Igor Clarot

A Complete Physicochemical Identity Card of S-nitrosoglutathione

S-nitrosoglutathione (GSNO) is one of low molecular weight S-nitrosothiols occuring in humans. Nowadays, it is widely used as a nitric oxide donor for in vitro, ex vivo and in vivo experiments related to the investigation of its pathophysiological role as well as in clinical trials, aimed at its potential therapeutic use. Despite numerous reports on this physiological molecule, its quality control does not match the criteria required by competent pharmaceutical authorities. Hereby, an extensive physicochemical characterisation of synthesised and purified GSNO is provided for the first time. Indeed, structural identification including spectrometric, thermal and elemental analyses was consistent with the GSNO structure. An ion-pairing reversed phase HPLC system was developed to assess (i) GSNO content with UV detection at 334 nm, and (ii) fingerprint of its impurities coming from synthesis process and/or storage conditions, at 220 nm. The assynthesised product showed a content of 102.5 %, with respect to a commercially available standard. The identified impurities, i.e. chloride, nitrite, nitrate, reduced glutathione and glutathione disulfide, were also quantified basing on pharmaceutical requirements. Main products released during various storage conditions (pH, temperature, dioxygen, ...) were disulfide glutathione and nitrite ion. Recommendations are given for the safe use of GSNO in biological and pharmacological experiments.

Characterization and stability of gold nanoparticles depending on their surface chemistry: Contribution of capillary zone electrophoresis to a quality control☆

Four kinds of gold nanoparticles (AuNP) quite similar in terms of gold core size (ca. 5nm) and shape (spherical) but differing by their surface chemistry (either negatively, or positively charged, or neutral) were synthesized. They were analyzed using both the classical physicochemical approach (spectrophotometry, dynamic light scattering coupled or not to electrophoresis and transmission electron microscopy) and capillary zone electrophoresis equipped with photodiode array detection. The results obtained by both methodologies (related to Surface Plasmon Band-maximal absorbance wavelength-, and zeta potential and electrophoretic mobilities) were well correlated. Moreover, taking advantage of the separation method, the sample heterogeneity was evaluated and an impurity profile was extracted. This allowed setting some specifications which were then applied on the one hand to a batch-to-batch survey to declare NP as conform or not after production and on the other hand to a stability study.

Glutathione: Antioxidant Properties Dedicated to Nanotechnologies

Which scientist has never heard of glutathione (GSH)? This well-known low-molecular-weight tripeptide is perhaps the most famous natural antioxidant. However, the interest in GSH should not be restricted to its redox properties. This multidisciplinary review aims to bring out some lesser-known aspects of GSH, for example, as an emerging tool in nanotechnologies to achieve targeted drug delivery. After recalling the biochemistry of GSH, including its metabolism pathways and redox properties, its involvement in cellular redox homeostasis and signaling is described. Analytical methods for the dosage and localization of GSH or glutathiolated proteins are also covered. Finally, the various therapeutic strategies to replenish GSH stocks are discussed, in parallel with its use as an addressing molecule in drug delivery.

One-week in vivo sustained release of a peptide formulated into in situ forming implants

The LR12 peptide has been reported to reduce the size of infarct and improve both cardiac function and survival in myocardial infarction in murine models, after daily repeated intraperitoneal injections. In order to protect peptide from degrading and to prolong its release, in situ implants based on biocompatible biodegradable polymers were prepared and both in vitro and in vivo releases were evaluated after subcutaneous administration to Wistar rats. A progressive and complete release was obtained in vitro in 3 weeks. In vivo, a 7-day sustained release was demonstrated after administrating the formulation once; bioavailability was improved by protecting the peptide against the degradation identified as a dimerization through disulfide bond formation. As a conclusion, in situ forming formulations are a suitable alternative for the therapeutic use of this peptide.

Labeling nitrogen species with the stable isotope 15N for their measurement by separative methods coupled with mass spectrometry: A review

Nitrogen and its numerous hydrogenated and oxygenated derivatives are of main importance in our environment and in living cells as well in both qualitative and quantitative aspects. Their monitoring is needed to evaluate all disturbances occurring in the nitrogen cycle and in pathophysiological events related to variations of nitric oxide (NO) bioavailability. Many analytical methods are devoted to the measurement of nitrogen species, especially those related to NO, in the environmental, biological and pharmacological fields, and they have already been compiled and discussed in numerous reviews. Nitrogen isotope (15N) is stable and has a low level of natural abundance. Labeling nitrogen species with 15N associated with mass spectrometry (MS) gives rise to more mechanistic information and improved analytical performances compared to conventional methods. The present review is dedicated to the 15N labeling of related nitrogen species to monitor their interconversion and metabolism, the different chemical probes used for their derivatization and the corresponding separative methods coupled with MS for analyzing resulting adducts. The fragmentation mode of the different adducts and the resulting selectivity and sensitivity are discussed.

Highly sensitive and simple liquid chromatography assay with ion-pairing extraction and visible detection for quantification of gold from nanoparticles

A simple isocratic HPLC method using visible detection was developed and validated for the quantification of gold in nanoparticles (AuNP). After a first step of oxidation of nanoparticles, an ion-pair between tetrachloroaurate anion and the cationic dye Rhodamine B was formed and extracted from the aqueous media with the help of an organic solvent. The corresponding Rhodamine B was finally quantified by reversed phase liquid chromatography using a Nucleosil C18 (150mm × 4.6mm, 3µm) column and with a mobile phase containing acetonitrile and 0.1% trifluoroacetic acid aqueous solution (25/75, V/V) at 1.0mLmin-1.and at a wavelength of 555nm. The method was validated using methodology described by the International Conference on Harmonization and was shown to be specific, precise (RSD < 11%), accurate and linear in the range of 0.1 - 30.0µM with a lower limit of quantification (LLOQ) of 0.1µM. This method was in a first time applied to AuNP quality control after their synthesis. In a second time, the absence of gold leakage (either as AuNP or gold salt form) from nanostructured multilayered polyelectrolyte films under shear stress was assessed.

Intestinal absorption of S-nitrosothiols: Permeability and transport mechanisms

Graphical abstract Figure. No caption available. &NA; S‐Nitrosothiols, a class of NO donors, demonstrate potential benefits for cardiovascular diseases. Drugs for such chronic diseases require long term administration preferentially through the oral route. However, the absorption of S‐nitrosothiols by the intestine, which is the first limiting barrier for their vascular bioavailability, is rarely evaluated. Using an in vitro model of intestinal barrier, based on human cells, the present work aimed at elucidating the mechanisms of intestinal transport (passive or active, paracellular or transcellular pathway) and at predicting the absorption site of three S‐nitrosothiols: S‐nitrosoglutathione (GSNO), S‐nitroso‐N‐acetyl‐l‐cysteine (NACNO) and S‐nitroso‐N‐acetyl‐d‐penicillamine (SNAP). These S‐nitrosothiols include different skeletons carrying the nitroso group, which confer different physico‐chemical characteristics and biological activities (antioxidant and anti‐inflammatory). According to the values of apparent permeability coefficient, the three S‐nitrosothiols belong to the medium class of permeability. The evaluation of the bidirectional apparent permeability demonstrated a passive diffusion of the three S‐nitrosothiols. GSNO and NACNO preferentially cross the intestinal barrier though the transcellular pathway, while SNAP followed both the trans‐ and paracellular pathways. Finally, the permeability of NACNO was favoured at pH 6.4, which is close to the pH of the jejunal part of the intestine. Through this study, we determined the absorption mechanisms of S‐nitrosothiols and postulated that they can be administrated through the oral route.