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. 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. 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. 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. 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. Furthermore, sterically stabilised liposomes intended for targeted BNCT have been characterised and optimised concerning loading and retention of boronated drugs.

Les cibles épigénétiques telles que les histones désacétylases (HDAC) sont étudiées comme nouveaux traitements anticancéreux, leur fonctionnement anormal étant associée aux processus cancéreux. De nombreux inhibiteurs de ces enzymes sont apparus tel que le SAHA ou le CI-994. Notre laboratoire a développé un nouvel analogue de la trichostatine A appelé NODH. Malgré leur activité anticancéreuse in vitro prometteuse, ces inhibiteurs souffrent de leur manque de solubilité, d'effets toxiques et de leur rapide métabolisation par l'organisme. Afin de pallier ces limitations, ces iHDAC peuvent être conjugués à des vecteurs capables de les transporter dans l'organisme et de les libérer une fois la cible tumorale atteinte. Ce travail de thèse a consisté dans un premier temps à développer des prodrogues acido-sensibles capable de libérer la substance active après endocytose par les cellules. Ces systèmes ont été appliqués aux trois iHDAC SAHA, CI-994 et NODH. Les prodrogues développées ont ensuite été accrochées sur des nanoparticules polymériques connues pour leur biocompatibilité et leur accumulation dans les tumeurs grâce à l'effet EPR. L'activité anticancéreuse de certaines nanoparticules a été validée par des tests in vitro et in vivo. Un deuxième travail a été entrepris concernant la préparation de prodrogues fluorescentes pH labiles capables de libérer la substance active et de moduler la fluorescence par la variation du pH. La dernière partie de ce travail a été consacrée à la mise au point d'une nouvelle méthode de synthèse par voie superacide potentiellement stéréosélective de benzofuranones nécessaires à la préparation d'analogues de NODH. / Epigenetic targets like histone deacetylases (HDAC) are studied as new anticancer treatments, their dysfunctioning being associated to cancer mechanisms. Several epigenetic target inhibitors have been developed like SAHA or CI-994. Our group has developed a new trichostatin A analogue called NODH. Despite their promising in vitro anticancer effect, these inhibitors suffer from lack of solubility, toxic effects and rapid clearance. These constrains make the HDACi good candidates for ligation to vectors able to carry them in the body and to release them when the tumor is reached. The first part of this thesis work has focused on the development of acid sensitive prodrugs able to release an active substance after cellular internalization by endocytosis. These systems were applied to three HDACi SAHA, CI-994 and NODH. These prodrugs were then conjugated to polymeric nanoparticles known for their biocompatibility and their accumulation in tumors exploiting the EPR effect. The anticancer activity of some nanoparticles has been validated by in vitro and in vivo tests. The second part of this work was carried out in order to prepare pH sensitive fluorescent prodrugs able to release the active molecule and to modulate the fluorescent effect by pH changes. The last part of this work was dedicated for the development of a new synthesis in suparacid potentially stereoselective of several benzofuranones required for the preparation of NODH analogs.

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

In the past, aminocyclohexanol rings have been successfully utilized as pH-triggered molecular switches in various trans-2-aminocyclohexanol derivatives. By changing the groups on the amine nitrogen, these models provided a wide pH range in which a switch can occur. The pH-induced switch of conformation was monitored by 1H-NMR spectroscopy. The models were also incorporated into the bilayer membrane of liposome structures and tested for their ability to disrupt their membrane upon their conformational flip induced by a decrease in pH. In this work, the amide bond has been studied as a molecular switch and various amide derivatives have been tested for their potential as lipid-like compounds that also exhibit a pH-sensitive conformational flip. The conformational analysis of these compounds was achieved by various NMR techniques and NMR acid-base titration studies were utilized to estimate the pKa of a number of the compounds described.

Les micelles apparaissent comme prometteuses dans le domaine de la vectorisation de médicaments. Afin d’améliorer leur biocompatibilité nous nous intéressons ici à des synthèses originales de copolymères amphiphiles comportant un bloc hydrophobe lipidique biosourcé. Un polymère intelligent constitue le bloc hydrophile. L’huile de lin subit une modification chimique afin d’introduire un site amorceur de polymérisation. Le bloc hydrophile est alors ajouté par polymérisation contrôlée. Deux copolymères sont obtenus, le lipide-b-poly(acide acrylique), pH-sensible et le lipide-b-poly(2-isopropyl-oxazoline), thermo-sensible. Une caractérisation physico-chimique complète révèle des concentrations micellaires critiques basses et des micelles de 10 nm. Un système mixte est préparé par mélange des deux copolymères. L’étude de ce système prouve une sensibilité aux deux stimuli. Afin d’améliorer la stabilité des micelles, nous proposons la photo-réticulation des insaturations de la chaîne lipidique. / Small micellar systems seem to be really promising candidates for drug delivery applications. In order to improve the bio-assimilation of our system, the original synthesis of amphiphilic copolymers from linseed oil is carried out. First, linseed oil is chemically modified in order to introduce a polymerization initiating site. Then, the lipoinitiator is engaged in the controlled polymerization of the hydrophilic block. Two amphiphilic copolymers are obtained through this strategy: a pH-sensitive lipid-b-poly(acrylic acid), and a thermo-sensitive lipid-b-poly(2-isopropyl-oxazoline). Both present a low critical micellar concentration and form small micelles (~10 nm). By mixing both copolymers, mixed micelles responding to both stimuli were obtained. In order to improve the system’s stability, the photo-cross-linking of the lipidic double bonds in the micelle’s core is finally realized.

Cancer is the second leading cause of death in the US and worldwide, accounting for 16% of deaths worldwide in 2015. Of more than 100 types of cancers affecting humans, breast cancer is the most common cancer among women and is the second leading cause of death in women. Triple negative breast cancer (TNBC) is a subtype of breast carcinomas defined by the lack of the expression of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor (HER2 /neu). The prognosis and survival of TNBC patients remains the poor due to the lack of effective targeted therapy. Nanotechnology-based drug delivery systems, such as liposomes, are widely investigated to enhance anticancer efficacy by concentrating the drug molecules in the tissues of interest and by altering the pharmacokinetic profile. Taking advantage of the pH gradient in the tumor microenvironment, pH-triggered release is a promising strategy to enhance the anticancer efficacy of drug delivery systems against TNBC. Previously, a strategy in our lab has been developed to render saturated and pegylated liposomes pH-sensitive: protonation-induced conformational switch of lipid tails, using trans-2-aminocyclohexanol lipids (TACH, flipids) as a molecular trigger. Based on previous work in our lab, pH-sensitive liposomes (fliposomes) composed of C-16 flipids with amine group of morpholine (MOR) and azetidine (AZE) demonstrated optimized triggered release in response to the tumor’s low pH microenvironment. In this study, different preparation methods were developed and optimized to produce viable fliposomes with high doxorubicin (DOX) encapsulation efficiency. In vitro release assays were established and validated to accurately reflect pH-triggered release of fliposomes. The physicochemical properties of DOX-loaded fliposomes were characterized and their pH-dependent release were investigated. Factors influencing the desirable attributes of liposomes, such as size, pH-sensitivity, stability and drug-loading capacity were explored. Based on these characterizations, central composite design (CCD) was utilized to optimize the formulation of fliposome with two critical factors, flipids and cholesterol. Cell viability assays on traditional monolayer and innovative three-dimensional multicellular spheroids (3D MCS) of TNBC cell lines were conducted to evaluate the anticancer efficacy of the resultant fliposomes in vitro. The constructed 3D MCS carried heterogeneously distributed live and apoptotic cells, as well as acidity inside the 3D MCS based on confocal microscopic imaging studies. The distribution and penetration of DOX-loaded fliposomes into 3D MCS was imaged by confocal microscopy in comparison to DOX-loaded non pH-sensitive liposomes and free DOX. As a result, fliposome manifested superior anticancer activity against TNBC 3D MCS by efficient penetration into 3D MCS, followed by tuning up the release rate of the anticancer agent DOX. A TNBC orthotopic xenograft model was established by transplanting TNBC into the murine mammalian fat pad, which maintains the organ-specific tumor microenvironment of the original organ . A pilot pharmacokinetic study was conducted in order to correlate the pH response and stability properties with the in vivo stability of the optimized AZE-C16 fliposome. The antitumor efficacy was comparable between free DOX and DOX-loaded stealth liposome with tumor volumes of ~ 80-90% of the control treatment 32 days post first dose. In contrast, the DOX-loaded fliposome, especially MOR-C16 fliposome, exhibited a significantly higher antitumor efficacy and delayed progression compared to free DOX and stealth liposome treatments. Taken together, DOX-loaded fliposomes were successfully prepared and optimized for in vivo application. They were able to achieve superior activity against TNBC in vitro and in vivo, facilitated by enhanced release of the anticancer drug DOX after penetration inside TNBC tumor.

No description available.

Σήμερα η μελέτη των βιοϋλικών προσανατολίζεται σε δύο κατευθύνσεις, την ανάπτυξη συστημάτων μεταφοράς φαρμάκων και συστημάτων κατάλληλων να διεγείρουν κυτταρικές λειτουργίες. Η έρευνά μας έχει σχέση με την συγκριτική μελέτη συστημάτων μεταφοράς φαρμάκων κατάλληλων για εφαρμογή σε οστικούς καρκίνους. Τέτοιου είδους συστήματα θα πρέπει, αρχικά να είναι ικανά να μεταφέρουν τα φαρμακευτικά μόρια και στη συνέχεια να μπορούν να επάγουν οστεογένεση. Η δεύτερη λειτουργικότητα είναι ιδιαιτέρως σημαντική καθώς έχει σαν αποτέλεσμα την πλήρωση του οστικού ελλείμματος που προκαλείται από την δράση των καρκινικών κυττάρων. Για τον σκοπό αυτό, διερευνήθηκε η ικανότητα του υαλώδους δικτύου να μεταφέρει φαρμακευτικά μόρια μέσω παραδοσιακών συστημάτων μεταφοράς. Συνεπώς, ακολούθησε η ex vitro μελέτη pH-ευαίσθητων τροποποιημένων πυριτικών ξηρών πηκτών στα οποία είχε συνδεθεί το αντικαρκινικό φάρμακο δοξορουπμυσίνη. Συγκεκριμένα, πυριτικά ξηρά πηκτώματα συντέθηκαν με την μέθοδο sol-gel και τροποποιήθηκαν περαιτέρω με χημεία καρβοδιϊμιδίου. Η τροποποίηση είχε σαν αποτέλεσμα την επιφανειακή σύνδεση υδροπηκτών δεξτράνης που παρουσιάζουν ευαισθησία στο pH. Στη συνέχεια, ακολούθησε η σύνθεση των ανόργανων βιοενεργών νανοσφαιρών. Για την σύνθεση των υαλωδών νανοσφαιρών με εσωτερική κοιλότητα ακολουθήθηκε η διαδικασία επικάλυψης sol-gel, κατά την οποία έγινε η ηλεκτροστατική επικάλυψη νανοσωματιδίων πολυστυρενίου με αποτέλεσμα την σύνθεση ανόργανων πυριτικών και φωσφοπυριτικών νανοσφαιρών. Επιπλέον, μελετήθηκε η εφαρμογή του συμπολυμερούς του πολυμεθακρυλικού μεθυλεστέρα – υδρομεθακρυλικού προπυλεστέρα ως υποστρώματος καθώς το PMMA αποτελεί βασικό συστατικό των οστικών τσιμέντων και παρουσιάζει βελτιωμένες μηχανικές ιδιότητες. Προκειμένου να ολοκληρωθεί η συγκριτική μελέτη μας, ακολούθησε η ex vitro ανάλυση των παραπάνω υβριδικών φωσφοπυριτικών νανοσφαιρών καθώς επίσης των πυριτικών νανοσφαιρών PS και PMMA-co-HPMA. Η επώαση σε διάλυμα SBF οδήγησε στον σχηματισμό ανθρακικού πυριτικού υδροξυαπατίτη με το μέγεθος των κρυσταλλιτών να μην υπερβαίνει τα 45 nm και έντονη παρουσία συσσωματωμάτων. Βέλτιστες ιδιότητες βιοενεργότητας παρουσιάζουν οι τροποποιημένες με αμίνες υβριδικές νανοσφαίρες PMMA-co-HPMA, οι οποίες έχουν επίσης την δυνατότητα να χρησιμοποιηθούν ως μεταφορείς φαρμακευτικών μορίων σε όξινο καθώς επίσης και σε φυσιολογικό pH με παρατεταμένη δυνατότητα αποδέσμευσης. / Recently the study of biomaterials has moved in two directions, the evolution of drug delivery systems and of systems that can stimulate specific cellular responses. Our investigation aims to the study of drug delivery systems for bone cancer therapy. These systems must fulfill two important functionalities. At first, they should be able to deliver drug molecules to bone cancer environment through loading or surface conjugation and subsequently to cause osteogenesis. Their second functionality is especially important since it leads to substitution of bone defects caused from the action of cancer cells. For this purpose, the ability of the glassy network to deliver drug molecules was studied. For this purpose, expanded ex vitro research was followed in DOX conjugated pH-sensitive functionalized silica xerogels. Specifically, silica xerogels were synthesized through a sol-gel process and further functionalized with carbodiimide chemistry. The functionalization process resulted in pH-sensitive dextran hydrogels. The study of the enhanced properties of glassy substrates was followed by the synthesis of amorphous bioactive nanospheres. Moreover, the change of the organic core and the use of PMMA-co-HPMA were advantageous due to the enhanced mechanical properties of PMMA-co-HPMA and its use in bone cements. In order to accomplish our comparative investigation, we followed the ex vitro study of the above hybrid binary silicate, ternary and quaternary phosphosilicate nanospheres as well as the silicate PS and PMMA-co-HPMA nanospheres. The incubation in SBF solution resulted in the formation of a silica-substituted carbonate hydroxyapatite (Si-HCA) a with crystallite size of around 45 nm and extended surface aggregates. The amino-modified PMMA-co-HPMA hybrid nanospheres present enhanced biocompatible properties, with prolonged release ability as drug delivery systems, in acidic as well as physiological pH.

Abstract ACCURATE METHODOLOGY FOR MONITORING BIOMEMBRANE EVENTS By Christine A. Winschel, Ph.D. A Dissertation submitted in partial fulfillment of the requirements for the degree of Doctorate of Philosophy in Chemistry at Virginia Commonwealth University. Virginia Commonwealth University, 2012 Major Director: Dr. Vladimir A. Sidorov ASSOCIATE PROFESSOR, DEPARTMENT OF CHEMISTRY This study describes the synthesis and characterization of a new receptor (cyclen 1) capable of strong selective binding of pyrene-based anionic dyes under near-physiological conditions. This receptor comprises four naphthylthiourea groups tethered to a cyclen core via an ester linkage. The most important finding was the ability of cyclen 1 to bind efficiently to a pH-sensitive pyranine dye, a dye that is commonly used in various biomembrane assays. The high affinity of cyclen 1 to pyranine, its impermeability to the lipid bilayer membrane, fast kinetics of binding, and ability to quench pyranine’s fluorescence were used as a basis for a new membrane leakage assay. This membrane leakage assay is fully compatible with the commonly applied pH-stat transport assay, and therefore it allows for differentiation of ion transport and nonselective leakage mechanisms within a single set of experiments. In the second part of this study a new methodology for the detection of lipid flip was developed. This methodology relies on the quenching of the fluorescence of a newly synthesized cascade-blue-labeled lipid through complex formation with cyclen 1. This receptor-dye complexation also has high affinity for binding at micromolar concentrations and can be reversed by either competitive displacement of the lipid probe or by enzymatic degradation of the receptor leading to the label release and fluorescence dequenching. This new methodology is suitable for the study of lipid flip in both model spherical bilayer membranes and in-vitro experiments, and is less invasive to the model and cell membranes than the commonly utilized 7-nitro-1,2,3-benzoxadiazol-4-yl (NBD)-dithionite methodology. Lastly, new pH-sensitive lipids were synthesized and utilized in the formulation of liposomes suitable for controlled drug release. These liposomes contain various amounts of internal NaCl and undergo internal acidification upon the exogenous addition of an HCl co-transporter in a physiologically relevant NaCl solution. Therefore, acidification ultimately leads to the hydrolysis of the pH-sensitive lipids and subsequent contents release. These liposomes were found to be insensitive to physiological concentrations of human serum albumin and to be non-toxic to cells at concentrations exceeding pharmacological relevance. These results render this new drug release model potentially suitable for in vivo applications.

<p>The stomach is frequently exposed to hazardous agents and to resist this harsh environment, several protective mechanisms exist. Of special interest is the gastric pathogen <i>Helicobacter pylori </i>which causes gastritis, ulcers and cancer but the mechanism leading to these diseases are still unclear. However it is very likely that <i>H. pylori </i>negatively influence the protection mechanisms that exist in the stomach. </p><p>The aims of the present investigation were first to develop an in vivo mouse model in which different protection mechanisms could be studied, and second to investigate the influence of <i>H. pylori</i> on these mechanisms. </p><p>An in vivo preparation of the gastric mucosa in mice was developed. This preparation allows studies of different gastric mucosal variables and can also be applied for studies in other gastro-intestinal organs. </p><p>Mice chronically infected with <i>H. pylori</i>, were shown to have a reduced ability of the mucosa to maintain a neutral pH at the epithelial cell surface. This could be due to the thinner inner, firmly adherent mucus gel layer, and/or to defective bicarbonate transport across the epithelium. The Cl^-/HCO₃^- exchanger SLC26A9 was inhibited by NH₄⁺, which also is produced by <i>H. pylori</i>. The mRNA levels of SLC26A9 were upregulated in infected mice, suggesting a way to overcome the inhibition of the transporter. Furthermore, the hyperemic response to acid pH 2 and 1.5 was abolished in these mice. The mechanisms by which the bacteria could alter the blood flow response might involve inhibition of the epithelial iNOS.</p><p>Water extracts of <i>H. pylori </i>(HPE) reduces the blood flow acutely through an iNOS and nerve-mediated pathway, possibly through the endogenous iNOS inhibitor ADMA. Furthermore, HPE alters the blood flow response to acid as the hyperemic response to acid pH 0.8 is accentuated in mice treated with HPE. </p>