Lipoplexes for effective in vitro delivery of microRNAs to adult human cardiac fibroblasts for perspective direct cardiac cell reprogramming

Design of nanocarriers for efficient miRNA delivery can significantly improve miRNA-based therapies. Lipoplexes based on helper lipid, dioleoyl phosphatidylethanolamine (DOPE) and cationic lipid [2-(2,3-didodecyloxypropyl)-hydroxyethyl] ammonium bromide (DE) were formulated to efficiently deliver miR-1 or a combination of four microRNAs (miRcombo) to adult human cardiac fibroblasts (AHCFs). Lipoplexes with amino-to-phosphate groups ratio of 3 (N/P 3) showed nanometric hydrodynamic size (372 nm), positive Z-potential (40 mV) and high stability under storage conditions. Compared to commercial Dharma FECT1 (DF), DE-DOPE/miRNA lipoplexes showed superior miRNA loading efficiency (99 % vs. 64 %), and faster miRNA release (99 % vs. 82 % at 48 h). DE-DOPE/miR-1 lipoplexes showed superior viability (80-100 % vs. 50 %) in AHCFs, a 2-fold higher miR-1 expression and Twinfilin-1 (TWF-1) mRNA down regulation. DE-DOPE/ miRcombo lipoplexes significantly enhanced AHCFs reprogramming into induced cardiomyocytes (iCMs), as shown by increased expression of CM markers compared to DF/miRcombo. (C) 2022 The Authors. Published by Elsevier Inc

L-Carnitine Functionalization to Increase Skeletal Muscle Tropism of PLGA Nanoparticles

Muscular dystrophies are a group of rare genetic pathologies, encompassing a variety of clinical phenotypes and mechanisms of disease. Several compounds have been proposed to treat compromised muscles, but it is known that pharmacokinetics and pharmacodynamics problems could occur. To solve these issues, it has been suggested that nanocarriers could be used to allow controlled and targeted drug release. Therefore, the aim of this study was to prepare actively targeted poly(lactide-co-glycolide) (PLGA) nanoparticles (NPs) for the treatment of muscular pathologies. By taking advantage of the high affinity for carnitine of skeletal muscle cells due to the expression of Na+-coupled carnitine transporter (OCTN), NPs have been actively targeted via association to an amphiphilic derivative of L-carnitine. Furthermore, pentamidine, an old drug repurposed for its positive effects on myotonic dystrophy type I, was incorporated into NPs. We obtained monodispersed targeted NPs, with a mean diameter of about 100 nm and a negative zeta potential. To assess the targeting ability of the NPs, cell uptake studies were performed on C2C12 myoblasts and myotubes using confocal and transmission electron microscopy. The results showed an increased uptake of carnitine-functionalized NPs compared to nontargeted carriers in myotubes, which was probably due to the interaction with OCTN receptors occurring in large amounts in these differentiated muscle cells

Hyaluronated and PEGylated Liposomes as a Potential Drug-Delivery Strategy to Specifically Target Liver Cancer and Inflammatory Cells

Hepatocellular carcinoma (HCC) is the most frequent primary liver cancer and is characterized by poor clinical outcomes, with the majority of patients not being eligible for curative therapy and treatments only being applicable for early-stage tumors. CD44 is a receptor for hyaluronic acid (HA) and is involved in HCC progression. The aim of this work is to propose HA- and PEGylated-liposomes as promising approaches for the treatment of HCC. It has been found, in this work, that CD44 transcripts are up-regulated in HCC patients, as well as in a murine model of NAFLD/NASH-related hepatocarcinogenesis. Cell culture experiments indicate that HA-liposomes are more rapidly and significantly internalized by Huh7 cells that over-express CD44, compared with HepG2 cells that express low levels of the receptor, in which the uptake seems due to endocytic events. By contrast, human and murine macrophage cell lines (THP-1, RAW264.7) show improved and rapid uptake of PEG-modified liposomes without the involvement of the CD44. Moreover, the internalization of PEG-modified liposomes seems to induce polarization of THP1 towards the M1 phenotype. In conclusion, data reported in this study indicate that this strategy can be proposed as an alternative for drug delivery and one that dually and specifically targets liver cancer cells and infiltrating tumor macrophages in order to counteract two crucial aspect of HCC progression

Nanosystèmes biocompatibles pour des applications innovantes de petites molécules thérapeutiques

La nanomédecine est une stratégie efficace pour améliorer le potentiel clinique de molécules actives par la formulation de systèmes d'administration de médicaments (DDSs). L'un des principaux obstacles au développement de ces systèmes réside dans leur toxicité éventuelle. L'objectif de ce travail était de développer des nanoparticules (NPs) biocompatibles pour des applications innovantes de petites molécules thérapeutiques. Des NPs à base de lipides et de polymères ont été conçus pour améliorer le potentiel thérapeutique de la pentamidine (PTM) en tant que médicament anticancéreux, en exploitant la technique classique de nanoprécipitation. Cependant, les difficultés de fabrication ont ouvert la voie au développement d’un processus de production innovant. Pour relever les défis de la formulation des NPs, la technologie microfluidique a été proposée pour ses avantages. Ainsi, une étude de transposition de la nanoprécipitation classique au processus microfluidique pour la fabrication de NPs à base d’acide poly(lactique-co-glycolique) (PLGA) a été mise en place, en choisissant un médicament modèle. En outre, l'évaluation biologique des NPs chargées, impliquée dans la réduction de l'inflammation et de la fibrose dans les dystrophies musculaires, a été évaluée. Les résultats biologiques ont démontré une réduction d'une cytokine pro-inflammatoire en présence du médicament. L'administration in vivo de NPs chargées a entraîné une amélioration de l'homéostasie musculaire. Dans la dernière partie du projet, la stéaroyl-L-carnitine (SC) a été utilisée pour marquer les NPs polymériques et cibler activement des récepteurs musculaires spécifiques, à savoir OCTN2. Des tests in vitro sur la lignée cellulaire C2C12 ont démontré une plus grande expression du récepteur OCTN2 sur les myotubes que sur les myoblastes, ce qui a entraîné une plus grande internalisation des NPs. Dans le cadre du repositionnement de médicaments, le PTM a été efficacement encapsulé dans des NPs marquées avec de la SC pour améliorer l'administration de médicaments aux cellules musculaires. Dans l'ensemble, cette thèse a permis d’étudier de manière approfondie les DDS à base de lipides et de polymères pour améliorer l’efficacité thérapeutique de deux petites molécules et a également souligné l'importance de la technique de fabrication sur les caractéristiques des NPs.Nanomedicine is a potential strategy to improve the clinical applicability of active molecules through the formulation of drug delivery systems (DDSs). One of the main hurdles for developing DDSs lies in their potential toxicity. The aim of the present work was the development of biocompatible nanoparticles (NPs) for innovative applications of small therapeutic molecules. Lipid and polymer-based nanocarriers were designed to improve the therapeutical applicability of pentamidine (PTM) as repurposed anticancer drug, avoiding drug derivatization and exploiting the classical nanoprecipitation technique. However, manufacturing limitations opened the way to the development of innovative production processes. To address challenges in NP formulation, the microfluidic technology has been proposed for its advantages. Thus, a manufacturing translation study of poly(lactic-co-glycolic acid) (PLGA)-based NPs from conventional nanoprecipitation to the microfluidic process was set up, choosing an hydrophobic model drug. Moreover, biological evaluation of loaded NPs, involved in reducing inflammation and fibrosis in muscular dystrophy, was assessed. Biological findings demonstrated a reduction in a pro-inflammatory cytokine in presence of drug. In vivo administration of loaded NPs resulted in an improvement of muscular homeostasis. In the last part of the project, stearoyl-L-carnitine (SC) was used to actively target polymer NPs toward muscular specific receptors, namely OCTN2. In vitro tests on C2C12 cell line demonstrated a higher expression of OCTN2 receptor on myotubes than myoblasts, occurring in increased NPs internalization. In the frame of drug repurposing, PTM has been efficiently encapsulated into SC-associated NPs to enhance drug delivery toward muscular cells. Overall, this thesis provided an extensive investigation of lipid and polymer-based DDSs to improve the therapeutic applicability of two small molecules and underlined the importance of the manufacturing technique for NP features