Although the application of sorafenib, a small inhibitor of tyrosine protein kinases, to cancer treatments remains a worldwide option in chemotherapy, novel strategies are needed to address the low ...water solubility (< 5 μM), toxicity, and side effects issues of this drug. In this context, the use of nanocarriers is currently investigated in order to overcome these drawbacks. In this contribution, we report a new type of sorafenib-based nanoparticles stabilized by hybrid nucleoside-lipids. The solid lipid nanoparticles (SLNs) showed negative or positive zeta potential values depending on the nucleoside-lipid charge. Transmission electron microscopy of sorafenib-loaded SLNs revealed parallelepiped nanoparticles of about 200 nm. Biological studies achieved on four different cell lines, including liver and breast cancers, revealed enhanced anticancer activities of Sorafenib-based SLNs compared to the free drug. Importantly, contrast phase microscopy images recorded after incubation of cancer cells in the presence of SLNs at high concentration in sorafenib (> 80 μM) revealed a total cancer cell death in all cases. These results highlight the potential of nucleoside-lipid-based SLNs as drug delivery systems.
Complex antigens require processing within antigen-presenting cells (APCs) to form T cell stimulatory complexes with CD1 antigen-presenting molecules. It remains unknown whether lipids with ...multi-acylated moieties also necessitate digestion by lipases to become capable of binding CD1 molecules and stimulate T cells. Here, we show that the mycobacterial tetra-acylated glycolipid antigens phosphatidyl-myo-inositol mannosides (PIM) are digested to di-acylated forms by pancreatic lipase-related protein 2 (PLRP2) and lysosomal phospholipase A2 (LPLA2) within APCs. Recombinant PLRP2 and LPLA2 removed the sn1- and sn2-bound fatty acids from the PIM glycerol moiety, as revealed by mass spectrometry and nuclear magnetic resonance studies. PLRP2 or LPLA2 gene silencing in APCs abolished PIM presentation to T cells, thus revealing an essential role of both lipases in vivo. These findings show that endosomal lipases participate in lipid antigen presentation by processing lipid antigens and have a role in T cell immunity against mycobacteria.
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•CD1b-restricted T cells recognize unusual di-acylated species of mycobacterial PIM•PLRP2 and LPLA2 lysosomal lipases digest tetra- into di-acylated PIM in vitro•PLRP2 and LPLA2 siRNA in APCs abrogate presentation of tetra-acylated PIM to T cells•PIM presentation to CD1b-restricted T cells requires both PLRP2 and LPLA2
Complex antigenic glycolipids require processing to form T cell stimulatory complexes with CD1 proteins. Gilleron et al. demonstrate that, in addition to the glycosidic part, the lipid moiety of multi-acylated mycobacterial glycolipids also needs to be processed by lysosomal lipases.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
•Study of poly(methacrylic acid) based double hydrophilic block copolymers.•Formation of micelles whatever their molecular weight.•Evidence that they could have destabilizing properties in acidic ...conditions.•Membrane destabilization requires small molecular weight and symmetric copolymers.•Copolymer-based micelles are vectors with different intracellular traffic pathways.
Poly(methacrylic acid)-b-poly(ethylene oxide) are double hydrophilic block copolymers, which are able to form micelles by complexation with a counter-polycation, such as poly-l-lysine. A study was carried out on the ability of the copolymers to interact with model membranes as a function of their molecular weights and as a function of pH. Different behaviors were observed: high molecular weight copolymers respect the membrane integrity, whereas low molecular weight copolymers with a well-chosen asymmetry degree can induce a membrane alteration. Hence by choosing the appropriate molecular weight, micelles with distinct membrane interaction behaviors can be obtained leading to different intracellular traffics with or without endosomal escape, making them interesting tools for cell engineering. Especially micelles constituted of low molecular weight copolymers could exhibit the endosomal escape property, which opens vast therapeutic applications. Moreover micelles possess a homogeneous nanometric size and show variable properties of disassembly at acidic pH, of stability in physiological conditions, and finally of cyto-tolerance.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
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Dendritic cells (DCs) are professional antigen-presenting cells that play a critical role in maintaining the balance between immunity and tolerance and, as such are a promising ...immunotherapy tool to induce immunity or to restore tolerance. The main challenge to harness the tolerogenic properties of DCs is to preserve their immature phenotype. We recently developed polyion complex micelles, formulated with double hydrophilic block copolymers of poly(methacrylic acid) and poly(ethylene oxide) blocks and able to entrap therapeutic molecules, which did not induce DC maturation. In the current study, the intrinsic destabilizing membrane properties of the polymers were used to optimize endosomal escape property of the micelles in order to propose various strategies to restore tolerance. On the first hand, we showed that high molecular weight (Mw) copolymer-based micelles were efficient to favor the release of the micelle-entrapped peptide into the endosomes, and thus to improve peptide presentation by immature (i) DCs. On the second hand, we put in evidence that low Mw copolymer-based micelles were able to favor the cytosolic release of micelle-entrapped small interfering RNAs, dampening the DCs immunogenicity. Therefore, we demonstrate the versatile use of polyionic complex micelles to preserve tolerogenic properties of DCs. Altogether, our results underscored the potential of such micelle-loaded iDCs as a therapeutic tool to restore tolerance in autoimmune diseases.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
For many years, a great deal of interest has been focusing on the optimization of peptide presentation by dendritic cells (DCs) using peptide-encapsulated particles, in order to enhance the immune ...response. Nowadays, DCs are also known to be involved in peripheral tolerance, inducing anergy or regulatory T lymphocytes. To preserve the plasticity of DCs, we formulated non-cytotoxic pH-sensitive polyion complex micelles based on an original tripartite association of polymethacrylic acid-b-polyethylene oxide, poly-
l-lysine and fluorescent-peptide: OVAFITC peptide, as a model drug. We demonstrated that the OVAFITC peptide was successfully entrapped into the micelles, released into DC endosomes thanks to the pH-sensitivity property of the micelles, and efficiently loaded onto MHC class II molecules. The phenotype as well as the cytokinic secretion profile of the mature and immature DCs loaded with peptide-encapsulated micelles was unaltered by the tripartite polyion micelles. The efficient loading of the peptide by immature and mature DCs was shown by the in vitro proliferation of OVA-specific transgenic T cells. Therefore, the present results show that the tripartite polyion complex micelles can be used as efficient peptide vectors immunogically inert for ex vivo DCs engineering without modifying their intrinsic immune plasticity.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
L'objectif de cette thèse repose sur le développement de micelles de polymères polyioniques, vecteurs de molécules thérapeutiques en immunothérapie avec des cellules dendritiques (DCs). Elles sont ...préparées à partir d'un copolymère à blocs double-hydrophiles, l'acide polyméthacrylique-b-polyoxyde d'éthylène (PMAA-b-POE) et d'un contre ion de charge opposée. De taille nanométrique, elles sont capables d'encapsuler des molécules thérapeutiques selon une association tripartite originale et de se désassembler à pH acide pour permettre leur libération dans le milieu endosomal.Le premier axe de travail a porté sur l'évaluation de la propriété des copolymères à induire un échappement endosomal en fonction de leur masse molaire en utilisant deux modèles membranaires (liposomes et globules rouges). La complexation des copolymères de masses molaires différentes avec la poly-L-lysine comme contre-ion a permis l'obtention de micelles avec des propriétés d'échappement endosomal variables. Cette propriété est intéressante car en fonction de la stratégie thérapeutique adoptée, elle orientera le choix de la masse molaire du copolymère pour la formulation des micelles.Le second axe a consisté en l'application de ces micelles pour la vectorisation d'un peptide modèle (peptide OVA) dans les DCs. La capacité des micelles à encapsuler le peptide et à le libérer au niveau des compartiments endosomaux a été évaluée par des techniques de spectrofluorimétrie et de microscopie confocale. Enfin, l'efficacité de présentation du peptide formulé dans les différentes micelles a été mise en évidence et a montré l'amélioration de la présentation par les DCs du peptide formulé dans les micelles comparé au peptide non formulé. Cette présentation est nettement supérieure en utilisant les micelles composées de copolymères de masse molaire élevée qui n'entraînent pas d'échappement endosomal.Le troisième axe de recherche a reposé sur la transfection des DCs avec des micelles de siRNA dirigés contre la protéine de surface CD86. Seules les micelles composées de copolymères de faible masse molaire ont permis l'encapsulation du siRNA et la baisse de l'expression de la protéine CD86 à la surface des DCs. Afin d'optimiser la capacité des micelles à encapsuler et transfecter les DCs, la formulation des micelles a été optimisée en remplaçant la PLL par un autre polycation la polyethylene imine PEI. Ces micelles polyioniques à base de copolymère PMAA-b-POE apparaissent donc comme des vecteurs de molécules d'intérêt thérapeutique prometteurs pour les cellules dendritiques en immunothérapie ou en thérapie génique.
The aim of the thesis work is based on the development of polymeric micelles vectors of therapeutic molecules in immunotherapy with dendritic cells (DCs). They are composed of a double hydrophilic blocks copolymers, poly(methacrylic acid)-b-poly(ethylene oxide) (PMAA-b-PEO) and an oppositely charged polyion. They are caracterized by a nanometric size, a capacity to encapsulate therapeutic molecules according to a tripartite association and are able to disassemble at acidic pH allowing the release of their cargo.The first part of this work has focused on the evaluation of the endosomal escape property of copolymers based on their molecular weight by using two membrane models (liposomes and red blood cells). Complexation of different molecular weight copolymers with poly- L- lysine as counter ion allowed the formation of micelles with variable endosomal escape properties. This property is interesting because according to the adopted therapeutic strategy, it will guide the choice of the copolymer micelles for formulation.The second part consisted of the application of these micelles for the vectorization of a model peptide (OVA peptide) in DCs. The ability of micelles to encapsulate and release this peptide in the endosomal compartments was assessed by fluorescence spectroscopy and confocal microscopy techniques. Finally, the effectiveness of the OVA presentation formulated in the different type of micelles has been demonstrated and shown that the peptide presentation by DCs was improved when it was formulated in micelles compared to unformulated peptide. This presentation was much higher using micelles composed of high molecular weight copolymers that do not involve endosomal escape.The third part of the research was based on the transfection of DCs with siRNA directed against CD86 protein surface. Only micelles composed of low molecular weight copolymers allowed the encapsulation of siRNA molecules and decreased the expression of CD86 protein on DCs surface. To increase the ability of micelles to encapsulate and transfect DCs, the micelle formulation was optimized by changing the PLL with another polycation PEI.These polyion micelles based PMAA-b-PEO copolymers appear as vectors of therapeutic molecules for promising strategies with dendritic cells such as vaccination and gene therapy.
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