Optimization of a vector for immunotherapy with dendritic cells : double hydrophilic block copolymer micelles

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

Polymeric micelles based on poly(methacrylic acid) block-containing copolymers with different membrane destabilizing properties for cellular drug delivery

International audiencePoly(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

Nucleoside-Lipid-Based Nanocarriers for Sorafenib Delivery

Abstract 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 ( 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

Versatile polyion complex micelles for peptide and siRNA vectorization to engineer tolerogenic dendritic cells

International audienc

Pancreatic lipase-related protein 2 and lysosomal phospholipase A2 process mycobacterial multiacylated lipid antigens

International audienc

Development of tripartite polyion micelles for efficient peptide delivery into dendritic cells without altering their plasticity

International audienceFor 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-Llysine 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 pHsensitivity property of themicelles, 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

Additional file 1: of Nucleoside-Lipid-Based Nanocarriers for Sorafenib Delivery

DLS data, zeta potentials, phase contrast microscopy, and TEM of SLNs synthetized in different experimental conditions, UHPLC dosages. (PDF 14710 kb

Assessment, endoscopy, and treatment in patients with acute severe ulcerative colitis during the COVID-19 pandemic (PROTECT-ASUC): a multicentre, observational, case-control study

BackgroundThere is a paucity of evidence to support safe and effective management of patients with acute severe ulcerative colitis during the COVID-19 pandemic. We sought to identify alterations to established conventional evidence-based management of acute severe ulcerative colitis during the early COVID-19 pandemic, the effect on outcomes, and any associations with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and severe COVID-19 outcomes. MethodsThe PROTECT-ASUC study was a multicentre, observational, case-control study in 60 acute secondary care hospitals throughout the UK. We included adults (≥18 years) with either ulcerative colitis or inflammatory bowel disease unclassified, who presented with acute severe ulcerative colitis and fulfilled the Truelove and Witts criteria. Cases and controls were identified as either admitted or managed in emergency ambulatory care settings between March 1, 2020, and June 30, 2020 (COVID-19 pandemic period cohort), or between Jan 1, 2019, and June 30, 2019 (historical control cohort), respectively. The primary outcome was the proportion of patients with acute severe ulcerative colitis receiving rescue therapy (including primary induction) or colectomy. The study is registered with ClinicalTrials.gov, NCT04411784. FindingsWe included 782 patients (398 in the pandemic period cohort and 384 in the historical control cohort) who met the Truelove and Witts criteria for acute severe ulcerative colitis. The proportion of patients receiving rescue therapy (including primary induction) or surgery was higher during the pandemic period than in the historical period (217 [55%] of 393 patients vs 159 [42%] of 380 patients; p=0·00024) and the time to rescue therapy was shorter in the pandemic cohort than in the historical cohort (p=0·0026). This difference was driven by a greater use of rescue and primary induction therapies with biologicals, ciclosporin, or tofacitinib in the COVID-19 pandemic period cohort than in the historical control period cohort (177 [46%] of 387 patients in the COVID-19 cohort vs 134 [36%] of 373 patients in the historical cohort; p=0·0064). During the pandemic, more patients received ambulatory (outpatient) intravenous steroids (51 [13%] of 385 patients vs 19 [5%] of 360 patients; p=0·00023). Fewer patients received thiopurines (29 [7%] of 398 patients vs 46 [12%] of 384; p=0·029) and 5-aminosalicylic acids (67 [17%] of 398 patients vs 98 [26%] of 384; p=0·0037) during the pandemic than in the historical control period. Colectomy rates were similar between the pandemic and historical control groups (64 [16%] of 389 vs 50 [13%] of 375; p=0·26); however, laparoscopic surgery was less frequently performed during the pandemic period (34 [53%] of 64] vs 38 [76%] of 50; p=0·018). Five (2%) of 253 patients tested positive for SARS-CoV-2 during hospital treatment. Two (2%) of 103 patients re-tested for SARS-CoV-2 during the 3-month follow-up were positive 5 days and 12 days, respectively, after discharge from index admission. Both recovered without serious outcomes. InterpretationThe COVID-19 pandemic altered practice patterns of gastroenterologists and colorectal surgeons in the management of acute severe ulcerative colitis but was associated with similar outcomes to a historical cohort. Despite continued use of high-dose corticosteroids and biologicals, the incidence of COVID-19 within 3 months was low and not associated with adverse COVID-19 outcomes