Hepatotropic Peptides Grafted onto Maleimide-Decorated Nanoparticles: Preparation, Characterization and In Vitro Uptake by Human HepaRG Hepatoma Cells.

We recently demonstrated the strong tropism of George Baker (GB) Virus A (GBVA10-9) and Plasmodium circumsporozoite protein (CPB) derived synthetic peptides towards hepatoma cells. In a first approach, these peptides were covalently bound to poly(benzyl malate) (PMLABe73) and poly(ethylene glycol)-block-PMLABe73 (PEG62-b-PMLABe73) (co)polymers, and corresponding peptide-decorated nanoparticles (NPs) were prepared by nanoprecipitation. We showed that peptide enhanced NPs internalization by hepatoma cells. In the present work, we set up a second strategy to functionalize NPs prepared from PMLABe73 derivates. First, maleimide-functionalized PMLABe73 (Mal-PMLABe73) and PEG62-b-PMLABe73 (Mal-PEG62-b-PMLABe73) were synthesized and corresponding NPs were prepared by nanoprecipitation. Then, peptides (GBVA10-9, CPB and their scramble controls GBVA10-9scr and CPBscr) with a thiol group were engrafted onto the NPs' maleimide groups using the Michael addition to obtain peptide functionalized NPs by post-formulation procedure. These peptide-modified NPs varied in diameter and dispersity depending on the considered peptides and/or (co)polymers but kept their spherical shape. The peptide-functionalized NPs were more efficiently internalized by HepaRG hepatoma cells than native and maleimide-NPs with various levels relying on the peptide's nature and the presence of PEG. We also observed important differences in internalization of NPs functionalized by the maleimide-thiol-peptide reaction compared to that of NPs prepared from peptide-functionalized PMLABe73 derivatives.

Synthesis of Poly(Malic Acid) Derivatives End-Functionalized with Peptides and Preparation of Biocompatible Nanoparticles to Target Hepatoma Cells.

Recently, short synthetic peptides have gained interest as targeting agents in the design of site-specific nanomedicines. In this context, our work aimed at developing new tools for the diagnosis and/or therapy of hepatocellular carcinoma (HCC) by grafting the hepatotropic George Baker (GB) virus A (GBVA10-9) and Plasmodium circumsporozoite protein (CPB)-derived peptides to the biocompatible poly(benzyl malate), PMLABe. We successfully synthesized PMLABe derivatives end-functionalized with peptides GBVA10-9, CPB, and their corresponding scrambled peptides through a thiol/maleimide reaction. The corresponding nanoparticles (NPs), varying by the nature of the peptide (GBVA10-9, CPB, and their scrambled peptides) and the absence or presence of poly(ethylene glycol) were also successfully formulated using nanoprecipitation technique. NPs were further characterized by dynamic light scattering (DLS), electrophoretic light scattering (ELS) and transmission electron microscopy (TEM), highlighting a diameter lower than 150 nm, a negative surface charge, and a more or less spherical shape. Moreover, a fluorescent probe (DiD Oil) has been encapsulated during the nanoprecipitation process. Finally, preliminary in vitro internalisation assays using HepaRG hepatoma cells demonstrated that CPB peptide-functionalized PMLABe NPs were efficiently internalized by endocytosis, and that such nanoobjects may be promising drug delivery systems for the theranostics of HCC.

Concentration of drugs in blood of suspected impaired drivers.

Analytical records concerning 440 living drivers suspected of driving under the influence of drug (DUID) were collected and examined during a 2 years period ranging from 2002 to 2003 in canton de Vaud, Valais, Jura and Fribourg (Switzerland). This study included 400 men (91%) and 40 women (9%). The average age of the drivers was 28+/-10 years (minimum 16 and maximum 81). One or more psychoactive drugs were found in 89% of blood samples. Half of cases (223 of 440, 50.7%) involved consumption of mixtures (from 2 to 6) of psychoactive drugs. The most commonly detected drugs in whole blood were cannabinoids (59%), ethanol (46%), benzodiazepines (13%), cocaine (13%), amphetamines (9%), opiates (9%) and methadone (7%). Among these 440 cases, 11-carboxy-THC (THCCOOH) was found in 59% (median 25 ng/ml (1-215 ng/ml)), Delta(9)-tetrahydrocannabinol (THC) in 53% (median 3 ng/ml (1-35 ng/ml)), ethanol in 46% (median 1.19 g/kg (0.14-2.95 g/kg)), benzoylecgonine in 13% (median 250 ng/ml (29-2430 ng/ml)), free morphine in 7% (median 10 ng/ml (1-111 ng/ml)), methadone in 7% (median 110 ng/ml (27-850 ng/ml)), 3,4-methylenedioxymethamphetamine (MDMA) in 6% (median 218 ng/ml (10-2480 ng/ml)), nordiazepam in 5% (median 305 ng/ml (30-1560 ng/ml)), free codeine in 5% (median 5 ng/ml (1-13 ng/ml)), midazolam in 5% (median 44 ng/ml (20-250 ng/ml)), cocaine in 5% (median 50 ng/ml (15-560 ng/ml)), amphetamine in 4% (median 54 ng/ml (10-183 ng/ml)), diazepam in 2% (median 200 ng/ml (80-630 ng/ml)) and oxazepam in 2% (median 230 ng/ml (165-3830 ng/ml)). Other drugs, such as lorazepam, zolpidem, mirtazapine, methaqualone, were found in less than 1% of the cases.

Determination and distribution of clotiapine (Entumine) in human plasma, post-mortem blood and tissue samples from clotiapine-treated patients and from autopsy cases.

The antipsychotic drug clotiapine (Entumine) has been marketed for more than 35 years, however there is little published data on the therapeutic and toxic concentrations of this drug. To fill this gap, two rapid and sensitive methods were developed for the determination of clotiapine (2-chloro-11-(4-methyl-1-piperazinyl)dibenzo-[b,f][1,4]-thiazepine), in human plasma and post-mortem blood and tissue samples. After simple liquid-liquid extraction at pH 9.5 with n-hexane/dichloromethane (85/15, v/v), clotiapine was quantitated by HPLC-DAD and by GC-NPD. The calibration curve was linear between 10 and 1000 microg/L. The limit of detection (LOD) and the limit of quantification (LOQ) were found to be 2 and 6 microg/L for the GC-NPD method and 5 and 15 microg/L for the HPLC-method, respectively. These methods were applied to 12 plasma samples from patients treated with clotiapine, to seven autopsy cases and to one case of driving under the influence of drugs (DUID). Concentrations ranged for the clotiapine-treated patients between 6 and 155 microg/L (mean 46 microg/L), and for the autopsy cases between 22 and 341 microg/L (mean 123 microg/L).