Les publications de l’UMR
Carbon dots (CDs) have been intensively investigated due to their unique photoluminescence (PL) properties that are improved through surface passivation with nitrogen-containing groups. Recently, gene delivery applications emerged as passivation of CDs may yield positively charged nanoparticles that can interact with negatively charged nucleic acids. However previous work in the field focused on the use of high molecular weight polyamines for CD passivation, posing the problem of the separation of nanoparticles from residual polymer that is harmful to cells. In this work, cationic CDs were prepared by pyrolysis of citric acid/bPEI600 (1/4, w/w) so unreacted low molecular weight reagents could be conveniently eliminated by extensive dialysis. Various reaction conditions and activation modes were evaluated and eleven CDs that exhibited superior solubility in water were produced. All the nanoparticles were characterized with respect to their physical, optical and PL properties and their ability to deliver plasmid DNA to mammal cells was evaluated. Despite their similar physical properties, the CDs displayed marked differences in their gene delivery efficiency. CDs produced under microwave irradiation in a domestic oven were revealed to be superior to all the other nanoparticles produced in this study and compared to the gold standard transfection reagent bPEI25k, with an optimal CD/pDNA w/w ratio that was significantly down shifted, as was the associated cytotoxicity.
A chronic antigenic stimulation is believed to sustain the leukemogenic development of chronic lymphocytic leukemia (CLL) and most of lymphoproliferative malignancies developed from mature B cells. Reproducing a proliferative stimulation ex vivo is critical to decipher the mechanisms of leukemogenesis in these malignancies. However, functional studies of CLL cells remains limited since current ex vivo B cell receptor (BCR) stimulation protocols are not sufficient to induce the proliferation of these cells, pointing out the need of mandatory BCR co-factors in this process. Here, we investigated benefits of several BCR co-stimulatory molecules (IL-2, IL-4, IL-15, IL-21 and CD40 ligand) in multiple culture conditions. Our results demonstrated that BCR engagement (anti-IgM ligation) concomitant to CD40 ligand, IL-4 and IL-21 stimulation allowed CLL cells proliferation ex vivo. In addition, we established a proliferative advantage for ZAP70 positive CLL cells, associated to an increased phosphorylation of ZAP70/SYK and STAT6. Moreover, the use of a tri-dimensional matrix of methylcellulose and the addition of TLR9 agonists further increased this proliferative response. This ex vivo model of BCR stimulation with T-derived cytokines is a relevant and efficient model for functional studies of CLL as well as lymphoproliferative malignancies.
Efficiency of polyethylenimine (PEI) for nucleic acid delivery is affected by the size of the carrier and length of the nucleic acids. For instance, PEIs with molecular weights between 10-30kDa provide optimal DNA delivery activity whereas PEIs with molecular weights below 1.8kDa are ineffective. The activity of PEI is also severely diminished by substitution of DNA for shorter nucleic acids such as mRNA or siRNA. Here, through chemical modification of the primary amines to aromatic domains we achieved nucleic acid delivery by the 1.8kDa polyethylenimine (PEI) particles. This modification did not affect the PEI buffering abilities but enhanced its pH-sensitive aggregation, enabling stabilization of the polyplex outside the cell while still allowing nucleic acid release following cellular entry. The aromatic PEIs were then evaluated for their gene, mRNA, siRNA and 2'O-methyl phosphorothioate oligonucleotide in vitro transfection abilities. The salicylamide-grafted PEI showed to be a reliable carrier for delivering nucleic acids with cytoplasmic activity such as the mRNA and siRNA or nuclear diffusible oligonucleotide. It was then further equipped with polyethyleneglycol (PEG) and the delivery efficiency of the copolymer was tested in vivo for regeneration of dystrophin in the muscle of mdx mouse through a 2'O-methyl phosphorothioate-mediated splicing modulation. Intramuscular administration of polyplexes resulted in dystrophin-positive fibers in a mouse model of Duchenne muscular dystrophy without apparent toxicity. These findings indicate that precise modifications of low molecular weight PEI improve its bio-responsiveness and yield delivery vehicles for nucleic acids of various types in vitro and in vivo.
Mcl-1, which is an anti-apoptotic member of the Bcl-2 protein family, is overexpressed in various cancers and promotes the aberrant survival of tumor cells. To inhibit Mcl-1, and initiate apoptosis, an interaction between BH3-only proteins and Mcl-1 anti-apoptotic protein is necessary. These protein-protein interactions exhibit some selectivity: Mcl-1 binds specifically to Noxa, whereas Bim and Puma bind strongly to all anti-apoptotic proteins. Even if the three-dimensional (3D) structures of several Mcl-1/BH3-only complexes have been solved, the BH3-only binding specificity to Mcl-1 is still not completely understood. In this study, molecular dynamics simulations were used to elucidate the molecular basis of the interactions with Mcl-1. Our results corroborate the importance of four conserved hydrophobic residues and a conserved aspartic acid on BH3-only as a common binding pattern. Furthermore, our results highlight the contribution of the fifth hydrophobic residue in the C-terminal part and a negatively charged patch in the N-terminal of BH3-only peptides as important for their fixation to Mcl-1. We hypothesize that this negatively charged patch will be an Mcl-1 specific binding pattern.
Medical application of siRNAs relies on methods for delivering nucleic acids into the cytosol. Synthetic carriers, which assemble with nucleic acids into delivery systems, show promises for cancer therapy but efficiency remains to be improved. In here, the effectiveness of pyridylthiourea-polyethylenimine (pi PEI), a siRNA carrier that favors both polyplex disassembly and endosome rupture upon sensing the acidic endosomal environment, in 3 experimental models of hepatocellular cancer is tested. The pi PEI-assisted delivery of a siRNA targeting the polo-like kinase 1 into Huh-7 monolayer produces a 90% cell death via a demonstrated RNA interference mechanism. Incubation of polyplex with Huh-7 spheroids leads to siRNA delivery into the superficial first cell layer and a 60% reduction in spheroid growth compared to untreated controls. Administration of polyplexes into mice bearing subcutaneous implanted Huh-7Luc tumors results in a reduced tumor progression, similar to the one observed in the spheroid model. Altogether, these results support the in vivo use of synthetic and dedicated polymers for increasing siRNA-mediated gene knockdown, and their clinical promise in cancer therapeutics.
Gene delivery to skeletal muscle is a promising strategy for the treatment of muscle disorders and for the systemic secretion of therapeutic proteins. In addition, muscle is an attractive target tissue because it is easily accessible. However, very few synthetic vectors proved capable of surpassing naked DNA mediated muscle gene transfer. In fact, only neutral copolymers, in particular poloxamers, demonstrated capacities to increase transgene expression in skeletal muscles. Here, we studied in vitro and in vivo behaviour of different bile salts. We report that sodium deoxycholate (DOC) and derivatives thereof increase after intramuscular injection by more than 100-fold the levels of the reporter gene luciferase compared to naked DNA. Using a LacZ expression cassette, we found that more than 20% of the muscle fibers expressed the reporter gene. Prolonged expression of a secreted reporter gene derived from a natural murine alkaline phosphatase enzyme could be documented. Altogether, our results demonstrate that bile salts belong to the most efficient chemicals identified so far for skeletal muscle gene transfer. Importantly, since these compounds are naturally found in the body, there is no risk of immune response against them and in addition several bile salts are already used in human medicine. Bile salt mediated muscle gene transfer may thus have broad applications in gene therapy.
The potent antitumor effect of alpha-galactosylceramide (alpha-GalCer) is based on its recognition by invariant Natural Killer T cells (iNKT) after its capture and presentation by antigen presenting cells including dendritic cells (DCs). Synthetic alpha-GalCer has already been tested in advanced cancer patients but no or only moderate clinical responses were obtained. To optimize alpha-GalCer efficacy, we have postulated that alternative formulations impacting its molecular organization in aqueous medium could modify DC uptake and iNKT-based immune responses. To this end, we have developed two strategies: (1) the formulation of alpha-GalCer in non-cationic liposomes and (2) the synthesis of a water-soluble alpha-GalCer analogue by anchoring a polyethyleneglycol moiety on its sugar head. The biological activities of these new preparations were compared to that induced by the classically used Polysorbate 20 alpha-GalCer micelles. Both formulations retained their uptake by DCs and activated iNKT cells both in vitro and in vivo. Despite a lower cytokine production, the formulations induced a potent immune response able to control lung murine carcinoma. In conclusion, it is possible to increase alpha-GalCer solubility in aqueous solution without limiting its antitumor properties.
The histidine-rich designer peptides of the LAH4 family exhibit potent antimicrobial, transfection, transduction and cell-penetrating properties. They form non-covalent complexes with their cargo, which often carry a negative overall charge at pH 7.4 and include a large range of molecules and structures such as oligonucleotides, including siRNA and DNA, peptides, proteins, nanodots and adeno-associated viruses. These complexes are thought to enter the cells through an endosomal pathway where the acidification of the organelle is essential for efficient endosomal escape. Biophysical measurements indicate that, upon acidification, almost half the peptides are released from DNA cargo, leading to the suggestion of a self-promoted uptake mechanism where the liberated peptides lyse the endosomal membranes. LAH4 derivatives also help in cellular transduction using lentiviruses. Here, we compare the DNA transfection activities of LAH4 derivatives, which vary in overall charge and/or the composition in the hydrophobic core region. In addition, LAH4 is shown to mediate the transport of functional beta-galactosidase, a large tetrameric protein of about 0.5 MDa, into the cell interior. Interestingly, the LAH1 peptide efficiently imports this protein, while it is inefficient during DNA transfection assays. Copyright (c) 2017 European Peptide Society and John Wiley & Sons, Ltd.
Physical oncology is an emerging paradigm which recognizes tissue mechanics, per se, as an active modulator of tumorigenesis, treatment resistance and clinical outcome, mediated by mechanosignaling pathways, matrix remodeling and physical barriers to drugs. The tumor microenvironment displays abnormal physical properties in comparison to healthy tissue which contribute to cancer progression and resistance to current treatments. Physical aberrancies comprise the chaotic organization of tumor vasculature, an increased interstitial pressure, an increased solid stress, hypoxia, an abundant extracellular matrix and a progressive stiffening of solid tumors. The physical barriers in tumors are of critical importance, as tissue mechanics compromises drug delivery, reduces immune cell infiltration and promotes disease aggressiveness. All these physical hallmarks of cancer, although not fully understood, are inspiring new anticancer strategies aiming to target and normalize the physical anomalies of solid tumors, particularly in the field of nanomedicine. Here we summarize the recent paradigm shift of physical oncology and review some of the proposed strategies using nanomaterials to tackle the tumor microenvironment and its aberrant physical properties. Nanomedicine might harness the features of the tumor microenvironment in order to improve nanoparticle and drug delivery, or propose nano-agents that can be activated on demand to achieve a tailored spatio-temporal modulation of the tumor microenvironment, reduce tumor pressure and stiffness and alleviate the resistance to current treatments.
The aim of the present research work was the synthesis of molecularly imprinted polymers (MIPs) with a rod-like geometry via "mesophase polymerization". The ternary lyotropic system consisting of sodium dodecyl sulfate (SDS), water, and decanol was chosen to prepare a hexagonal mesophase to direct the morphology of the synthesized imprinted polymers using theophylline, methacrylic acid, and ethylene glycol dimethacrylate as a drug model template, a functional monomer, and a crosslinker, respectively. The obtained molecularly imprinted microrods (MIMs) were assessed by performing binding experiments and in vitro release studies, and the obtained results highlighted good selective recognition abilities and sustained release properties. In conclusion, the adopted synthetic strategy involving a lyotropic mesophase system allows for the preparation of effective MIPs characterized by a rod-like morphology.