Application to several suspected poisoning cases of a validated analytical method for the determination of muscarine in human biological matrices using liquid chromatography with high-resolution mass spectrometry detection.

Despite prevention efforts, many cases of mushroom poisoning are reported around the world every year. Among the different toxins implicated in these poisonings, muscarine may induce parasympathetic neurological damage. Muscarine poisonings are poorly reported in the current literature, implying a lack of available data on muscarine concentrations in human matrices. A validated liquid chromatography with high-resolution mass spectrometry detection (Orbitrap technology) method was developed to determine muscarine concentrations in human urine, plasma, and whole blood samples. Muscarine was determined using 100 µL of biological fluids, and precipitation was used for sample preparation. Liquid chromatography-mass spectrometry was performed using an Accucore Phenyl-X analytical column with the electrospray source in positive ion mode. Muscarine was quantitated in parallel reaction monitoring (PRM) mode with D9-muscarine as the internal standard. The method was validated successfully over the concentration range 0.1-100 µg/L for plasma and whole blood and 1-100 µg/L for urine, with acceptable precision and accuracy (<13.5%), including the lower limit of quantification. Ten real cases of suspected muscarine poisoning were successfully confirmed with this validated method. Muscarine concentrations in these cases ranged from 0.12 to 14 µg/L in whole blood,

Determination of Orellanine in Human Biological Matrices Using Liquid Chromatography with High-Resolution Mass Spectrometry Detection: A Validated Method Applied to Suspected Poisoning Cases.

Consumption of mushrooms can become unsafe for the consumer in case of confusion. Some fungi of Cortinarius genus contain the nephrotoxic mycotoxin orellanine responsible for their toxicity. Related case poisoning diagnosis is a challenge for both clinicians and analysts because of a long latency period between intake and toxic syndrome, the lack of available information in literature and the numerous pitfalls of orellanine identification/quantification in biological samples. In this situation, we propose an analytical method designed for the orellanine detection and/or quantification in biological matrices such as plasma, urine and whole blood, in a context of related intoxication suspected case. Using 1 mL biological sample volume, this liquid chromatographic with high-resolution mass spectrometry detection method (i) exhibits a limit of quantification for orellanine of 0.5 µg/L in plasma and urine and (ii) enables orellanine detection in whole blood with a limit of detection of 0.5 µg/L. This validated analytical method was successfully applied to 10 suspected intoxication cases.

Preparation and characterization of mTHPC-loaded solid lipid nanoparticles for photodynamic therapy.

Among various attempts to enhance the therapeutic efficacy of photodynamic therapy (PDT), the specific delivery of photosensitizer (PS) in the tumor tissue is expected to improve its clinical applications. The aim of this study was to engineer lipid nanoparticles (LNP) with different sizes and various PS contents, using simple solvent-free and easily scale up manufacturing processes. Meso-(tetrahydroxyphenyl) chlorin (mTHPC) is one of the most potent photoactive compounds for clinical use. We demonstrated that mTHPC was efficiently incorporated into the lipid core of LNP, leading to a large range of stable and reproducible mTHPC-loaded LNP with narrow size distribution. Photophysical and physico-chemical properties of mTHPC-loaded LNP were assessed as well as absorption spectra and singlet oxygen emission, colloidal stability, particle size and zeta potential. The photocytotoxicity of selected mTHPC-loaded solid LNP was demonstrated on MCF-7 cells under irradiation at 652nm with a range of light fluence from 1.0 to 10J/cm(2). All physico-chemical, photophysical and biological results allow us to conclude that solid LNP appear as a very promising nano-mTHPC delivery system for PDT.

Phospholipid-detergent conjugates as novel tools for siRNA delivery.

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.

Novel glycolipid TLR2 ligands of the type Pam2Cys-α-Gal: synthesis and biological properties.

A more complete understanding of the mechanism of action of TLR agonists has fueled the investigation of new synthetic immunoadjuvants. In this context, we designed and synthesized glycolipids of the type Pam(2)Cys-α-Galactose as novel immunoadjuvants. Their synthesis required modifying a hydrophobic tBoc-[2,3-bispalmitoyloxy-(2R)-propyl]-R-cysteinyl moiety, i.e. the minimal structure required for TLR2 agonist activity, by addition of a hydrophilic head, either an α-Galactosylpyranose or an α-Galactosylfuranose to gain respectively Pam(2)CGalp and Pam(2)CGalf. While preparing a carbohydrate building block, an unexpected stereoselectivity was observed during a halide ion-catalytic process on a protected galactofuranose: the alpha anomer was obtained with surprisingly high selectivity (α/ß ratio>9) and with good isolated yield (51%). The TLR2 binding properties of Pam(2)CGalp and Pam(2)CGalf were then fully evaluated. Their efficiency in triggering the proliferation of BALB/c mouse splenocytes was also compared to that of Pam(2)CAG and Pam(3)CAG, two well-established ligands of TLRs. Moreover, the maturation state of murine dendritic cells previously incubated with either Pam(2)CGalp or Pam(2)CGalf was monitored by flow cytometry and compared to that induced by lipopolysaccharide. Pam(2)CGalp and Pam(2)CGalf were found to be equivalent TLR2 agonists, and induced splenocyte proliferation and DC maturation. With very similar activity, Pam(2)CGalp and Pam(2)CGalf were also 10-fold to 100-fold better than Pam(2)CAG and Pam(3)CAG at inducing B cell proliferation. This represents the first time a glucidic head has been added to the tBoc-[2,3-bispalmitoyloxy-(2R)-propyl]-R-cysteinyl moiety whilst maintaining the immunomodulating activity. This should greatly enrich the data available on Pam(2)C structure/activity relationships.

Pyridylthiourea-grafted polyethylenimine offers an effective assistance to siRNA-mediated gene silencing in vitro and in vivo.

Success of synthetic interfering nucleic acids (siRNAs)-based therapy relies almost exclusively on effective, safe and preferably nanometric delivery systems which can be easily prepared, even at high concentrations. We prepared by chemical synthesis various self-assembling polymers to entrap siRNAs into stable polyplexes outside cells but with a disassembly potential upon sensing endosomal acidity. Our results revealed that pyridylthiourea-grafted polyethylenimine (πPΕΙ) followed the above-mentioned principles. It led to above 90% siRNA-mediated gene silencing in vitro on U87 cells at 10 nM siRNA concentration and did not have a hemolytic activity. Assembly of siRNA/πPΕΙ at high concentration was then studied and 4.5% glucose solution, pH 6.0, yielded stable colloidal solutions with sizes slightly below 100 nm for several hours. A single injection of these concentrated siRNA polyplexes into luciferase-expressing human glioblastoma tumors, which were subcutaneously xenografted into nude mice, led to a significant 30% siRNA-mediated luciferase gene silencing 4 days post-injection. Our results altogether substantiate the potential of self-assembling cationic polymers with a pH-sensitive disassembly switch for siRNA delivery in vitro and also in vivo experiments.

Proton sponge trick for pH-sensitive disassembly of polyethylenimine-based siRNA delivery systems.

Small interfering RNAs offer novel opportunities to inhibit gene expression in a highly selective and efficient manner but depend on cytosolic translocation with synthetic delivery systems. The polyethylenimine (PEI) is widely used for plasmid DNA transfection. However, the water-soluble PEI does not form siRNA polyplexes stable enough in extracellular media for effective delivery. We previously showed that rendering PEI insoluble in physiological media, without modifying drastically its overall cationic charge density, by simple conjugation with natural hydrophobic alpha-amino acids, can lead to effective siRNA delivery in mammalian cells. In here, we comprehensively investigated the mechanism behind the excellent efficacy of the leading PEIY vector. Our data revealed that the underlining proton sponge property is key to the effectiveness of the tyrosine-polyethylenimine conjugate as it may allow both endosomal rupture and siRNA liberation via an optimal pH-sensitive dissolution of the PEIY self-aggregates. Altogether, these results should facilitate the development of novel and more sophisticated siRNA delivery systems.

Tyrosine-modified PEI: a novel and highly efficient vector for siRNA delivery in mammalian cells.

Delivery of synthetic small interfering RNA (siRNA) into cell remains the major obstacle to its biological activity. Cationic polymers, and in particular the "proton sponge" polyethylenimine (PEI), has shown promise for cancer gene therapy but appears inefficient for siRNA delivery. Here we report that modifying branched PEI with amino acids led to efficient siRNA delivery into mammalian cell lines, even in the presence of serum, and at dose as low as 1 nM.

Sticky overhangs enhance siRNA-mediated gene silencing.

siRNA delivery to cells offers a convenient and powerful means of gene silencing that bypasses several barriers met by gene delivery. However, nonviral vectors, and especially polymers, form looser complexes with siRNA than with plasmid DNA. As a consequence, exchange of siRNA for larger polymeric anions such as proteoglycans found outside cells and at their surface may occur and lower delivery. We show here that making siRNAs "gene-like," via short complementary A(5-8)/T(5-8) 3' overhangs, increases complex stability, and hence RNase protection and gene silencing in vitro up to 10-fold. After decomplexation in the cytoplasm, sticky siRNA (ssiRNA) concatemers fall apart. ssiRNAs are therefore not inducing antiviral responses, as shown by the absence of IFN-beta production. Finally, transfection experiments in the mouse lung show that ssiRNA should be particularly suited to silencing with linear polyethylenimine in vivo.