Conformation-dependent membrane permeabilization by neurotoxic PrP oligomers: The role of the H2H3 oligomerization domain.

The relationship between prion propagation and the generation of neurotoxic species and clinical onset remains unclear. Several converging lines of evidence suggest that interactions with lipids promote various precursors to form aggregation-prone states that are involved in amyloid fibrils. Here, we compared the cytotoxicities of different soluble isolated oligomeric constructs from murine full-length PrP and from the restricted helical H2H3 domain with their effects on lipid vesicles. The helical H2H3 domain is suggested to be the minimal region of PrP involved in the oligomerization process. The discrete PrP oligomers of both the full-length sequence and the H2H3 domain have de novo ß-sheeted structure when interacting with the membrane. They were shown to permeabilize synthetic negatively charged vesicles in a dose-dependent manner. Restricting the polymerization domain of the full-length PrP to the H2H3 helices strongly diminished the ability of the corresponding oligomers to associate with the lipid vesicles. Furthermore, the membrane impairment mechanism occurs differently for the full-length PrP oligomers and the H2H3 helices, as shown by dye-release and black lipid membrane experiments. The membrane damage caused by the full-length PrP oligomers is correlated to their neuronal toxicity at submicromolar concentrations, as shown by cell culture assays. Although oligomers of synthetic H2H3 could compromise in vitro cell homeostasis, they followed a membrane-disruptive pattern that was different from the full-length oligomers, as revealed by the role of PrPC in cell viability assays.

Polyglycidol-Stabilized Nanoparticles as a Promising Alternative to Nanoparticle PEGylation: Polymer Synthesis and Protein Fouling Considerations.

We herein demonstrate the outstanding protein-repelling characteristic of star-like micelles and polymersomes manufactured from amphiphilic block copolymers made by poly(butylene oxide) (PBO) hydrophobic segments and polyglycidol (PGL) hydrophilic outer shells. Although positively charged proteins (herein modeled by lysozyme) may adsorb onto the surface of micelles and polymersomes where the assemblies are stabilized by short PGL chains (degree of polymerization smaller than 15), the protein adsorption vanishes when the degree of polymerization of the hydrophilic segment (PGL) is higher than ∼20, regardless the morphology. This has been probed by using three different model proteins which are remarkably different concerning molecular weight, size, and zeta potential (bovine serum albumin (BSA), lysozyme, and immunoglobulin G (IgG)). Indeed, the adsorption of the most abundant plasma protein (herein modeled as BSA) is circumvented even by using very short PGL shells due to the highly negative zeta potential of the produced assemblies which presumably promote protein-nanoparticle electrostatic repulsion. The negative zeta potential, on the other hand, enables lysozyme adsorption, and the phenomenon is governed by electrostatic forces as evidenced by isothermal titration calorimetry. Nevertheless, the protein coating can be circumvented by slightly increasing the degree of polymerization of the hydrophilic segment. Notably, the PGL length required to circumvent protein fouling is significantly smaller than the one required for PEO. This feature and the safety concerns regarding the synthetic procedures on the preparation of poly(ethylene oxide)-based amphiphilic copolymers might make polyglycidol a promising alternative toward the production of nonfouling spherical particles.

Topology and Electronic Density Driven Generation of Alkali Cation Complexes.

The formation and characterization of K+ and Cs+ complexes originating from the cooperativity of three non-covalent interactions is explored. The tridimensional preorganization of the naphthothiophene platform displays a favorable well-defined bay region combining a π fragment and a carbonyl moiety flanking a central sulfur atom. A joint theoretical and experimental infrared multiple photon dissociation (IRMPD) study allowed deciphering the key contribution of the orthogonal phenyl fragment to the elaboration of alkali metal complexes. In combination with S and CO interactions, the π-cation interaction significantly enhances the binding energies of naphthothiophene derivatives.

Polynucleotide transport through lipid membrane in the presence of starburst cyclodextrin-based poly(ethylene glycol)s.

Symmetrical cyclodextrin-based 14-arm star polymers with poly(ethylene glycol) PEG branches were synthesized and characterized. Interactions of the star polymers with lipid bilayers were studied by the "black lipid membrane" technique in order to demonstrate the formation of monomolecular artificial channels. The conditions for the insertion are mainly based on dimensions and amphiphilic properties of the star polymers, in particular the molar mass of the water-soluble polymer branches. Translocation of single-strand DNA (ssDNA) through those synthetic nanopores was investigated, and the close dimension between the cross-section of ssDNA and the cyclodextrin cavity led to an energy barrier that slowed down the translocation process.

Biomimetic Nanotubes Based on Cyclodextrins for Ion-Channel Applications.

Biomimetic membrane channels offer a great potential for fundamental studies and applications. Here, we report the fabrication and characterization of short cyclodextrin nanotubes, their insertion into membranes, and cytotoxicity assay. Mass spectrometry and high-resolution transmission electron microscopy were used to confirm the synthesis pathway leading to the formation of short nanotubes and to describe their structural parameters in terms of length, diameter, and number of cyclodextrins. Our results show the control of the number of cyclodextrins threaded on the polyrotaxane leading to nanotube synthesis. Structural parameters obtained by electron microscopy are consistent with the distribution of the number of cyclodextrins evaluated by mass spectrometry from the initial polymer distribution. An electrophysiological study at single molecule level demonstrates the ion channel formation into lipid bilayers, and the energy penalty for the entry of ions into the confined nanotube. In the presence of nanotubes, the cell physiology is not altered.

Poly(2-methyl-2-oxazoline)-b-poly(tetrahydrofuran)-b-poly(2-methyl-2-oxazoline) amphiphilic triblock copolymers: synthesis, physicochemical characterizations, and hydrosolubilizing properties.

Block copolymers assembled into micelles have gained a lot of attention to improve drug delivery. The recent drawbacks of the poly(ethylene oxide) blocks (PEO) contained in amphiphilic pluronics derivatives made of a central poly(propylene oxide) block surrounded by two PEO blocks were recently revealed, opening the way to the design of new amphiphilic block copolymers able to self-assemble in water and to entrap molecules of interest. Here, a family of p(methyloxazoline)-b-p(tetrahydrofuran)-b-p(methyloxazoline) triblock copolymers (called TBCP) is synthesized using cationic ring opening polymerization. Studies of micelle formation using dynamic light scattering, isothermal titration calorimetry (ITC), NMR diffusion-ordered spectroscopy (DOSY), and fluorescence experiments lead us to draw a relationship between copolymer structure and the physicochemical properties of the block copolymers (critical micellar concentration (CMC), Nagg, core diameter, shell thickness, etc.). The packing parameter of the block copolymers indicates the formation of a core-corona structure. Hydrosolubilizing properties of TBCPs were exemplified with curcumin selected as a highly insoluble drug model. Curcumin, a natural polyphenolic compound, has shown a large spectrum of biological and pharmacological activity, including anti-inflammatory, antimicrobial, antioxidant, and anticarcinogenic activities. An optimized formulation process reveals that the aggregation number is the parameter affecting drug encapsulation. Patch clamp experiments carried out to study the interaction of TBCP with the cell membrane demonstrate their permeation property suitable to promote the cellular internalization of curcumin.

Neutral Block Copolymer Assisted Gene Delivery using Hydrodynamic Limb Vein Injection.

Three different amphiphilic block copolymer families are synthesized to investigate new opportunities to enhance gene delivery via Hydrodynamic Limb Vein (HLV) injections. First a polyoxazoline-based family containing mostly one poly(2-methyl-2-oxazoline) (PMeOx) block and a second block POx with an ethyl (EtOx), isopropyl (iPrOx) or phenyl substituent (PhOx) is synthesized. Then an ABC poly(2-ethyl-2-oxazoline)-b-poly(2-n-propyl-2-oxazoline)-b-poly(2-methyl-2-oxazoline) triblock copolymer is synthesized, with a thermosensitive middle block. Finally, polyglycidol-b-polybutylenoxide-b-polyglycidol copolymers with various molar masses and amphiphilic balance are produced. The simple architecture of neutral amphiphilic triblock copolymer is not sufficient to obtain enhanced in vivo gene transfection. Double or triple amphiphilic neutral block copolymers are improving the in vivo transfection performances through HLV administration as far as a block having an lower critical solution temperature is incorporated in the vector. The molar mass of the copolymer does not seem to affect the vector performances in a significant manner.

Engineering of ion permeable planar membranes and polymersomes based on ß-cyclodextrin-cored star copolymers.

For polymersome-based nanoreactor purposes, we herein present the synthesis and characterization of well-defined star amphiphilic copolymers composed of a beta-cyclodextrin (ßCD) core and seven poly(butylene oxide)-block-polyglycidol (PBO-PGL) arms per side (ßCD-(PBO-PGL)14). The self-assembly behavior of 14-armed ßCD-(PBO-PGL)14 and PGL-PBO-PGL (linear analogues without the ßCD segment) was investigated using scattering techniques for comparison. The morphologies, including vesicles and micelles, are governed by the hydrophobic-to-hydrophilic (weight) ratio, regardless of the polymer architecture (linear or star). Interestingly, despite notable differences in polymer conformation, the produced supramolecular structures were evidenced to be fairly similar on the structural point of view. We subsequently investigated the ion permeability of the membranes of the self-assemblies focusing on the impact of the presence of ßCD. The results demonstrated that the ßCD-containing vesicular membranes are less permeable to H+, compared with ßCD-free vesicular membranes. The presence of ßCD in planar membranes also influences the K+Cl- permeability to some extent. Thus, ßCD-containing membranes can be considered as potential candidates in designing nano-containers towards applications where precise changes in environmental pH are required.

Evidence of DNA transfer across a model membrane by a neutral amphiphilic block copolymer.

BACKGROUND: Neutral amphiphilic triblock copolymers have been shown to be efficient for gene transfection in vivo, especially by direct injection into the muscle. To contribute to a better understanding of the underlying mechanisms, in the present study, we investigated the properties of a poly(ethylene oxide-b-4-vinylpyridine) diblock copolymer as vector for nucleic acid transfer, with the particular aim of shedding some light on a possible mechanism explaining the internalization of DNA by the transfected cells. METHODS: Complexation of plasmid DNA with the PEO-b-P4VP diblock copolymer was investigated by ethidium bromide exclusion and gel electrophoresis assays. Interaction of the copolymer with a lipid model membrane was evaluated by electrophysiological assays and quantification of plasmid DNA was performed by quantitative polymerase chain reaction. In vivo luciferase transfection assays were finally performed. RESULTS: The diblock copolymer was found to poorly interact with DNA up to a mass ratio (copolymer/DNA) as high as 150. At a concentration of 36 µg/ml, it induced the formation of mainly transient (but sometimes permanent) pores and the formation of those pores allowed the translocation of plasmid DNA across the model membrane. However, only low transgene expression was obtained; the luciferase levels observed with the diblock being of the same order of magnitude as those observed with the corresponding PEO and P4VP homopolymers. CONCLUSIONS: These results strongly suggest that gene transfection by neutral block copolymers may involve the formation of cellular pores; in addition, they also highlight that in vivo gene transfection requires the use of adequately soluble block copolymers.

Helically shaped cation receptor: design, synthesis, characterisation and first application to ion transport.

The methyl ester of 8-oxo-8H-indeno[2',1':7,8]naphtho[1,2-b]thiophene-2-carboxylic acid (1) and its corresponding PEGylated ester were synthesised and fully characterised. X-ray diffraction studies on (1) confirmed the helical structure of the receptor and that it is self-assembled into layers by π-π interactions. An in-depth study by DFT calculations and MS experiments (ESI-MS, MS/MS, IMRPD and ESI-IMS-MS) was carried out between (1) and the physiological cation K+. The formation of supramolecular complexes between (1) and K+ with different stoichiometries was demonstrated and the cation K+ preferentially interacts with the oxygen atoms of the carbonyl bond of the ketone and ester groups and the sulphur atom of the heterocycle. The ability of the two synthesized aromatic architectures to transport ions across a model lipid membrane has been studied by electrophysiology experiments. The formation of pores was observed, even at nanomolar concentrations. Since the PEGylated molecule showed more regular pore definitions than the hydrophobic molecule, the introduction of a polar hydrophilic chain made it possible to control the orientation of the aromatic architectures within the membrane.

Temperature-Sensitive Amphiphilic Non-Ionic Triblock Copolymers for Enhanced In Vivo Skeletal Muscle Transfection.

It is reported that low concentration of amphiphilic triblock copolymers of pMeOx-b-pTHF-b-pMeOx structure (TBCPs) improves gene expression in skeletal muscle upon intramuscular co-injection with plasmid DNA. Physicochemical studies carried out to understand the involved mechanism show that a phase transition of TBCPs under their unimer state is induced when the temperature is elevated from 25 to 37 °C, the body temperature. Several lines of evidences suggest that TBCP insertion in a lipid bilayer causes enough lipid bilayer destabilization and even pore formation, a phenomenon heightened during the phase transition of TBCPs. Interestingly, this property allows DNA translocation across the lipid bilayer model. Overall, the results indicate that TBCPs exhibiting a phase transition at the body temperature is promising to favor in vivo pDNA translocation in skeletal muscle cells for gene therapy applications.

Expression of PPARgamma is reduced by medium supplementation with L-glutamine in human colorectal Caco-2 cells.

Peroxisome proliferator-activated receptor-gamma (PPARgamma) belongs to the nuclear hormone receptor family. This receptor is implicated in colon cell differentiation and in colon cancer. Receptor activation by specific agonists has been shown to protect against colon cancer progression. PPARgamma protein content within cells is modulated by several mechanisms, including proteasome degradation, activation of Wnt signalling pathways and presence of fermentation products such as butyrate. Herein, we investigated the impact of L-glutamine on PPARgamma expression during the differentiation of Caco-2 cells grown in medium containing dialyzed fetal calf serum supplemented or not with L-glutamine. Using RT-PCR and Western blotting, we demonstrated that PPARgamma expression was decreased when L-glutamine was added to the medium. Using immunohistochemistry, we demonstrated that PPARgamma immunostaining was mainly found in cytoplasm when cells were cultured with L-glutamine while it was found in nuclei and cytoplasm when cells were grown without the addition of L-glutamine. Supershift retardation assays demonstrated a decrease of PPARgamma binding onto consensus peroxisome proliferator response element. We concluded that L-glutamine modulated PPARgamma expression in Caco-2 cells.

Versatile cyclodextrin nanotube synthesis with functional anchors for efficient ion channel formation: design, characterization and ion conductance.

Biomimetic ion channels with different materials have been extensively designed to study the dynamics in a confined medium. These channels allow the development of several applications, such as ultra-fast sequencing and biomarker detection. When considering their synthesis, the use of cheap, non-cytotoxic and readily available materials is an increasing priority. Cyclodextrins, in supramolecular architectures, are widely utilized for pharmaceutical and biotechnological applications. Recent work has shown that short nanotubes (NTs) based on alpha-cyclodextrin (α-CD) assemble transient ion channels into membranes without cytotoxicity. In this study, we probe the influence of new cyclodextrin NT structural parameters and chemical modifications on channel formation, stability and electrical conductance. We report the successful synthesis of ß- and γ-cyclodextrin nanotubes (ß-CDNTs and γ-CDNTs), as evidenced by mass-spectrometry and high-resolution transmission electron microscopy. CDNTs were characterized by their length, diameter and number of CDs. Two hydrophobic groups, silylated or vinylated, were attached along the γ-CDNTs, improving the insertion time into the membrane. All NTs synthesized form spontaneous biomimetic ion channels. The hydrophobic NTs exhibit higher stability in membranes. Electrophysiological measurements show that ion transport is the main contribution of NT conductance and that the ion energy penalty for the entry into these NTs is similar to that of biological channels.

Curcumin/poly(2-methyl-2-oxazoline-b-tetrahydrofuran-b-2-methyl-2-oxazoline) formulation: An improved penetration and biological effect of curcumin in F508del-CFTR cell lines.

Neutral amphiphilic triblock ABA copolymers are of great interest to solubilize hydrophobic drugs. We reported that a triblock ABA copolymer consisting of methyl-2-oxazoline (MeOx) and tetrahydrofuran (THF) (MeOx6-THF19-MeOx6) (TBCP2) can solubilize curcumin (Cur) a very hydrophobic molecule exhibiting multiple therapeutic effects but whose insolubility and low stability in water is a major drawback for clinical applications. Here, we provide evidences by flow cytometry and confocal microscopy that Cur penetration in normal and ΔF508-CFTR human airway epithelial cell lines is facilitated by TBCP2. When used on ΔF508-CFTR cell lines, the Cur/TBCP2 formulation promotes the restoration of the expression of the CFTR protein in the plasma membrane. Furthermore, patch-clamp and MQAE fluorescence experiments show that this effect is associated with a correction of a Cl- selective current at the membrane surface of F508del-CFTR cells. The results show the great potential of the neutral amphiphilic triblock copolymer MeOx6-THF19-MeOx6 as carrier for curcumin in a Cystic Fibrosis context. We anticipate that other MeOxn-THFm-MeOxn copolymers could have similar behaviours for other highly insoluble therapeutic drugs or cosmetic active ingredients.

Genetic analysis of peroxisome proliferator-activated receptor gamma1 splice variants in human colorectal cell lines.

Peroxisome proliferator-activated receptor gamma (PPARgamma) is a member of the nuclear hormone receptor family. In colon, this transcription factor is involved in differentiation of absorptive cells. PPARgamma participates also in colon carcinogenesis and cancer progression. Two isoforms, namely PPARgamma1 and PPARgamma2, have been described. Recently, new PPARgamma1 transcripts whose translation raises PPARgamma1 protein have been characterised. They differ from each other by combination of untranslated exons localised in the 5' UTR of the PPARG gene. Here, we studied whether such a diversity of PPARgamma transcripts occurs in human colon cell models. Based on bioinformatic analysis, putative untranslated exons were identified in the human PPARG gene. By RT-PCR analysis, we have demonstrated that several of these untranslated exons are included in PPARgamma transcripts from colon-derived cell lines or in those derived from other tissue. Using HT-29 cells, changes in PPARgamma1 mRNA levels were observed after treatment with PPARgamma agonists such as pioglitazone and troglitazone. These modifications correlated with particular PPARgamma transcripts excluding the untranslated exon A2. HT-29 cells treatment with actinomycin D or cycloheximide showed that the presence of PPARgamma mRNA including exon A2 was dependent on de novo protein synthesis. We concluded that diverse PPARgamma1 mRNA exist in colorectal cells. Levels of PPARgamma1 transcript varied according to the phenotype of colon cell model used. We suggest that regulation of PPARgamma1 mRNA levels could be dependent in part on the composition of untranslated exon(s) in the 5' UTR of PPARgamma1 mRNA.

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.

Biomimetic artificial ion channels based on beta-cyclodextrin.

Star polymers based on ß-cyclodextrin were synthesized by a ''click-chemistry'' process and characterized by NMR, SEC and BLM. The resulting triazole functional groups create, at a specific pH range, electrostatic hindrance between pores inserted in lipid bilayers, preventing their aggregation, allowing formation of well-defined isolated unitary pores and mimicking biological natural channels.

Regioselective allylation of cyclomaltoheptaose (ß-cyclodextrin) leading to per(2,6-di-O-hydroxypropyl-3-O-methyl)-ß-cyclodextrin.

The selective modification of cyclodextrins remains a real challenge to obtain well-defined structures. The targeted cycloheptakis-(1→4)-2,6-di-O-hydroxypropyl-3-O-methyl-α-D-glucopyranosyl [per(2,6-di-O-hydroxypropyl-3-O-methyl)-ß-CD] was obtained by a three-step procedure. The selective allylation of the hydroxyl functions at the positions 2 and 6 was used as a first step. This reaction was revisited then enlarged to α and γ-CDs to determine new conditions for a one-step synthesis in high yield. The per(2,6-di-O-allyl)-ß-CD derivative was then reacted with iodomethane to provide per(2,6-di-O-allyl-3-O-methyl)-ß-CD. Oxidative hydroboration of the allyl functions was then carried out in order to obtain a new CD derivative with seven primary hydroxyl functions on each side of the truncated cone, having a similar reactivity.

Expression of peroxisome proliferator-activated receptors alpha and gamma in differentiating human colon carcinoma Caco-2 cells.

The expression of peroxisome proliferator-activated receptors alpha (PPARalpha) and gamma (PPARgamma) was studied in the human adenocarcinoma Caco-2 cells induced to differentiate by long term culture (15 days). The differentiation of Caco-2 cells was attested by increases in the activities of sucrase-isomaltase and alkaline phosphatase (two brush border enzymes), fatty acyl-CoA oxidase (AOX) and catalase (two peroxisomal enzymes), by an elevation in the protein levels of villin (a brush border molecular marker), AOX, peroxisomal bifunctional enzyme (PBE), catalase and peroxisomal membrane protein of 70 kDa (PMP70). and by the appearance of peroxisomes. The expression of PPARalpha and PPARgamma was investigated by Western blotting, immunocytochemistry, Northern blotting and S1 nuclease protection assay during the differentiation of Caco-2 cells. The protein levels of PPARalpha, PPARgamma, and PPARgamma2 increased gradually during the time-course of Caco-2 cell differentiation. Immunocytochemistry revealed that PPARalpha and gamma were localized in cell nuclei. The PPARgamma1 protein was encoded by PPARgamma3 mRNA because no signal was obtained for PPARgamma1 mRNA using a specific probe in S1 nuclease protection assay. The amount of PPARgamma3 mRNA increased concomitantly to the resulting PPARgamma1 protein. On the other hand, the mRNA of PPARalpha and PPARgamma2 were not significantly changed, suggesting that the increase in their respective protein was due to an elevation of the translational rate. The role played by the PPAR subtypes in Caco-2 cell differentiation is discussed.