboré pour estimer la vitesse de libération du calcéine à travers la bicouche liposomale, son coefficient de partage, en utilisant l'AFM et la méthode STED. Les résultats obtenus ont confirmé que la calcéine diffuse lentement à travers la membrane liposomale sans pour autant déstructurer le liposome
From a molecular point of view, transport of small molecules across lipid bilayers is a fundamental and functional process. The release of efficacious dose of bioactive-entrapped in liposome depends on different parameters such as liposome permeability, bioactive structural properties and strength of liposome / bioactive interaction. The aim of this study was investigation the possible mechanisms of hydrophilic molecules transfer through liposomal bilayer. Calcein was chosen as model of hydrophilic drugs. In the first step, we optimized liposome formulation by considering its physicochemical properties (size, encapsulation efficiency, fluidity and etc.) by different methods such as DSC, TEM, SAXS, DLS, NMR and Spectroufluremtere. The reported results show that mean size, zeta potential, Tc, entrapment efficiency and fluidity were influenced by liposome lipid composition. Then, we tried to investigate hydrophilic bioactive agents? interaction with liposome by Raman Spectroscopy, Langmuir Balance and Differential Scanning Calorimetry. The obtained results indicated that calcein is being able to interact with the choline polar-head group of the lipids but probability it could intercalate into the acyl chains and disturb the chain order. Finally, the permeability of calcein across some liposome membranes was first evaluated on the basis of the first-order kinetics by spectrofluorometer. Second, the composition/fluidity effect of liposome as well as the incubation temperature/pH effect was investigated. Furthermore, a model simulating the conditions of digestion was developed to estimate the partition coefficient and to determine the mechanism transfer through liposomal bilayer by using AFM and STED methods. The results confirmed that calcein permeates slowly through liposomal membrane by diffusion without liposome disruption

The use of liposomes as drug delivery units necessitates that they come into contact with a number of plasma proteins and cells. This dissertation investigates the interaction of liposomes with two plasma reaction cascades: the complement and the coagulant cascade. The first section of the studies involves the investigation of the contribution of immunoglobulin to the liposome-induced activation of complement in human serum. Liposomes containing the negatively charged phospholipids cardiolipin, phosphatidyiglycerol or phosphatidylinositol, in addition to the neutral charged lipids phosphatidylcholine and cholesterol, were used to activate complement in a whole serum system. The contribution of immunoglobulin was studied by comparing normal human serum to serum depleted of lgG and IgM. Using hemolytic assays of complement function, greater concentrations of phospholipids were required to activate complement in the absence of immunoglobulins. Activation of the classical pathway was confirmed using a C1q consumption assay which showed that activation was dependent on the presence of C1q and confirmed that greater concentrations of phospholipids were required to activate complement in the absence of immunoglobulins. Complement activation was also assessed using crossed immunoelectrophoresis of C3 activation fragments. Using immunoblot analysis, iC3b was detected on the surface of liposomes exposed to normal human serum or immunoglobulin-depleted serum. Having demonstrated that specific antibody was not required, the second section of the studies involves the investigation of the contribution of mannose binding protein to the liposome-induced activation of complement in human serum. The ligand to which mannose binding protein binds was also identified. Mannose binding protein is an activator of the classical complement pathway in the absence of immunoglobulin and C1q. Using immunoaffinity chromatography it was demonstrated that mannose binding protein copurified with beta-2-glycoprotein-1 when beta-2-glycoprotein-1 was immobilized by specific antibody. Using immunoblot analysis mannose binding protein was shown to bind to PC:CH:CL (35:45:20 mol%) through beta-2-glycoprotein-1 which in itself binds avidly to anionic liposomes. This binding was specific as it was inhibited using N-acetyl- mannosamine, an inhibitor of mannose binding protein. Using complement hemolytic assays in the presence of mannose binding protein inhibitors, it was demonstrated that the route of activation of the classical pathway was primarily through C1q in the absence or presence of immunoglobulins. Preliminary competitive binding studies confirmed the presence of the competition between C1q and mannose binding protein that was previously observed using the functional complement hemolytic assays. The competitive binding study demonstrated that various concentrations of mannose binding protein, including physiological concentration, could compete with and inhibit binding of ¹²⁵l-labeled C1q to PC:CH:CL (35:45:20 mol%). There was no binding of ¹²⁵l-labeled C1q detected with the neutral composition PC:CH (55:45 mol%). During the course of the immunoaffinity chromatography studies conducted in section two, it was demonstrated that a number of other proteins copurified with beta-2-glycoprotein-1. Protein S was one of five proteins that copurified with beta-2-glycoprotein-1 when the latter was immobilized using immunoaffinity chromatography. The final section of the studies deals with the investigation of the contribution of protein S on the anticoagulant ability of plasma as the removal of free protein S from circulation may disrupt the hemostatic balance. Using Western blot analysis, protein S was detected on the surface of anionic liposomes. Furthermore, free protein S was deposited on the surface of increasing concentrations of anionic liposomes, quantitated using immunological assays. This effect translated into a lower functional activity of protein S for plasma exposed to anionic liposomes, demonstrated using a commercial assay to measure the functional activity of protein S. The removal of free protein S from the plasma was due to the depletion of beta-2-glycoprotein-1. These studies suggest that the incorporation of glycosides and polyethyleneglycol into the liposomes would be beneficial since they may reduce the observed interactions demonstrated in this study. This in turn could potentially increase the half-life of the circulating liposomes.

碩士
高雄醫學大學
香粧品學系碩士班
104
Among the dendrimers, melamine based dendrimer (also called triazine dendrimer) is potential and considering in biomedical application owing to the simplicity and variety in its synthetic route and various products could be easily obtained by temperature-controlling process. In the recent past, dendrimers are being studied as drug carriers for drug delivery. In this research, the triazine dendrimers with Piperazine and 4- (aminomethyl)piperidine as linkers were prepared through solid phase synthesis processes to offer generation zero (G0) to generation two (G2). In general, our approach significantly reduced the product complexity and also achieved the yield enhancement to 70%. In the drug encapsulation test, there are two groups of compounds were used as guest molecules to determine the interaction between them with host molecules, triazine dendrimers. Group one is three common sunscreens with benzene ring and carbochain in their structures. Another group is non-sunscreen compounds containing thymine, kojic acid and (R)-(-)-Mandelic acid. The binding were evaluated by isothermal titration calorimeter (iTC 200 ) and Nuclear Magnetic Resonance (NMR), however, the binding capacities were only determined by the chemical shift of guest molecules in NMR. The chemical shift of sunscreens changes around -0.0010-0.013 ppm and another group changes around -0.005-0.052 ppm. Among them, the thymine binding with PITD-G2 through hydrogen bonding gave +0.052 ppm change to present the strongest interaction. Therefore, it demonstrates the interaction ability between thymine and the rigid PITD-G2 is best. This study has proved that the design of the triazine dendrimers can be combined with the drug molecules, and NMR can clearly determine a combination of Host-and- Guest reaction.

碩士
國立清華大學
工程與系統科學系
99
Abstract Small-Angle X-ray scattering (SAXS) was used to investigate the dispersion of thiolated gold nanoparticles (Au-16C and Au-8C NPs) with DPPC, DiC7PC, and their combination of molar ratio 1:1. It was found that adding small amounts of phospholipids to disperse the synthesized thiolated small Au NPs in water phase would induce the formation of small thiolated-Au NP clusters that presumably are wrapped by a monolayer of phospholipids on the surface of the cluster, resembling the swollen micellar structure. However, when larger amounts of phospholipids (DPPC) were added, the morphology is transformed from thiolated-Au clusters into vesicle forms. The structural transition could be induced by the presence of lipid bilayers (liposomes) that could engulf the lipid wrapped thiolated-Au NP clusters to form such lipid bilayer vesicles loaded with several thiolated-Au NP clusters in the lipid membrane. The in-situ time-resolved study on the structural transformation by mixing lipid wrapped thiolated-Au NPs with liposomes showed that such a process would take hours to complete. The further observation of Au-8C NP dispersed by DPPC shows there’re two phases occurred as Au-8C aggregation, amorphous and crystalline. The phase can only be influenced by the initial concentration of sample chloroform state instead of thermal treatment or adding 8C thiol. And we believe that the crystalline phase will be superlattice structure made by Au-8C packing.

No
Incorporating edge activators (surfactants) into liposomes was shown previously to improve estradiol vesicular skin delivery; this phenomenon was concentration dependent with low or high concentrations being less effective. Replacing surfactants with limonene produced similar behaviour, but oleic acid effects were linear with concentration up to 16% (w/w), beyond which it was incompatible with the phospholipid. This present study thus employed high sensitivity differential scanning calorimetry to probe interactions of additives with dipalmitoylphosphatidylcholine (DPPC) membranes to explain such results. Cholesterol was included as an example of a membrane stabiliser that removed the DPPC pre-transition and produced vesicles with a higher transition temperature (Tm). Surfactants also removed the lipid pre-transition but reduced Tm and co-operativity of the main peak. At higher concentrations, surfactants also formed new species, possibly mixed micelles with a lower Tm. The formation of mixed micelles may explain reduced skin delivery from liposomes containing high concentrations of surfactants. Limonene did not remove the pre-transition but reduced Tm and co-operativity of the main peak, apparently forming new species at high concentrations, again correlating with vesicular delivery of estradiol. Oleic acid obliterated the pre-transition. The Tm and the co-operativity of the main peak were reduced with oleic acid concentrations up to 33.2 mol%, above which there was no further change. At higher concentrations, phase separation was evident, confirming previous skin transport findings.

碩士
國立交通大學
分子科學研究所
95
During the two-year study, I built and employed two experimental systems: fluorescence correlation spectroscopy (FCS) and fluorescence tweezers to study different aspects of the interaction between proteins and model membrane systems. Specifically, I used FCS to characterize the binding between MARCKS, liposomes, and calmodulin. Besides, I applied a fluorescence optical tweezers system to study the pore-forming and pore-healing of cobra toxins on liposomes. FCS allows the extraction of information on molecular motion by monitoring the spontaneous fluctuation of fluorescence intensity followed. In general, all processes that cause signal fluctuation are accessible with FCS; diffusion, photobleaching, and chemical reaction are just a few examples. In chapter 2 details my study on MARCKS, liposomes, and calmodulin. A peptide corresponding to the effector domain of MARCKS was synthesized. The association of the MARCKS peptide with liposome and the following dissociation induced by calmodulin are observed with fluorescence correlation measurement. Our results show that: 1) The binding of MARCKSs to liposomes as the ratio of the negative charged lipids increases in the liposomes, 2) MARCKS binds to liposomes more easily due to the phase change of lipids in the liposomes as observed in the heating process, 3) Modulation of MARCKS binding with liposomes by calmodulin was directly observed with FCS. Cobra cardiotoxin were known to bind with cell membranes, form holes on membranes and eventually lead to the death of cells. I am particularly interested in the dynamics of the pore-forming and hearing on membranes. For this purpose, I built a fluorescence tweezers system and did some preliminary study. The results are included in chapter 3. Single liposomes fluorescent molecules embedded in it were successfully captured and the fluorescence intensity of the single liposome was monitored. At this stage, I have evidence that I have observed the change of fluorescence intensity induced by cobratoxin. The decrease of the fluorescence was explained by leakage of embedded fluorescent molecules due to holes formed by cobratoxin.

The thermodynamic properties of bioactive peptides determine how they interact with cellular assemblies. Ultrasonic velocity and density measurements were used to analyse the volumetric properties in aqueous solution of 3 different materials: KCl, bioactive peptides (from hemp seed and dairy proteins), and liposomes (cell membrane models), as well as the interaction between peptides and liposomes. Serial dilutions of the three different materials showed linear relationships between density and concentration and between ultrasonic velocity and concentration. The apparent specific volume and apparent specific compressibility in solution of all materials showed concentration dependence as a result of increased electrostriction as solutions were diluted. The experimental ultrasonic velocities of liposome-dairy peptide mixes were higher than the theoretical additive value, due to interactions between liposomes and peptides. My research demonstrates the benefits of precise volumetric assessments in biological assays.

abstract: To mimic the membrane environment for the photosynthetic reaction center of the photoheterotrophic Heliobacterium modesticaldum, a proteoliposome system was developed using the lipids found in native membranes, as well as a lipid possessing a Ni(II)-NTA head group. The liposomes were also saturated with menaquinone-9 to provide further native conditions, given that menaquinone is active within the heliobacterial reaction center in some way. Purified heliobacterial reaction center was reconstituted into the liposomes and a recombinant cytochrome c553 was decorated onto the liposome surface. The native lipid-attachment sequence of cytochrome c553 was truncated and replaced with a hexahistidine tag. Thus, the membrane-anchoring observed in vivo was simulated through the histidine tag of the recombinant cytochrome binding to the Ni(II)-NTA lipid's head group. The kinetics of electron transfer in this system was measured and compared to native membranes using transient absorption spectroscopy. The preferential-orientation of reconstituted heliobacterial reaction center was also measured by monitoring the proteoliposome system's ability to reduce a soluble acceptor, flavodoxin, in both whole and detergent-solubilized proteoliposome conditions. These data demonstrate that this proteoliposome system is reliable, biomimetic, and efficient for selectively testing the function of the photosynthetic reaction center of Heliobacterium modesticaldum and its interactions with both donors and acceptors. The recombinant cytochrome c553 performs similarly to native cytochrome c553 in heliobacterial membranes. These data also support the hypothesis that the orientation of the reconstituted reaction center is inherently selective for its bacteriochlorophyll special pair directed to the outer-leaflet of the liposome.
Dissertation/Thesis
Masters Thesis Chemistry 2018

碩士
國立中央大學
化學工程研究所
89
This study investigated the behavior of mixed DMPC/PF-127 Gibbs’ adsorption monolayers (the weight ratio are 34: 4 , 34:14 and 34:34)and the PF-127 absorbed on different monolayers(the composition are DMPC, DMPC/Vit-E and DMPC/cholesterol)at the air/liquid interface at 25 . For mixed DMPC/PF-127 adsorption monolayers system at the air/water interface, the measurements of equilibrium surface tensions and surface pressure-area per molecule(Π-A) isotherms were carried out. The results of the mixed monolayer, the equilibrium surface tensions increased with the PF-127 concentration. But the excess of surface concentration is non- linear. TheΠ-A isotherms of the behavior of mixed DMPC/PF-127 monolayers indicate that the addition of the PF-127increased the shift percentages and the transition phases were obviously. The behavior of the PF-127 adsorbed on different monolayers were investigated from the measurements of the change of surface pressure with time andΠ-A isotherms. In the PF-127 adsorption of monolayer systems, the equilibrium adsorption surface pressure increased with the PF-127 concentration and the initial surface pressure. The behavior of PF-127 adsorbed on DMPC/Vit-E and DMPC/cholesterol mixed monolayers, the ranges of equilibrium adsorption surface pressures are 15.8~28.3mN/m and 20.3~27.7mN/m, indicated that the addition of α-tocopherol and cholesterol change the distance of mixed monolayers. TheΠ-A isotherms of the PF-127 adsorbed to balance indicate that the transition phases were carried out and the PF-127 might be insert or absorb to monolayers. However, at higher surface pressure, the PF-127 molecules are squeezed into the subphase. From the DSC (differential Scanning Calorimetry ) experiments of liposome, the change of temperatures of phase transition regardless of the PF-127 incorporated or adsorbed to liposomes, which suggest the PF-127 does not insert or absorb deeply. Finally, the results obtained in this study support that the PF-127 can be readily absorbed or inserted onto liposome, providing steric barrier , preventing the fusion or aggregation of liposomes.

Les peptides et protéines extracteurs de lipides (PEL) se lient aux membranes lipidiques puis en extraient des lipides en formant de plus petits auto-assemblages, un phénomène qui peut aller jusqu'à la fragmentation des membranes. Dans la nature, cette extraction se produit sur une gamme de cellules et entraîne des conséquences variées, comme la modification de la composition de la membrane et la mort de la cellule. Cette thèse se penche sur l’extraction lipidique, ou fragmentation, induite par le peptide mélittine et la protéine Binder-of-SPerm 1 (BSP1) sur des membranes lipidiques modèles. Pour ce faire, des liposomes de différentes compositions sont préparés et incubés avec la mélittine ou la BSP1. L'association aux membranes est déterminée par la fluorescence intrinsèque des PEL, tandis que l'extraction est caractérisée par une plateforme analytique combinant des tests colorimétriques et des analyses en chromatographie en phase liquide et spectrométrie de masse (LCMS). La mélittine fait partie des peptides antimicrobiens cationiques, un groupe de PEL très répandu chez les organismes vivants. Ces peptides sont intéressants du point du vue médical étant donné leur mode d’action qui vise directement les lipides des membranes. Plusieurs de ceux-ci agissent sur les membranes des bactéries selon le mécanisme dit « en tapis », par lequel ils s’adsorbent à leur surface, forment des pores et ultimement causent leur fragmentation. Dans cette thèse, la mélittine est utilisée comme peptide modèle afin d’étudier le mécanisme par lequel les peptides antimicrobiens cationiques fragmentent les membranes. Les résultats montrent que la fragmentation des membranes de phosphatidylcholines (PC) est réduite par une déméthylation graduelle de leur groupement ammonium. L'analyse du matériel fragmenté révèle que les PC sont préférentiellement extraites des membranes, dû à un enrichissement local en PC autour de la mélittine à l'intérieur de la membrane. De plus, un analogue de la mélittine, dont la majorité des résidus cationiques sont neutralisés, est utilisé pour évaluer le rôle du caractère cationique de la mélittine native. La neutralisation augmente l'affinité du peptide pour les membranes neutres et anioniques, réduit la fragmentation des membranes neutres et augmente la fragmentation des membranes anioniques. Malgré les interactions électrostatiques entre le peptide cationique et les lipides anioniques, aucune spécificité lipidique n'est observée dans l'extraction. La BSP1 est la protéine la plus abondante du liquide séminal bovin et constitue un autre exemple de PEL naturel important. Elle se mélange aux spermatozoïdes lors de l’éjaculation et extrait des lipides de leur membrane, notamment le cholestérol et les phosphatidylcholines. Cette étape cruciale modifie la composition lipidique de la membrane du spermatozoïde, ce qui faciliterait par la suite la fécondation de l’ovule. Cependant, le contact prolongé de la protéine avec les spermatozoïdes endommagerait la semence. Cette thèse cherche donc à approfondir notre compréhension de ce délicat phénomène en étudiant le mécanisme moléculaire par lequel la protéine fragmente les membranes lipidiques. Les résultats des présents travaux permettent de proposer un mécanisme d’extraction lipidique en 3 étapes : 1) L'association à l’interface des membranes; 2) La relocalisation de l’interface vers le cœur lipidique; 3) La fragmentation des membranes. La BSP1 se lie directement à deux PC à l'interface; une quantité suffisante de PC dans les membranes est nécessaire pour permettre l'association et la fragmentation. Cette liaison spécifique ne mène généralement pas à une extraction lipidique sélective. L'impact des insaturations des chaînes lipidiques, de la présence de lysophosphatidylcholines, de phosphatidyléthanolamine, de cholestérol et de lipides anioniques est également évalué. Les présentes observations soulignent la complexe relation entre l'affinité d'un PEL pour une membrane et le niveau de fragmentation qu'il induit. L'importance de la relocalisation des PEL de l'interface vers le cœur hydrophobe des membranes pour permettre leur fragmentation est réitérée. Cette fragmentation semble s'accompagner d'une extraction lipidique préférentielle seulement lorsqu'une séparation de phase est induite au niveau de la membrane, nonobstant les interactions spécifiques PEL-lipide. Les prévalences des structures amphiphiles chez certains PEL, ainsi que de la fragmentation en auto-assemblages discoïdaux sont discutées. Finalement, le rôle des interactions électrostatiques entre les peptides antimicrobiens cationiques et les membranes bactériennes anioniques est nuancé : les résidus chargés diminueraient l'association des peptides aux membranes neutres suite à l'augmentation de leur énergie de solvatation.
Lipid-extracting peptides and proteins (LEPs) bind to lipid membranes, extract lipids in the form of smaller auto-assemblies, and ultimately fragment membranes. In nature, this lipid extraction occurs in many different cell systems and causes various consequences, such as a modification of the membrane lipid composition or the cell death. This thesis focuses on the lipid extraction, or fragmentation, induced by the peptide melittin and the protein Binder-of-SPerm 1 (BSP1) on model lipid membranes. To this end, liposomes of different composition are prepared and incubated with melittin or BSP1. The association to membranes is determined by the LEPs intrinsic fluorescence, while the extraction is characterized by a combination of colorimetric phosphorus assays and liquid chromatography-mass spectrometry analyses (LCMS). Melittin is a cationic antimicrobial peptide, a very common category of LEP found in living organisms. Cationic antimicrobial peptides are interesting to medicine because they directly target membrane lipids. The action of many of these peptides is described by the carpet-like mechanism, by which they adsorb to membrane surface, induce the formation of pores and then cause the fragmentation of the membranes. In this thesis, melittin is used as a model peptide in order to study the mechanism by which cationic antimicrobial peptides fragment lipid membranes. Results show that the phosphocholine (PC) membrane fragmentation is reduced by a gradual demethylation of the ammonium group. Analysis of the fragmented material reveals that PC are preferentially extracted from membranes, due to a local enrichment in PC near melittin in the membrane. Furthermore, a melittin analogue, for which a majority of its cationic residues were neutralized, is used to investigate the role of the cationic character of native melittin. The neutralization increases the peptide affinity for neutral and anionic membranes, reduces fragmentation of neutral membranes and increases fragmentation of anionic membranes. Despite electrostatic interactions between the cationic peptide and the anionic lipids, no lipid specificity is observed in the extraction. BSP1 is the most abundant protein of the bovine seminal plasma and constitutes another example of important LEP found in nature. Upon ejaculation, it mixes with spermatozoa and extracts membrane lipids, such as cholesterol and phosphatidylcholines. This crucial process modulates the lipid composition of sperm membranes, which would then facilitate egg fertilization. However, a prolonged contact between the protein and spermatozoa could damage the semen. This thesis is looking to deepen our understanding of this delicate phenomenon by studying the molecular mechanism by which this protein fragments lipid membranes. Results of the present work suggest a 3-step mechanism for the extraction: 1) Association to membrane interface; 2) Relocation towards the lipid core; 3) Fragmentation of membranes. BSP1 binds directly to two interfacial PC; a sufficient quantity of PC in membranes is necessary for protein association and fragmentation. This specific binding generally does not lead to specificity in the lipid extraction. The impact of unsaturation of the lipid chains, of the presence of lysophosphatidylcholines, of phosphatidylethanolamines, of cholesterol and of anionic lipids is also studied. The present observations underline the complex relationship between a LEP affinity for membranes and the level of fragmentation it induces. The importance of LEP relocation, from the interface to the hydrophobic core of the membranes, for fragmentation is reiterated. This fragmentation seems to be lipid specific only when a phase separation of the lipids occurs in the membrane, notwithstanding specific LEP-lipid interactions. The prevalence of amphipathic structures in certain LEPs, as well as of the auto-assembled discoidal structures resulting from fragmentation is discussed. Finally, the role of electrostatic interactions between cationic antimicrobial peptides and anionic bacterial membranes is detailed: charged residues lower peptide association to neutral membrane due to an increase of their free energy of solvation.