Philippe Guégan

Gene transfer by chemical vectors, and endocytosis routes of polyplexes, lipoplexes and lipopolyplexes in a myoblast cell line.

Chemical vectors are widely developed for providing safe DNA delivery systems. It is well admitted that their endocytosis and intracellular trafficking are critical for the transfection efficiency. Here, we have compared the endocytic pathways of lipoplexes, polyplexes and lipopolyplexes formed with carriers of various chemical compositions. Engineered C2C12 mouse myoblast cells expressing Rab5-EGFP, Rab7-EGFP or Cav1-GFP were used to monitor the location of the plasmid DNA into the endocytic compartments by real time fluorescence confocal microscopy. We observed that (i) DNA complexes made with dioleyl succinyl paromomycin:O,O-dioleyl-N-histamine phosphoramidate (DOSP/MM27) liposomes or histidinylated lPEI (His-lPEI) allowing the highest transfection efficiency displayed a positive ζ potential and were internalized by clathrin-mediated endocytosis, (ii) DOSP/MM27 lipoplexes were 6-times more internalized than His-lPEI polyplexes, (iii) all negatively charged DNA complexes lead to less efficient transfection and entered the cells via caveolae and (iv) lipopolyplexes allowing high transfection efficiency were weakly internalized via caveolae. Our results indicate that the transfection efficiency is better correlated with the nature of the endocytic pathway than with the uptake efficacy. This study shows also that engineered cells expressing specific fluorescent compartments are convenient tools to monitor endocytosis of a fluorescent plasmid DNA by real time fluorescence confocal microscopy.

Histidinylated linear PEI: a new efficient non-toxic polymer for gene transfer

A series of linear polyethylenimine (lPEI) substituted with histidine residue (His-lPEI) was synthesized using the Michael reaction in order to provide new highly efficient vectors for gene therapy applications (up to 95% of transfected cells) with remarkable low cytotoxicity compared to lPEI-based polyplexes.

Variation of the mesh size of PEO-based networks filled with TFSILi: from an Arrhenius to WLF type conductivity behavior

The properties of ionically conducting membranes based on polyethylene oxide (PEO) networks crosslinked with urethane functions, containing lithium bis(trifluoromethane sulfone imide) (TFSILi) were studied. The temperature and salt concentration influence on the ionic conductivity of these materials was analyzed. The best conductivity value measured at 30°C was 7×10−5 S cm−1 at O/Li=43 for a PEO molecular weight between crosslinks of 2000 g mol−1. The curves obtained in the Arrhenius coordinates were typical of a William Landel Ferry (WLF) like behavior when the molecular weight of the PEO between crosslinks was 1000 or 2000 g mol−1. However, the network based on PEO600 followed an Arrhenius behavior, typical of a network where segmental movements are prevented. A strong interaction between TFSILi, ethylene oxide units and urethane crosslinks functions at high urethane concentration explains these results. This finding demonstrates that the two types of conductivity behavior (Arrhenius or WLF) can be found for the same type of polymer electrolyte depending on the predominant elementary conductivity process.

Lipopolyplexes comprising imidazole/imidazolium lipophosphoramidate, histidinylated polyethyleneimine and siRNA as efficient formulation for siRNA transfection

Lipopolyplexes formulations resulting from association of nucleic acid, cationic liposomes and a cationic polymer are attracting formulations for siRNA delivery. Herein, imidazole- and imidazolium-based liposomes in association with histidinylated polymers are studied to produce siRNA lipopoplyplexes (LPRi) subsequently used for gene silencing. Several kinds of imidazole/histidine liposomes and cationic polymers are tested. The gene silencing effect is evaluated with synthetic siRNA directed against EGFP or luciferase mRNA, in HeLa cells stably expressing EGFP or B16F10 melanoma cells stably expressing luciferase, respectively. SiRNA formulations are compared with those prepared using some commercial transfection reagents. One formulation called His-lPEI LPRi100 comprising siRNA, histidinylated lPEI (His-lPEI) and liposomes 100 made with O,O-dioleyl-N-[3N-(N-methylimidazolium iodide)propylene] phosphoramidate and O,O-dioleyl-N-histamine phosphoramidate appears to give the best specific inhibition of gene expression at 10nM siRNA in a dose-dependent manner with low cytotoxicity. This formulation exhibits a size and a zeta potential of 60 nm and +84 mV, respectively. According to our previous works, histidinylated lipopolyplexes appears as a versatile formulation for DNA, mRNA and siRNA transfection.

Ultrasensitive QRS made by supramolecular assembly of functionalized cyclodextrins and graphene for the detection of lung cancer VOC biomarkers

A novel electronic nose system comprising functionalized β-cyclodextrin wrapped reduced graphene oxide (RGO) sensors with distinct ability of discrimination of a set of volatile organic compounds has been developed. Non-covalent modification of chemically functionalized cyclodextrin with RGO is carried out by using pyrene adamantane as a linker wherever necessary, in order to construct a supramolecular assembly. The chemical functionality on cyclodextrin is varied utilising the principle of selective chemical modification of cyclodextrin. In the present study, the combined benefits of the host-guest inclusion complex formation ability and tunable chemical functionality of cyclodextrin, as well as the high surface area and electrical conductivity of graphene, are utilized for the development of a set of highly selective quantum resistive chemical vapour sensors (QRS), which can be assembled in an electronic nose.

Synthesis of Poly(2-methyl-2-oxazoline) Star Polymers with a β-Cyclodextrin Core

Synthesis of star polymers with a β-cyclodextrin (CD) core was undertaken using the arm-first, then the core-first strategy. Cationic ring opening polymerisation (CROP) of 2-methyl-2-oxazoline (MeOx) was first initiated by allyl bromide, and then quenched with heptakis(6-deoxy-6-amino)β-CD in order to get a 7-arm star polymer. Then heptakis(6-deoxy-6-iodo-2,3-di-O-acetyl)β-CD was synthesised in order to get an initiator for the CROP of MeOx. Initiation and propagation kinetic measurements were undertaken and the ratio kp/ki was found to be too high to provide a controlled polymerisation. Using iodine as co-initiator allowed a decrease of the kp/ki ratio that gave better control of the polymerisation. DOSY NMR and viscosity characterisations were undertaken, and both techniques lead to the demonstration of a lower hydrodynamic volume of the star polymers versus the linear counterparts, for compounds of the same molecular weight.

Synthesis and Evaluation of Amphiphilic Poly(tetrahydrofuran-b-ethylene oxide) Copolymers for DNA Delivery into Skeletal Muscle

PurposeAmphiphilic triblock copolymers such as the pluronic poly(ethylene oxide-b-propylene oxide-b-ethylene oxide) L64 (pEO13-pPO30-pEO13) have been shown to mediate more efficient gene transfer in muscle as compared to naked DNA. We were interested in studying the effect of a chemical change of the central block of pluronic polymers on the transfection activity.MethodsWe synthesized new amphiphilic copolymers in which the hydrophobic pPO block was replaced by poly(tetrahydrofuran) (pTHF) chains. The resulting triblock pEO–pTHF–pEO polymers have been characterized by NMR and SEC and assayed for in vitro and in vivo gene transfer.ResultsThe animal experiments showed that the new copolymers are able to significantly increase the transfection efficiency of plasmid DNA after intramuscular injection.ConclusionsThese results indicate that the capacity to enhance plasmid DNA transfection in skeletal muscle is not restricted to pEO–pPO–pEO arrangements.

Synthesis and electrochemical characterization of crosslinked poly(ethylene oxide) containing LiN(CF3SO2)2

Abstract The ionic conductivity of polymeric electrolyte systems comprising a network of poly(ethylene oxide) (PEO) crosslinked with urethane functions and containing LiN(CF3SO2)2 (TFSILi), was studied. Polyurethanes based on three different PEO molecular weights were prepared. Diffusion studies of TFSILi in swollen PEO-urethane membranes with acetonitrile were performed showing a diffusion coefficient for PEO2000 higher than that observed with LiClO4 in the same material, and were correlated with the variation of Tgs of the dried membranes which were determined as a function of salt concentration and crosslink density. The influence of a salt molecule on the Tg (for the TFSILi charged membranes) is equivalent to 25% of the influence of a covalent crosslink in the range of molecular weight between crosslinks used in this study. The relatively high slope for the plot Tg−1 vs salt concentration for the network in PEO600 allows to consider a high chain rigidification due to the introduction of salt. The conductivity of network based on PEO2000 with a molar concentration ratio O Li = 43 reached 10−5 S cm−1.

Anionic ring-opening polymerization of ethylene oxide in DMF with cyclodextrin derivatives as new initiators

Anionic polymerization initiated by cyclodextrins suffers from a poor solubility of those derivatives in standard polymerization solvents. The possibility to perform ethylene oxide polymerization initiated by monofunctional initiators (allyl alcohol, 2-methoxyethanol) by living ring opening polymerization in DMF, a good solvent for any CD derivative, was demonstrated by SEC, (1)H and (13)C NMR analyses. The study was extended to the use of native CD as initiator, leading to the synthesis of ill-defined structures, explained by the reactivity scale of the various hydroxyl functions. Two selectively modified CD derivatives are then used to synthesize a new family of star-shaped poly(ethylene oxide) polymers with CD core, having 14 or 21 arms. The polymerization was found to be living and DOSY experiments confirmed the well-defined structures for the synthesized star-polymers.

Far beyond primary poly(vinylamine)s through free radical copolymerization and amide hydrolysis

Due to their affinity for many supports, their pH responsiveness, metal binding capacity and polyelectrolyte complexation, poly(vinylamine) derivatives have attracted attention for many applications including coatings, water purification, or gas membrane separation. Nevertheless, most of them possess only pendant primary amines despite the possible benefits of incorporating different amino groups along the chain. In this work, a straightforward and scalable synthesis route towards polymers bearing primary and secondary amines, as well as imidazole groups, is reported. The general strategy relies on the radical copolymerization of different vinylamides and vinyl imidazoles followed by the hydrolysis of the resulting poly(vinylamide) derivatives. Binary and ternary free radical copolymerizations of N-vinylacetamide (NVA), N-methyl vinylacetamide (NMVA) and 1-vinylimidazole (VIm) were investigated and the reactivity ratios for each copolymerization system were determined. Thanks to these values a series of statistical copolymers with predictable composition and low deviation over the chain distribution could then be synthesized. Finally, the acidic hydrolysis of the acetamide functions towards the corresponding amine was performed and optimized. Copolymers containing various pendant amino groups and with low dispersity in the chain composition could be obtained, which opens new perspectives for the above mentioned applications.