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O presente trabalho tem como intuito avaliar e otimizar o processo de confecção e as propriedades de nanopartículas poliméricas (NPs) e de lipossomos (LPs) para o transporte de substâncias de interesse visando o tratamento e prevenção de doenças que atingem o sistema nervoso central. As NPs de PLGA com polissorbato-80 (T-80) adsorvido foram otimizadas quanto ao tipo de tensoativo, regime de sonicação, volume de solução de T-80 e tipo de agitador sobre o tamanho, polidispersão e potencial zeta das NPs; ao passo que LPs funcionalizados com anticorpo anti-transferrina também tiveram sua confecção otimizada e suas propriedades foram avaliadas para diferentes misturas de lipídeos contendo DPPC ou DOPC. Após a otimização dos nanocarreadores, ensaios celulares em células Neuro-2a e SH-SY5Y foram realizados de modo comparar a citotoxicidade e a cinética de internalização de tais sistemas. Demonstra-se que o processo de confecção dos carreadores para aplicação no sistema nervoso central requer investigação pormenorizada para melhores resultados na aplicação biológica. / This study aims at analyzing and optimizing the obtainment process and properties of polymeric nanoparticles (NPs) and liposomes (LPs) for the transport of substance of interest with the purpose of treatment and prevention of various central nervous system diseases. PLGA NPs coated with polissorbate-80 (T-80) were optimized regarding the type of surfactant, sonication regime, T-80 solution dispersion volume and size of stirrer on the size, polidispersity and zeta potencial of NPs; whilst anti-transferrin antibody functionalized LPs also had their synthesis optimized and their properties were also compared for different lipid mixtures containing DPPC or DOPC. After optimization, the nanocarriers were submitted to in vitro assays with Neuro-2a and SH-SY5Y cells in order to compare their cytotoxicity and internalization kinetics. It is demonstrated that the process to obtain nanocarriers for central nervous system applications require detailed analysis when seeking the most favorable biological results.

O presente trabalho tem como intuito avaliar e otimizar o processo de confecção e as propriedades de nanopartículas poliméricas (NPs) e de lipossomos (LPs) para o transporte de substâncias de interesse visando o tratamento e prevenção de doenças que atingem o sistema nervoso central. As NPs de PLGA com polissorbato-80 (T-80) adsorvido foram otimizadas quanto ao tipo de tensoativo, regime de sonicação, volume de solução de T-80 e tipo de agitador sobre o tamanho, polidispersão e potencial zeta das NPs; ao passo que LPs funcionalizados com anticorpo anti-transferrina também tiveram sua confecção otimizada e suas propriedades foram avaliadas para diferentes misturas de lipídeos contendo DPPC ou DOPC. Após a otimização dos nanocarreadores, ensaios celulares em células Neuro-2a e SH-SY5Y foram realizados de modo comparar a citotoxicidade e a cinética de internalização de tais sistemas. Demonstra-se que o processo de confecção dos carreadores para aplicação no sistema nervoso central requer investigação pormenorizada para melhores resultados na aplicação biológica. / This study aims at analyzing and optimizing the obtainment process and properties of polymeric nanoparticles (NPs) and liposomes (LPs) for the transport of substance of interest with the purpose of treatment and prevention of various central nervous system diseases. PLGA NPs coated with polissorbate-80 (T-80) were optimized regarding the type of surfactant, sonication regime, T-80 solution dispersion volume and size of stirrer on the size, polidispersity and zeta potencial of NPs; whilst anti-transferrin antibody functionalized LPs also had their synthesis optimized and their properties were also compared for different lipid mixtures containing DPPC or DOPC. After optimization, the nanocarriers were submitted to in vitro assays with Neuro-2a and SH-SY5Y cells in order to compare their cytotoxicity and internalization kinetics. It is demonstrated that the process to obtain nanocarriers for central nervous system applications require detailed analysis when seeking the most favorable biological results.

Das Humane Immundefizienz-Virus 1 (HIV-1) rekrutiert Wirtszellproteine und -Signalwege für seinen eigenen Lebenszyklus und beeinträchtigt viele Funktionen von Immunzellen, darunter die Phagozytose von Pathogenen durch Makrophagen. Diese Schwächung der Immunabwehr verursacht das acquired immunodeficiency syndrome (AIDS).Der Zusammenbau von HIV-1 geschieht sowohl an der Plasmamembran als auch in endosomalen Kompartimenten und wird durch das Struktur-Polyprotein Gag kontrolliert, das auch für die Freisetzung virusähnlicher Partikel (VLPs) aus der Wirtszelle sorgt. Vorangegangene Studie haben gezeigt, dass durch hohe induzierte Calciumkonzentrationen im Zytoplasma die Anzahl an VLPs in endolysosomalen Kompartimenten steigt und die VLP-freisetzung drastisch zunimmt. Der verantwortliche Mechanismus ist jedoch bisher unbekannt. Die vorliegende Arbeit zeigt zunächst, dass Calciumausschüttung aus Lysosomen die Fusion von Endosomen und Lysosomen - und damit die Exozytose der in Lysosomen enthaltenen VLPs - auslöst. Dieser Prozess wird durch Synaptotagmin VII reguliert und verhindert den Abbau eines Teils der in späten Endosomen und Lysosomen eingschlossenen VLPs. Der zweite Teil dieser Arbeit beschreibt die Entwicklung eines nanobiotechnologischen Systems zur Eliminierung von Env/Gag-VLPs (HIV-VLPs) mit Hilfe von Makrophagen. Dieses basiert auf Immunoliposomen, die HIV-VLPs über anti-Env-Antikörper binden und von Makrophagen dank membranständigem Phosphatidylserin (PS), einem apoptotischen Signal, phagozytiert werden. Die Lipososmen imitieren apoptotische Zellen und induzieren ihre Internalisierung und die lysosomale Aufnahme von HIV-VLPs. Das System nutzt einen effizienten Internalisierungsweg, der während der HIV-1-Infektion nicht beeinträchtigt ist. Diese Ergebnisse bieten neue Einblicke in die intrazellulären Prozesse der HIV-1 Freisetzung und präsentieren PS-Immunoliposomen als neuen potentiellen nanomedizinischen Ansatz zur Virusbeseitigung und HIV-Antigenpräsentation. / Human immunodeficiency virus 1 (HIV-1) hijacks proteins and signaling pathways of the host cell for its own life cycle, thereby impairing many functions of immune cells, including pathogen-phagocytosis by macrophages. The overall weakening of immune functions eventually results in the development of acquired immunodeficiency syndrome (AIDS). HIV-1 assembly takes place at the plasma membrane as well as in endosomal compartments and is governed by the structural polyprotein Gag, which is also sufficient for the release of virus-like particles (VLPs) from the host cell. It was shown that an induced high cytoplasmic calcium concentration increases the amount of VLPs in endo-lysosomal compartments and results in a dramatic enhancement of VLP release [1,2]. However, the mechanism by which calcium can promote the release of VLPs remains to be determined. The first part of this work shows that release of calcium from lysosomes causes fusion between endosomes and lysosomes as well as exocytosis of lysosomeentrapped VLPs. This mechanism is regulated by Synaptotagmin VII and prevents degradation of a part of the late endosome- and lysosome-entrapped VLPs. The second part focuses on the development of a nanobiotechnological system for the clearance of Env/Gag-VLPs (HIV-VLPs) by macrophages. This system is based on immunoliposomes that (1) bind HIV-VLPs via anti-Env antibodies and (2) are phagocytosed by macrophages due to the presence of phosphatidylserine (PS), an apoptotic signal. Essentially, the PS-liposomes mimic apoptotic cells thereby inducing internalization and lysosomal delivery of bound HIV-VLPs. These immunoliposomes exploit an efficient internalization pathway not impaired upon HIV-1 infection. The results of this thesis provide new insights into the intracellular pathways controlling HIV-1 release and demonstrate that PS-immunoliposomes can represent a novel nanomedical approach for viral clearance and HIV antigen presentation.

La 1-β-D-arabinofuranosylcytosine (ara-C) demeure l’agent anticancéreux principalement utilisé dans le traitement de la leucémie myéloblastique aiguë (LMA), malgré sa dégradation et son élimination rapide après une administration parentérale. Son encapsulation dans des vecteurs pharmaceutiques, majoritairement des liposomes, a permis de surmonter ces inconvénients. L’objectif général de ce projet de doctorat était de développer deux systèmes à libération prolongée, à base de phospholipides, de cholestérol et de poly(éthylène glycol) (PEG) afin d’encapsuler l’ara-C et ultimement, d’améliorer son efficacité dans le traitement de la LMA. Des Sphérulites® (vésicules multilamellaires d’un type particulier) ont d’abord été étudiées pour leur forte capacité d’encapsulation, due à leur mode de préparation. Par la suite, une formulation liposomale capable, d’une part de cibler spécifiquement les cellules leucémiques et, d’autre part, de promouvoir la libération intracellulaire de l’ara-C grâce à sa sensibilité au pH, a été mise au point. Les deux formulations se devaient d’avoir un faible diamètre, une stabilité en présence de fluides biologiques et des temps de circulation prolongés chez l’animal. Une préparation de Sphérulites®, composée de Phospholipon 90G, de Solutol HS15 et de cholestérol, a permis d’obtenir des vésicules de 300 nm de diamètre. Un dérivé lipidique de PEG a pu être fixé à leur surface, sans modifier la disposition concentrique des lamelles, ni changer leur stabilité. Les Sphérulites® PEGylées ont été chargées d’ara-C et injectées chez le rat par la voie intraveineuse. Elles ont démontré des temps de circulation significativement prolongés comparativement aux Sphérulites® sans PEG. Cependant, l’ara-C s’est retrouvée éliminée de la circulation sanguine très rapidement, révélant une libération précoce du principe actif à partir de ces vésicules. Les liposomes sensibles au pH (~150 nm) ont été obtenus suite à l’insertion d’un copolymère à base de dioctadécyle, de N-isopropylacrylamide (NIPAM) et d’acide méthacrylique. L’anticorps anti-CD33, soit complet soit son fragment Fab’, a été fixé à la surface des liposomes afin de cibler les cellules leucémiques. Les essais in vitro ont démontré la spécificité de la formulation pour différentes cellules leucémiques (CD33+), sa stabilité en présence de protéines plasmatiques et la libération intracellulaire d’un marqueur fluorescent et de l’ara-C. Enfin, des études menées chez la souris saine et immunodéprimée inoculée de cellules HL60 ont montré que la formulation exposant le fragment Fab’ possédait un profil pharmacocinétique et une biodistribution semblables à ceux des liposomes contrôles non-ciblés. L’encapsulation de l’ara-C a permis d’améliorer grandement ses temps de circulation après une administration intraveineuse. Cependant, bien que les immunoliposomes ont permis de prolonger la survie des souris leucémiques comparativement à l’ara-C libre, l’addition du polymère sensible au pH n’a pas permis d’apporter de réel avantage à la formulation lorsque administrée in vivo. Les résultats obtenus dans ce travail de thèse ont, dans un premier temps, mis en évidence que les Sphérulites® pourraient s’avérer utiles dans la vectorisation d’agents anticancéreux si leur capacité à retenir le principe actif in vivo était améliorée. Dans un second temps, les données présentées avec les immunoliposomes suggèrent qu’ils pourraient apporter un bénéfice notable dans le traitement de la LMA. / Despite its rapid degradation and fast elimination in vivo, 1-β-D-arabinofuranosylcytosine (ara-C) is the main anticancer agent used in the treatment of acute myeloid leukemia (AML). The encapsulation of this drug into nanocarriers such as liposomes has been shown to improve its stability, pharmacokinetic profile and, consequently, the treatment efficacy. The purpose of this doctoral work was to develop two nanocarriers employing phospholipids, cholesterol and poly(ethylene glycol) (PEG) to encapsulate ara-C, with the ultimate goal of developing more efficient treatments for AML. The first formulation relied on Spherulites®, which are multilamellar vesicles possessing high entrapment yields due to their fabrication method. In a second part, pH-sensitive immunoliposomes were optimized to target specifically the leukemia cells and promote the release of the loaded ara-C at the desired intracellular site. Both formulations required a small diameter, stability in the presence of biological fluids and long circulation time properties when injected intravenously. An optimized formulation of Spherulites® was developed. It was composed of Phospholipon 90G, Solutol HS15 and cholesterol. The vesicles (300 nm) were able to accommodate PEG-lipid derivatives at their surface without altering their concentric lamellar shape and their in vitro stability. The PEGylated Spherulites® were loaded with ara-C and injected intravenously into rats. The surface-modified vesicles exhibited longer circulation times compared to uncoated Spherulites®. However, most of the loaded-drug was cleared from the systemic circulation very rapidly, reflecting rapid leakage of ara-C from the vesicles. The pH-sensitive immunoliposomes (~150 nm) were obtained by including a terminally-alkylated copolymer made of dioctadecyl, N-isopropylacrylamide (NIPAM) and methacrylic acid in the liposome bilayer. The whole monoclonal antibody anti-CD33 or its Fab’ fragment were grafted on liposomes to target leukemic cells. In vitro assays revealed that this formulation was really specific for the various CD33+ leukemic cell lines, stable in presence of blood proteins, and able to promote the intracellular release of an encapsulated fluorescent probe as well as ara-C. In vivo studies in naïve Balb/c and immunodeprimed (SCID) mice inoculated with HL60 cells confirmed that the anti-CD33 Fab’ targeted formulation possessed pharmacokinetic and biodistribution profiles similar to those of the non-targeted liposomes. The encapsulation of ara-C in this formulation improved substantially its circulation time after intravenous injection. However, although ara-C-loaded immunoliposomes were able to prolong the survival of leukemic mice compared to the free drug, the addition of pH-sensitive polymer did not add any benefit to the formulation. Although these formulations require some optimization, the first part of this work pointed out that Spherulites® could be used to deliver anticancer agents provided that leakage is reduced in vivo. On the other hand, the data obtained with the targeted immunoliposomes suggest that these carriers could be beneficial in the treatment of AML.

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