Christelle Mansuy

Peptide-grafted nanodiamonds: preparation, cytotoxicity and uptake in cells.

Nanodiamonds that were prepared by high pressure/high temperature were functionalised with biomolecules for biological applications. Nanodiamonds (NDs, ≤35 nm) that were coated by silanisation or with polyelectrolyte layers were grafted with a fluorescent thiolated peptide via a maleimido function; this led to an aqueous colloidal suspension that was stable for months. These substituted NDs were not cytotoxic for CHO cells. Their capacity to enter mammalian cells, and their localisation inside were ascertained after labelling the nucleus and actin, by examining the cells by confocal, reflected light and fluorescence microscopy.

Molecular design of inorganic scintillators: from alkoxides to scintillating materials

In this paper we report on preliminary results obtained on sol–gel derived inorganic scintillators. A new general scheme based on the use of alkoxide chemistry is proposed for the preparation of various scintillating materials including LuBO3, GdBO3, LuPO4 and Lu2SiO5. The materials are obtained as monophasic powders and as homogeneous transparent thin films for borates and silicate. Thermal behaviour is analysed and a study of the oxidation state of cerium ions is proposed by combining X-ray photoelectron spectroscopy and X-ray absorption near edge structure spectroscopy. A mixture of Ce3+ and Ce4+ is observed in proportions depending on the material. This presence of Ce4+ ions does not seem to greatly affect the observed scintillation yields of the materials. Efficient sol–gel derived scintillators have been prepared both as powders and thin films. The preparation of optical quality scintillator thin films could be the starting point for a new generation of scintillating materials. Finally, the versatility of the proposed synthesis route will allow extension of this study towards various other materials.

Sol–gel derived YPO4 and LuPO4 phosphors, a spectroscopic study

Abstract LnPO 4 (Ln=Y, Lu) orthophosphates have been prepared by a new sol–gel route. Eu 3+ doped materials have also been synthesized and studied. X-ray diffraction performed on all samples showed that pure xenotime phase is obtained in all cases even for doped samples. The morphology of the powders has been determined and small grains with narrow size distribution are evidenced. Finally the optical properties of the doped materials have been studied, the substitution of Eu 3+ for Ln 3+ is confirmed and the radiative lifetimes are compatible with potential applications of these phosphors.

The Efficacies of Cell-Penetrating Peptides in Accumulating in Large Unilamellar Vesicles Depend on their Ability To Form Inverted Micelles

In this study, the direct translocation of cell‐penetrating peptides (CPPs) into large unilamellar vesicles (LUVs) was shown to be rapid for all the most commonly used CPPs. This translocation led within a few minutes to intravesicular accumulation up to 0.5 mM, with no need for a transbilayer potential. The accumulation of CPPs inside LUVs was found to depend on CPP sequence, CPP extravesicular concentration and phospholipid (PL) composition, either in binary or ternary mixtures of PLs. More interestingly, the role of anionic phospholipid flip‐flopping in the translocation process was ascertained. CPPs enhanced the flipping of PLs, and the intravesicular CPP accumulation directly correlated with the amount of anionic PLs that had been transferred from the external to the internal leaflet of the LUV bilayer, thus demonstrating the transport of peptide/lipid complexes as inverted micelles.

Preferential site substitution in sol-gel derived Eu3+ doped Lu2SiO5: a combined study by X-ray absorption and luminescence spectroscopies

Eu3+ doped Lu2SiO5 (LSO) has been prepared by sol-gel chemistry. X-Ray diffraction showed that only the monoclinic phase corresponding to LSO is observed, even for doped samples. X-Ray absorption measurements were performed at both Lu and Eu LIII edges to investigate the substitution of Eu3+ for Lu3+ in LSO. Lutetium and europium oxides were used as references for XAFS signal extraction. The validity of XAFS measurements for studying the substitution of Eu3+ for Lu3+ at low doping levels (5at.%) was confirmed on a LuBO3 sample which has a simpler crystalline structure. The XAFS spectrum recorded at the Eu LIII edge for Eu3+ doped LSO demonstrates the effective substitution of Eu3+ for Lu3+ and refined inter-atomic distances involving Eu3+ are consistent with a preferential substitution on the higher coordination site labelled A2 [LuO7]. Luminescence measurements performed on doped samples show the existence of an impurity phase identified as Lu2O3. Site selective excitation measurements can distinguish between the two crystallographic sites of (Lu,Eu) ions in LSO and the numbering of the Eu3+ emission lines is in good agreement with the expected C1 symmetry. De-convolution of the 7F0 → 5D0 excitation spectrum recorded at room temperature allowed quantification of the populations of the two sites: 37% on A1 [LuO6] and 63% on A2 [LuO7]. This result agrees with structural considerations taking into account the difference in the ionic radii of Eu3+ and Lu3+.

How to unveil self-quenched fluorophores and subsequently map the subcellular distribution of exogenous peptides

Confocal laser scanning microscopy (CLSM) is the most popular technique for mapping the subcellular distribution of a fluorescent molecule and is widely used to investigate the penetration properties of exogenous macromolecules, such as cell-penetrating peptides (CPPs), within cells. Despite the membrane-association propensity of all these CPPs, the signal of the fluorescently labeled CPPs did not colocalize with the plasma membrane. We studied the origin of this fluorescence extinction and the overall consequence on the interpretation of intracellular localizations from CLSM pictures. We demonstrated that this discrepancy originated from fluorescence self-quenching. The fluorescence was unveiled by a “dilution” protocol, i.e. by varying the ratio fluorescent/non-fluorescent CPP. This strategy allowed us to rank with confidence the subcellular distribution of several CPPs, contributing to the elucidation of the penetration mechanism. More generally, this study proposes a broadly applicable and reliable method to study the subcellular distribution of any fluorescently labeled molecules.

Accumulation of cell‐penetrating peptides in large unilamellar vesicles: A straightforward screening assay for investigating the internalization mechanism

The internalization of cell‐penetrating peptides (CPPs) into liposomes (large unilamellar vesicles, LUVs) was studied with a rapid and robust procedure based on the quenching of a small fluorescent probe, 7‐nitrobenz‐2‐oxa‐1,3‐diazole (NBD). Quenching can be achieved by reduction with dithionite or by pH jump. LUVs with different compositions of phospholipids (PLs) were used to screen the efficacy of different CPPs. In order to “validate” the composition of the membrane models, a control cationic peptide, which does not enter eukaryotic cells, was included in the study. It was found that pure DOPG or DOPG within ternary mixtures with cholesterol are the most appropriate models for studying CPP translocation. An anionic lipid, such as DOPG, is required for the adsorption of the basic peptides on the surface of LUVs. In addition, it acts as transfer agent through the lipid bilayer. A fluid phase and/or the presence of phase defects also appear mandatory for the internalization to occur. The neutralization of charges within an inverted micelle demonstrated in the case of DOPG and also proposed for a ternary mixture of PLs might not be the only mechanism for the CPP translocation. Finally, it is shown that oleic acid facilitates the entry inside LUVs in gel phase of a series of cationic peptides including CPPs and also the negative control peptide PKCi.

Antibody Recognition in multiple sclerosis and rett syndrome using a collection of linear and cyclic N‐glucosylated antigenic probes

Antibody detection in autoimmune disorders, such as multiple sclerosis (MS) and Rett syndrome (RTT) can be achieved more efficiently using synthetic peptides. The previously developed synthetic antigenic probe CSF114(Glc), a type I′ β‐turn N‐glucosylated peptide structure, is able to recognize antibodies in MS and RTT patients’ sera as a sign of immune system derangement. We report herein the design, synthesis, conformational analysis, and immunological evaluation of a collection of glycopeptide analogs of CSF114(Glc) to characterize the specific role of secondary structures in MS and RTT antibody recognition. Therefore, we synthesized a series of linear and cyclic short glucosylated sequences, mimicking different β‐turn conformations, which were evaluated in inhibition enzyme‐linked immunosorbent assays (ELISA). Calculated IC50 ranking analysis allowed the selection of the candidate octapeptide containing two (S)−2‐amino‐4‐pentynoic acid (L‐Pra) residues Ac‐Pra‐RRN(Glc)GHT‐Pra‐NH2, with an IC50 in the nanomolar range. This peptide was adequately modified for solid‐phase ELISA (SP‐ELISA) and surface plasmon resonance (SPR) experiments. Pra‐RRN(Glc)GHT‐Pra‐NH2 peptide was modified with an alkyl chain linked to the N‐terminus, favoring immobilization on solid phase in SP‐ELISA and differentiating IgG antibody recognition between patients and healthy blood donors with a high specificity. However, this peptide displayed a loss in IgM specificity and sensitivity. Moreover, an analog was obtained after modification of the octapeptide candidate Ac‐Pra‐RRN(Glc)GHT‐Pra‐NH2 to favor immobilization on SPR sensor chips. SPR technology allowed us to determine its affinity (KD = 16.4 nM), 2.3 times lower than the affinity of the original glucopeptide CSF114(Glc) (KD = 7.1 nM).

Unsaturated acyl chains dramatically enhanced cellular uptake by direct translocation of a minimalist oligo-arginine lipopeptide

The recurring issue with cell penetrating peptides is how to increase direct translocation vs. endocytosis, to avoid premature degradation. Acylation by a cis unsaturated chain (C22:6) of a short cationic peptide provides a new rational design to favour diffuse cytosolic and dense Golgi localisations.

Surface characterization of sol-gel derived scintillating rare-earth doped Lu2SiO5 thin films

Rare earth doped Lu2SiO5 thin films have been prepared by combining sol-gel process and spin coating. Annealing treatment results in the crystallization of the film and efficient incorporation of rare earth doping ions. XPS and RBS spectrocopies showed that the composition of the films is close to the nominal one. Adventitious carbon has been observed and attributed to incomplete pyrolysis of metal-organic precursors. XPS concentrations profiles show a good homogeneity for the films. RBS demonstrated some inter-diffusion between amorphous carbon substrate and silicate films resulting in a gradient of carbon at the interface between the substrate and the film itself.