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Carbon dots are emerging nanoparticles (NPs) with tremendous applications, especially in the biomedical field. Herein is reported the first quantitative proteomic analysis of the protein corona formed on CDs with different surface charge properties. Four CDs were synthesized from citric acid and various amine group-containing passivation reagents, resulting in cationic NPs with increasing zeta (ζ)-potential and density of positive charges. After CD contact with serum, we show that protein corona identity is influenced by CD surface charge properties, which in turn impacts CD uptake and viability loss in macrophages. In particular, CDs with high ζ-potential (+30 mV) and charge density (2 μmol mg

In this work, we used an original experimental setup to examine the behavior of insoluble monolayers made with pH-sensitive lipids. Two kinds of unsaturated lipids were chosen: a cationic one (lipid 1) bearing an ammonium headgroup and an anionic one (lipid 2) terminated with an acidic phenol group. The lipids were deposited onto an air bubble interface maintained in an aqueous phase and, after stabilization, were subjected to a series of compressions performed at different pH values. These experiments disclosed a gradual increase in the specific area per molecule when lipids were neutralized. Imposing a pH variation at constant bubble volume also provided surface pressure profiles that confirmed this molecular behavior. As complementary characterization, dilatational rheology disclosed a phase transition from a purely elastic monophasic system to a viscoelastic two-phase system. We hypothesized that this unexpected increase in the specific area with lipid neutralization is related to the presence of unsaturations in each of the two branches of the hydrophobic tails that induce disorder, thereby increasing the molecular area at the interface. Application of the two-dimensional Volmer equation of state allowed the generation of quantitative values for the specific areas that showed variations with pH. It also allowed the determination of apparent pKa values, which are affected by both the electrostatic potential within the monolayer and the affinity of the lipid polar head for the aqueous phase.

International audience; Alkylphospholipids (APLs) have elicited great interest as antitumor agents due to their unique mode of action on cell membranes. However, their clinical applications have been limited so far by high hemolytic activity. Recently, cationic prodrugs of erufosine, a most promising APL, have been shown to mediate efficient intracellular gene delivery, while preserving the antiproliferative properties of the parent APL. Here, cationic prodrugs of the two APLs that are currently used in the clinic, miltefosine, and perifosine, are investigated and compared to the erufosine prodrugs. Their synthesis, stability, gene delivery and self-assembly properties, and hemolytic activity are discussed in detail. Finally, the potential of the pro-miltefosine and pro-perifosine compounds M E12 and P E12 in combined antitumor therapy is demonstrated using pUNO1-hTRAIL, a plasmid DNA encoding TRAIL, a member of the TNF superfamily. With these pro-APL compounds, we provide a proof of concept for a new promising strategy for cancer therapy combining gene therapy and APL-based chemotherapy.

International audience; Purpose Hemolysis is a serious side effect of antitumor alkylphospholipids (APLs) that limits dose levels and is a constraint in their use in therapeutic regimen. Nine prodrugs of promising APLs (miltefosine, perifosine, and erufosine) were synthesized so as to decrease their membrane activity and improve their toxicity profile while preserving their antineoplastic potency. Methods The synthesis of the pro-APLs was straightforwardly achieved in one step starting from the parent APLs. The critical aggregation concentration of the prodrugs, their hydrolytic stability under various pH conditions, their blood compatibility and cytotoxicity in three different cell lines were determined and compared to those of the parent antitumor lipids. Results The APL prodrugs display antitumor activity which is similar to that of the parent alkylphospholipids but without associated hemolytic toxicity. Conclusion The pro-APL compounds may be considered as intravenously injectable derivatives of APLs. They could thus address one of the major issues met in cancer therapies involving antitumor lipids and restricting their utilization to oral and topical administration because of limited maximum tolerated dose.

International audience; Carbon dots (CDs) are emerging nanomaterial in medicine and pharmacy. To explore the impact of physicochemical characteristics on their safety, we synthesized a library of 35 CDs exhibiting different size, charge, chemical composition and surface coating, using various starting materials (carbon source and passivation reagent) and carbonization procedures. The 35 CDs triggered different levels of viability loss when incubated with human macrophages at 3-200 µg/mL for 24 h. The smaller NPs (10-20 nm) were more toxic that larger ones (40-100 nm), whereas NPs that aggregated in culture medium were more toxic than dispersed ones. A positive correlation was found between CD charge or nitrogen content and toxicity. Furthermore, a greater toxicity was observed for CDs prepared from high molecular weight polyamines, suggesting a role of the CD global density of positive charges, rather than the charge at the CD surface, in the CD toxicity. At last, PEG decoration decreased the toxicity of cationic NPs. In conclusion, the size, aggregation in culture medium, charge, nitrogen content, nature of the passivation agent and synthesis procedure were found to influence CD toxicity, making it difficult to predict CD safety from a single characteristic.

International audience; Sixteen cationic prodrugs of the antitumor alkylphospholipid (APL) erufosine were rationally synthesized to provide original gene delivery reagents with improved cytotoxicity profile. The DNA complexation properties of these cationic lipids were determined and associated transfection rates were measured. Furthermore, the self‐assembly properties of the pro‐erufosine compounds were investigated and their critical aggregation concentration was determined. Their hydrolytic stability under pH conditions mimicking the extracellular environment and the late endosome milieu was measured. Hemolytic activity and cytotoxicity of the compounds were investigated. The results obtained in various cell lines demonstrate that the prodrugs of erufosine display antineoplastic activity similar to that of the parent antitumor drug but are not associated with hemolytic toxicity, which is a dose‐limiting side effect of APLs and a major obstacle to their use in anticancer therapeutic regimen. Furthermore, by using lipoplexes prepared from a prodrug of erufosine and a plasmid DNA encoding a pro‐apoptotic protein (TRAIL), evidence was provided for selective cytotoxicity towards tumor cells while nontumor cells were resistant. This study demonstrates that the combination approach involving well tolerated erufosine cationic prodrugs and cancer gene therapy holds significant promise in tumor therapy.

International audience

Phospholipid-detergent conjugates are proposed as fusogenic carriers for gene delivery. Eleven compounds are prepared and their properties are investigated. The ability of the conjugates to promote fusion with a negatively charged model membrane is determined. Their DNA delivery efficiency and cytotoxicity are assessed in vitro. Lipoplexes are administered in the mouse lung, and transgene expression Indeterminate inflammatory activity are measured. The results show that conjugation of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) with C12 E4 produces a carrier that can efficiently deliver DNA to cells, with negligible -associated toxicity. Fusogenicity of the conjugates shows good correlation with in vitro transfection efficiency and crucially depends on the length of the polyether moiety of the detergent. Finally, DOPC-C12 E4 reveals highly potent for in vivo DNA delivery and favorably compares to GL67A, the current golden standard for gene delivery to the airway, opening the way for further promising developments.

International audience

Cationic carbon dots (CDs) have been recently described as nucleic acid carriers with high in vitro and in vivo transfection efficiency and imaging properties. However, developing nanoparticles (NPs) for bio-medical applications requires assessing their safety. In the present study, we characterized the cell uptake and trafficking, as well as the cell viability loss, oxidative stress, inflammation, and mitochondrial and lysosomal perturbations evoked by cationic CDs prepared by microwave-assisted pyrolysis of citric acid and high molecular weight branched polyethyleneimine (bPEI25k), using THP-1-derived macrophages. CDs were rapidly internalized by cells and addressed to the lysosomes after their cell entry. The NPs induced a dose-and time-dependent loss in cell viability that was associated with oxidative stress and IL-8 release. The CDs triggered also a dose-dependent loss in lysosome integrity , mitochondrial dysfunction, and NLRP3 inflammasome activation. Inhibition of the lysosomal protease cathepsin B significantly reduced CD-induced mitochondrial dysfunction and NLRP3 inflammasome activation, suggesting a pivotal role of the lysosome in the toxicological effects of the NPs. Our study provides for the first time a mechanistic pathway for the toxicological effects of bPEI25k-based cationic CDs.

Lung diseases are among the more representative causes of mortality and morbidity worldwide and gene therapy is considered as a promising therapeutic approach for their treatment. However the design of efficient nucleic acid carriers for airway administration still is a challenge and there is a pressing need for new developments in this field. Herein, new synthetic DNA carriers based on the conjugation of a phospholipid and C12E4, a nonionic detergent, are developed. DNA complexes with phosphatidylcholine-detergent conjugates are administered in mouse airways, and transgene expression and inflammatory activity as an index of toxicity are investigated as a function of time, DNA dose, and presence of helper and stealth lipids. Introduction of a biodegradable linker between the phosphatidylcholine and detergent moieties significantly attenuates the severity of inflammatory response that characterizes cationic lipid-mediated gene transfer. Concurrent introduction of polyunsaturated fatty acid chains in the carrier scaffold improves transgene expression and further reduces airway inflammation. Finally, the biodegradable phosphatidylcholine-detergent conjugates favorably compare to GL67A, the gold standard for DNA delivery to the airway that is currently under clinical evaluation. Our findings indicate that the lipid formulations described herein may have great potential as nucleic acid carriers for gene therapy.

The ability of a nonviral nucleic acid carrier to deliver its cargo to cells with low associated toxicity is a critical issue for clinical applications of gene therapy. We describe biodegradable cationic DOPC-C12 E4 conjugates in which transfection efficiency is based on a Trojan horse strategy. In situ production of the detergent compound C12 E4 through conjugate hydrolysis within the acidic endosome compartment was expected to promote endosome membrane destabilization and subsequent release of the lipoplexes into cytosol. The transfection efficiency of the conjugates has been assessed in vitro, and associated cytotoxicity was determined. Cellular uptake and intracellular distribution of the lipoplexes have been investigated. The results show that direct conjugation of DOPC with C12 E4 produces a versatile carrier that can deliver both DNA and siRNA to cells in vitro with high efficiency and low cytotoxicity. SAR studies suggest that this compound might represent a reasonable compromise between the membrane activity of the released detergent and susceptibility of the conjugate to degradation enzymes in vitro. Although biodegradability of the conjugates had low impact on carrier efficiency in vitro, it proved critical in vivo. Significant improvement of transgene expression was obtained in the mouse lung tuning biodegradability of the carrier. Importantly, this also allowed reduction of the inflammatory response that invariably characterizes cationic-lipid-mediated gene transfer in animals.

Nucleic acids have a huge intrinsic therapeutic potential that, however, relies heavily on the development of methods for their delivery to their intracellular target site. Cationic lipids and polymers were introduced more than twenty years ago for the delivery of DNA molecules to cells. Since then, they have become useful tools for biomedical research and have also been adopted for siRNA delivery. Their mode of action has been roughly determined. It is based on the property of adherent cell lines to easily internalize large quantities of cationic complexes in endocytic compartments, and on the incorporation of membrane-perturbing elements within internalized complexes to trigger subsequent release of the nucleic acid payload into the cytosol. Cationic lipids forming hexagonal phase or formulations of cationic lipids with the fusogenic lipid 1,2-dioleoyl-snglycero-3-phosphoethanolamine (DOPE) enable both the formation of cationic nucleic acid complexes (lipoplexes) and rupture of the endosome membrane. Yet, the endosomolytic activity of the current effective synthetic nucleic acid delivery systems remains low and effective delivery is only achieved by using cell entry pathways with a high flow rate. The membrane-disruptive properties of detergents have been considered for improving nucleic acid delivery. However, the use of detergents remains delicate because direct plasma membrane permeation might have an irreversible impact on cell viability. Cationic detergents have been shown to provoke DNA condensation but the resulting lipoplexes have turned out to be inactive or only poorly active, in vitro, when particles are prepared from cationic detergent mixed with DOPE. This is explained by the rapid exchange of the detergent molecules between the complex and the aqueous environment, which results in an irreversible decondensation of the nucleic acid even before entry into the cells. On the other hand, preparation of DNA lipoplexes from cationic lipids in the presence of Tween-80, a non-ionic detergent, has led to systems with enhanced gene transfection properties both in vitro and in vivo. These results led us to explore the consequences on nucleic acid delivery of the conjugation of a detergent molecule to a cationic lipid so that the detergent cannot be depleted from the transfection particle. We selected Triton X-100 (TX100) for its well-known detergent properties and for synthetic convenience. Furthermore, TX100 does not solubilize cholesterol-enriched raft domains, which might be involved in cationic lipoplexes internalization. TX100 was covalently linked to the phosphate group of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), a major component of biological membranes (Figure 1). Diacylglycerophosphocholines (PCs) are normal cellular metabolites and offer the opportunity to easily generate cationic lipids by O-alkylation of the phosphate group. Gorman et al. first demonstrated that a PC-derived phosphotriester (1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine, EDMPC) can mediate efficient gene transfer. Later on, derivatives of DOPC (i.e., ethyl-DOPC, EDOPC, Figure 1) and other PCs were developed by MacDonald et al. , the physical and transfection properties of which were extensively investigated. We hypothesized that the covalent attachment of a detergent molecule and a natural phospholipid to form a cationic phospholipid–detergent conjugate might improve cell uptake and transfection efficiency of lipoplexes prepared thereof. TX100 conjugation to DOPC was realized according to Scheme 1. Detergent activation with trifluoromethanesulfonyl anhydride provided the corresponding sulfonyl ester. This compound is unstable and decomposes in a few hours on standing at room temperature in chloroform. Analysis of degradation products is consistent with a deoligomerization process leading to the formation of dioxane as reported previously with other PEG derivatives. Consequently, the sulfonyl ester (1 equiv) was directly submitted to nucleophilic displacement by the anionic phosphate in DOPC (3 equiv). Conjugate 1 was obtained in 32% yield and unreacted DOPC (61%) was recovered after purification. The use of a larger excess of activated TX100 (7 equiv) improved the DOPC conversion but finally proved detrimental as the purification of the product became difficult (1: 22%; recovered DOPC: 24%). Control compound 2 was prepared similarly, by replacing Triton X-100 by polyethylene glycol mono[a] Dr. P. Pierrat, Dr. G. Creusat, Dr. G. Laverny, Prof. F. Pons, Dr. G. Zuber, Dr. L. Lebeau Laboratoire de Conception et Application de Mol!cules Bioactives CNRS, Universit! de Strasbourg, Facult! de Pharmacie 74 Route du Rhin, BP 60024, Illkirch (France) Fax: (+33)333-6885-4306 E-mail : llebeau@unistra.fr Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/chem.201103645.

Correction for ‘Influence of carbonization conditions on luminescence and gene delivery properties of nitrogen-doped carbon dots’ by Mickaël Claudel et al., RSC Adv., 2019, 9, 3493–3502.

International audience; Understanding the ionization behavior of lipid membranes is a key parameter for successful development of lipid-based drug delivery systems. Accurate determination of the ionization state of a titratable species incorporated in a lipid bilayer however requires special care. Herein we investigated the behavior of titratable lipids in liposomes by fluorescence spectroscopy and determined which extrinsic parameters-i.e., besides those directly related to their molecular structure-determine their ionization state. Two fluorescent dyes, TNS and R18, have been used to investigate basic and acidic titratable lipids, respectively. Our results suggest that the titration behavior of the ionizable lipid in the membrane is more sensitive to the composition of the membrane and to its physical state than to the presence of solutes in the aqueous phase. Essentially overlooked in earlier studies on ionizable lipid assemblies, the concentration of the titratable lipid in the membrane was found to have a major effect on the ionization state of the lipid polar head. This may result in a shift in the apparent plc value which may be as large as two pK(a) units and cannot be satisfactorily predicted.

International audience; Cationic carbon dots were fabricated by pyrolysis of citric acid and bPEI25k under microwave radiation. Various nanoparticles were produced in a 20-30 % yield through straightforward modifications of the reaction parameters (stoichiometry of the reactants and energy supply regime). Particular attention was paid to the purification of the reaction products to ensure satisfactory elimination of the residual starting polyamine. Intrinsic properties of the particles (size, surface charge, photoluminescence and quantum yield) were measured and their ability to form stable complexes with nucleic acid was determined. Their potential to deliver plasmid DNA or small interfering RNA to various cell lines was investigated and compared to that of bPEI25k. The pDNA in vitro transfection efficiency of these carbon dots was similar to that of the parent PEI, as was their cytotoxicity. The higher cytotoxicity of bPEI25k/siRNA complexes when compared to that of the CD/siRNA complexes however had marked consequences on the gene silencing efficiency of the two carriers. These results are not fully consistent with those in some earlier reports on similar nanoparticles, revealing that toxicity of the carbon dots strongly depends on their protocol of fabrication. Finally, these carriers were evaluated for in vivo gene delivery through the non-invasive pulmonary route in mice. High transgene expression was obtained in the lung that was similar to that obtained with the golden standard formulation GL67A, but was associated with significantly lower toxicity. Post-functionalization of these carbon dots with PEG or targeting moieties should significantly broaden their scope and practical implications in improving their in vivo and biocompatibility.

Aza-Michael addition of optically pure 4-phenyl-2-oxazolidinone to 3,3,3-trifluoro-1-nitropropene proceeds smoothly at low temperature with a high yield. Diastereoselectivity of the addition depends on the base used and lithiated species proved to be highly efficient affording 92% de. Optically pure 1,2-diamino-3,3,3-trifluoropropane is prepared in 58% yield from the aza-Michael addition product through a three-step procedure.

International audience; Human Eg5, responsible for the formation of the bipolar mitotic spindle, has been identified recently as one of the targets of S-trityl-L-cysteine, a potent tumor growth inhibitor in the NCI 60 tumor cell line screen. Here we show that in cell-based assays S-trityl-L-cysteine does not prevent cell cycle progression at the S or G(2) phases but inhibits both separation of the duplicated centrosomes and bipolar spindle formation, thereby blocking cells specifically in the M phase of the cell cycle with monoastral spindles. Following removal of S-trityl-L-cysteine, mitotically arrested cells exit mitosis normally. In vitro, S-trityl-L-cysteine targets the catalytic domain of Eg5 and inhibits Eg5 basal and microtubule-activated ATPase activity as well as mant-ADP release. S-trityl-L-cysteine is a tight binding inhibitor (estimation of K(i,app)

Human Eg5, a member of the kinesin superfamily, plays a key role in mitosis, as it is required for the formation of a bipolar spindle. We describe here the first in vitro microtubule-activated ATPase-based assay for the identification of small-molecule inhibitors of Eg5. We screened preselected libraries obtained from the National Cancer Institute and identified S-trityl-l-cysteine as the most effective Eg5 inhibitor with an IC50 of 1.0 μmol/L for the inhibition of basal ATPase activity and 140 nmol/L for the microtubule-activated ATPase activity. Subsequent cell-based assays revealed that S-trityl-l-cysteine induced mitotic arrest in HeLa cells (IC50, 700 nmol/L) with characteristic monoastral spindles. S-trityl-l-cysteine is 36 times more potent for inducing mitotic arrest than the well-studied inhibitor, monastrol. Gossypol, flexeril, and two phenothiazine analogues were also identified as Eg5 inhibitors, and we found that they all result in monoastral spindles in HeLa cells. It is notable that all the Eg5 inhibitors identified here have been shown previously to inhibit tumor cell line growth in the NCI 60 tumor cell line screen, and we conclude that their antitumor activity may at least in part be explained by their ability to inhibit Eg5 activity.

Thiamine triphosphate (ThTP) is found in most living organisms and it may act as a phosphate donor for protein phosphorylation. We have recently cloned the cDNA coding for a highly specific mammalian 25 kDa thiamine triphosphatase (ThTPase; EC 3.6.1.28). As the enzyme has a high catalytic efficiency and no sequence homology with known phosphohydrolases, it was worth investigating its structure and catalytic properties. For this purpose, we expressed the untagged recombinant human ThTPase (hThTPase) in E. coli, produced the protein on a large scale and purified it to homogeneity. Its kinetic properties were similar to those of the genuine human enzyme, indicating that the recombinant hThTPase is completely functional. Mg2+ ions were required for activity and Ca2+ inhibited the enzyme by competition with Mg2+. With ATP as substrate, the catalytic efficiency was 10(-4)-fold lower than with ThTP, confirming the nearly absolute specificity of the 25 kDa ThTPase for ThTP. The activity was maximum at pH 8.5 and very low at pH 6.0. Zn2+ ions were inhibitory at micromolar concentrations at pH 8.0 but activated at pH 6.0. Kinetic analysis suggests an activator site for Mg2+ and a separate regulatory site for Zn2+. The effects of group-specific reagents such as Woodward's reagent K and diethylpyrocarbonate suggest that at least one carboxyl group in the active site is essential for catalysis, while a positively charged amino group may be involved in substrate binding. The secondary structure of the enzyme, as determined by Fourier-transform infrared spectroscopy, was predominantly beta-sheet and alpha-helix.

Dinucleoside polyphosphates are ubiquitous compounds tightly involved in the regulation of a number of key biological processes. Hydrolysis-resistant analogues of Ap(3)A and Gp(3)G, two important members of that family of nucleotides, have been synthesized. P(1),P(2):P(2),P(3)-Bis-methylene diadenosine and diguanosine triphosphates were prepared from O,O-dialkyl methaneselenophosphonates using an original methodology. Whereas the 2-fold addition of the methanephosphonate anion to the activated phosphorus species cannot be performed, multiple condensation of lithiated methaneselenophosphonate with electrophilic trivalent phosphorus compounds is revealed to be very effective. A one-pot condensation/esterification/oxidation sequence involving O,O-dialkyl methaneselenophosphonates provides a highly efficient route to the PCH(2)PCH(2)P backbone. This new development in selenophosphonate chemistry offers a great potential for further regioselective functionalization of polyphosphate mimics.

A new route to bis-methylene analogs of triphosphate esters involving carbanion chemistry is described. Lithiated methaneselenophosphonate anion is condensed several times with chlorophosphites and phosphoramidous chlorides prior to esterification/transesterification and selenation or oxidation.

An affinity study between the G protein of the visual photoreceptor, transducin, and eight different non-hydrolyzable GDP analogues is described. Imidodiphosphate derivatives have been shown to exhibit good affinities to transducin. This very important heterotrimeric G protein is shown to be highly restrictive with regard to structural modifications of the nucleotide at the pyrophosphate moiety, at the 3′ position on ribose, as well as at the N 1 position of the purine.

The synthesis of the reduced form of AZT-triphosphate, 3′-amino-3′-deoxythymidine-5′-triphosphate, and of its stable analog, 3′-amino-3′-deoxythymidine-5′-(α,β:β,γ-bis-methylene)triphosphate, is described. AMT-TP and AMT-PCPCP were prepared to develop a specific immunometric assay for AZT-TP.

An enzymatically stable analogue of AZT-triphosphate is prepared in 7 steps starting from thymidine. The thymine moiety is functionalized at N 3 to allow the further cross-linking to a carrier protein for subsequent immunization of rabbits in order to raise specific antibodies directed against AZT-TP. An enzymatically stable analogue of AZT-triphosphate is prepared in 7 steps starting from thymidine. Download full-size image

Two complementary procedures are described for the preparation of N, N-dibenzyl formamidines from primary amines in high yield. The primary amine protective group can be easily introduced on polyhydroxylated molecules and is removable either by hydrolysis or hydrogenolysis.

Biolabile cationic lipids were developed for efficient intracellular delivery of DNA and siRNA.The compounds have been designed starting from the membrane lipid DOPC in a way they may loose their cationic charge when exposed to an acidic and/or enzymatic stimulus, such as those met during the journey of a lipoplex in biological media.They demonstrated remarkable efficiency to deliver DNA in various cell lines (BHK-21, Calu-3, NCI-H292, and A549), with no significant cytotoxicity. Furthermore, two of the compounds (carbonate-based DOPC derivatives) revealed able to deliver small interfering RNA in U87Luc and A549Luc cancer cells and to mediate a selective 70-80% knockdown of the stably transfected luciferase gene.The results show that the described bioresponsive cationic lipids have high DNA and siARN delivery activity which is encouraging in view of delivering a therapeutic nucleic acid to pulmonary tissues in vivo.

The synthesis of glycero-lipids linked to colchicine derivatives is reported. The lipid structures are designed to perform two-dimensional crystallization experiments with tubulin, the structural subunit protein of microtubules.

Benzyl phosphites were used in the Micaelis-Arbuzov reaction. Special experimental conditions allowed preparation of a set of phosphonate analogs of mono-, di-, and triphosphate. Furthermore, regioselective monodeprotection makes these molecules useful building blocks for the synthesis of analogs of polyphosphorylated compounds of biological interest (e.g. nucleotides), after removal of all phosphonate benzyl ester groups under very mild conditions and high yields.

This method of two-dimensional crystallization of proteins on a lipid monolayer aims at producing 2D crystals of membrane proteins, which can provide structural information at high resolution by electron crystallography. A lipid monolayer is spread over the whole air-water interface of a drop, which provides a substrate for protein binding. The protein of interest is then adsorbed onto the lipid monolayer and forms a closely packed layer. The reconstitution step of the membrane protein into a lipid bilayer is realized by elimination of detergent. The combined effect of the elevated protein concentration, the alignment of the protein on the lipid monolayer, and the fluid monolayer film properties are conducive, in some cases, to the formation of 2D crystals. The use of locally fluorinated lipids in avoiding solubilization of the lipid monolayer by detergents is presented and discussed.

This method of two-dimensional crystallization of proteins on a lipid monolayer aims at producing 2D crystals of membrane proteins, which can provide structural information at high resolution by electron crystallography. A lipid monolayer is spread over the whole air-water interface of a drop, which provides a substrate for protein binding. The protein of interest is then adsorbed onto the lipid monolayer and forms a closely packed layer. The reconstitution step of the membrane protein into a lipid bilayer is realized by elimination of detergent. The combined effect of the elevated protein concentration, the alignment of the protein on the lipid monolayer, and the fluid monolayer film properties are conducive, in some cases, to the formation of 2D crystals. The use of locally fluorinated lipids in avoiding solubilization of the lipid monolayer by detergents is presented and discussed.

One of the potential benefits of drug delivery systems in medicine is the creation of nanoparticle-based vectors that deliver a therapeutic cargo in sufficient quantity to a target site to enable a selective effect, width of the therapeutic window depending on the toxicity of the vector and the cargo. In this work, we intended to improve the siRNA delivery efficiency of a new kind of nucleic acid carrier, which is the result of the conjugation of the membrane phospholipid 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) to the membrane-active species Triton X-100 (TX100). We hypothesized that by improving the biodegradability the cytotoxicity of the conjugate might by reduced, whereas its original transfection potential would be tentatively preserved. DOPC was conjugated to Triton X-100 through spacers displaying various resistance to chemical hydrolysis and enzyme degradation. The results obtained through in vitro siRNA delivery experiments showed that the initial phosphoester bond can be replaced with a phospho(alkyl)enecarbonate group with no loss in the transfection activity, whereas the associated cytotoxicity was significantly decreased, as assessed by metabolic activity and membrane integrity measurements. The toxicity of the conjugates incorporating a phospho(alkyl)enesuccinnate moiety proved even lower but was clearly balanced with a reduction of the siRNA delivery efficiency. Hydrolytic stability and intracellular degradation of the conjugates were investigated by NMR spectroscopy and mass spectrometry. A general trend was that the more readily degraded conjugates were those with the lower toxicity. Otherwise, the phospho(alkyl)enecarbonate conjugates revealed some hemolytic activity, whereas the parent phosphoester did not. The reason why these conjugates behave differently with respect to hemolysis might be a consequence of unusual fusogenic properties and probably reflects the difference in the stability of the conjugates in the intracellular environment.

International audience; In the present work, we elaborated a synthetic lung surfactant composed of dipalmitoyl phosphatidylcholine (DPPC), phosphatidylglycerol, cholesterol and bovine serum albumin (BSA), as a vehicle to study the lung toxicity of pristine multi-walled carbon nanotubes (MWCNT). MWCNT were dispersed in surfactant, saline or saline containing DPPC, BSA, Pluronic(®) F68 or sodium dodecyl sulfate, for comparison. Dispersions were characterized visually, and by light microscopy, dynamic light scattering and transmission electronic microscopy (TEM). Deposition of surfactant-dispersed MWCNT in the lung of BALB/c mice upon single or repeated administrations was analyzed by histology and TEM. Inflammation and airway remodeling were assessed in bronchoalveolar lavage fluid (BALF) or lung tissue of mice by counting cells and quantifying cytokines, tumor growth factor (TGF)-β1 and collagen, and by histology. We found that the elaborated surfactant is more effective in dispersing MWCNT when compared to the other agents, while being biocompatible. Surfactant-dispersed MWCNT distributed all throughout the mouse airways upon single and repeated administrations and were observed in alveolar macrophages and epithelial cells, and in infiltrated neutrophils. Mice that received a single administration of MWCNT showed neutrophil infiltrate and greater concentrations of tumor necrosis factor (TNF)-α, keratinocyte-derived chemokine (KC) and interleukin (IL)-17 in BALF when compared to controls. After repeated MWCNT administrations, increases in macrophage number, KC and TGF-β1 levels in BALF, and collagen deposition and mucus hyperplasia in lung tissue were observed. Altogether, the elaborated lung surfactant could be a valuable tool to further study the toxicological impact of pristine MWCNT in laboratory animals.

One important function of conventional dendritic cells (cDC) is their high capacity to capture, process and present Ag to T lymphocytes. Mouse splenic cDC subtypes, including CD8α(+) and CD8α(-) cDC, are not identical in their Ag presenting and T cell priming functions. Surprisingly, few studies have reported functional differences between CD4(-) and CD4(+) CD8α(-) cDC subsets. We show that, when loaded in vitro with OVA peptide or whole protein, and in steady-state conditions, splenic CD4(-) and CD4(+) cDC are equivalent in their capacity to prime and direct CD4(+) and CD8(+) T cell differentiation. In contrast, in response to α-galactosylceramide (α-GalCer), CD4(-) and CD4(+) cDC differentially activate invariant Natural Killer T (iNKT) cells, a population of lipid-reactive non-conventional T lymphocytes. Both cDC subsets equally take up α-GalCer in vitro and in vivo to stimulate the iNKT hybridoma DN32.D3, the activation of which depends solely on TCR triggering. On the other hand, and relative to their CD4(+) counterparts, CD4(-) cDC more efficiently stimulate primary iNKT cells, a phenomenon likely due to differential production of co-factors (including IL-12) by cDC. Our data reveal a novel functional difference between splenic CD4(+) and CD4(-) cDC subsets that may be important in immune responses.

A series of 14 new 3-alkoxycarbonyl 6-aza-, 7-aza-, and 6,8-diaza-coumarins was prepared using various strategies involving either a Knoevenagel or a Pechmann condensation reaction. The coumarin nucleus displays different reactive functional groups allowing straightforward derivatization. The optical properties of the new azacoumarins were measured in methanol.

α-Chlorination of phosphonates, and O,O -dialkyl thio- and selenophosphonates involving the direct reaction of their lithiated anion with phosphorus oxychloride is described. The reaction gives good results where previously known methods fail. The role of the chalcogen atom, and the influence of the nature of the alkyl chain with respect to the reactivity are discussed.

A series of 14 new 3-alkoxycarbonyl 6-aza-, 7-aza-, and 6,8-diaza-coumarins was prepared using various strategies involving either a Knoevenagel or a Pechmann condensation reaction. The coumarin nucleus displays different reactive functional groups allowing straightforward derivatization. The optical properties of the new azacoumarins were measured in methanol.

The synthesis of phospholipids 1n–3n, rationally designed for two-dimensional crystallization of progesterone and estradiol receptors, is reported. The structure of these lipids provides them with essential properties such as fluidity and stability when spread into monolayers at the air/H2O interface, affinity for the protein to be crystallized, and accessibility of the ligand under the lipid monolayer.