Ariane Boudier

PLGA in situ implants formed by phase inversion: critical physicochemical parameters to modulate drug release.

In situ forming implants (ISI) based on phase separation by solvent exchange represent an attractive alternative to conventional preformed implants and microparticles for parenteral applications. They are indeed easier to manufacture and their administration does not require surgery, therefore improving patient compliance. They consist of polymeric solutions precipitating at the site of injection and thus forming a drug eluting depot. Drug release from ISI is typically divided into three phases: burst during precipitation of the depot, diffusion of drug through the polymeric matrix and finally drug release by system degradation. This review gives a comprehensive overview on (i) the theoretical bases of these three phases, (ii) the parameters influencing them and (iii) the remaining drawbacks which have to be addressed to enlarge their commercial opportunities. Indeed, although some of them are already commercialized, ISI still suffer from limitations: mainly lack of reproducibility in depot shape, burst during solidification and potential toxicity. Nevertheless, depending on the targeted therapeutic application, these shortcomings may be transformed into advantages. As a result, keys are given in order to tailor these formulations in view of the desired application so that ISI could gain further clinical importance in the following years.

Pitfalls of assays devoted to evaluation of oxidative stress induced by inorganic nanoparticles

During the last years, there has been a remarkable increase in the use of inorganic nanoparticles (NP) in different applications, including consumer and medical products. Despite these promising applications, the extremely small size of NP allows them to penetrate cells, in which they can interact with intracellular structures causing serious side effects. A number of studies showed that NP cause adverse effects predominantly via induction of an oxidative stress - an imbalance between damaging oxidants and protective antioxidants - resulting in inflammation, immune response, cell damages, genotoxicity, etc … Most of the in vitro methods used for measurement of oxidative stress biomarkers were designed and standardized for conventional organic, inorganic and biochemical compounds. More recently, these methods have been adapted to studies related to various nanomaterials. Thus, this review is an attempt to highlight some current methods employed in and to provide a critical analysis of the major challenges and issues faced in this emerging field.

S-nitrosation/Denitrosation in Cardiovascular Pathologies: Facts and Concepts for the Rational Design of S-nitrosothiols

Nitric oxide (•NO) is a physiological mediator of vasorelaxation constitutively synthesized by endothelial nitric oxide synthase. Because •NO has a short half-life, it is stored by proteins through S-nitrosation reactions. S-nitrosation was recently defined as a post-translational modification of proteins for cellular signalling, as important as glycosylation and phosphorylation. Disulfide forming/ isomerizing enzymes like thioredoxin (Trx), protein disulfide isomerase (PDI), which are chaperone proteins, are implicated into transnitrosation reactions, which are the transfer of •NO from one cysteine residue to another one. Furthermore, Trx has been shown to denitrosate S-nitrosoproteins depending on its redox status. S-nitrosation of Trx on Cys residues apart from active site, under nitrosative or oxidative stresses, enhances its activity, thereby reducing intracellular reactive oxygen species. Trx and PDI have therefore an essential role for cell signalling control which leads, among other actions, to cardio and vasculo-protection. The diminution of either •NO synthesis or bioavailability is implicated into a large number of cardiovascular pathologies associated to hypoxia or vasoconstriction like, endothelial dysfunction, arterial hypertension and atherosclerosis. In order to mimic the physiological storage of •NO as S-nitrosothiols, the development of •NO donors should be based on the covalent S-NO bond. The chemical stabilisation of the S-NO bond and protection against enzymatically active proteins such as PDI//Trx are major points for the design of stable compounds. S-nitrosothiols entrapment in innovative formulations (films, gels, microparticles, nanoparticles) is an emerging field in order to stabilise and protect them, and to deliver •NO under a sustained release at the targeted site.

Impact of gold nanoparticle coating on redox homeostasis

Gold nanoparticles (AuNP) hold great potential for biomedical applications. This study was aimed at examination of the effect of AuNP coating on the redox status of their environment. Two kinds of AuNP were tested, similar by shape and size, but with different surface coatings: either stabilized with citrate or functionalized with dihydrolipoic acid (Au@DHLA NP). Interestingly, whereas citrate-stabilized AuNP interact in vitro with reduced glutathione (GSH) and S-nitrosoglutathione, Au@DHLA NP do not interfere with both biomolecules. Albumin exhibits higher affinity toward citrate-stabilized AuNP than Au@DHLA NP, increasing their hydrodynamic diameter (8.0- and 1.3-fold, respectively). Furthermore, the AuNP coating affects also their internalization by macrophages (which was two fold higher for citrate-stabilized AuNP), following an exposure to a subtoxic NP concentration (10 nM, 80% viability). Citrate-stabilized AuNP were found to decrease the intracellular GSH level (ca. 20%), with no increase in reactive oxygen species production. Furthermore, these AuNP did not induce apoptosis (as shown by caspase-3 activity and nfkb2 transcription factor), and also did not activate gene expression related to oxidative stress (ncf1) and inflammatory response (tnfα). The present data highlight that the functionalization of AuNP with DHLA decreases their reactivity with biomolecules and cells, resulting in a promising medical platform.

Simple spectrophotocolorimetric method for quantitative determination of gold in nanoparticles.

A simple spectrophotocolorimetric method devoted to the measurement of gold content in nanoparticles (NPs) was developed. It includes two steps: (i) metal gold NPs (Au NPs) are oxidized into the AuCl(4)(-) anion using a 5×10(-2) M HCl-1.5×10(-2) M NaCl-7×10(-4) M Br(2) solution, next (ii) AuCl(4)(-) concentration is measured using a spectrophotometric assay based on the reaction of AuCl(4)(-) with the cationic form of Rhodamine B to give a violet ion pair complex. This latter is extracted with diisopropyl ether and the absorbance of the organic complex is measured at 565 nm. The method is linear in the range 6-29 μM of AuCl(4)(-) with a limit of detection of 4.5μM. The analytical method was optimized with respect of bromine excess to obtain complete Au NPs oxidation. The method was applied to two types of Au NPs currently under investigation: citrate-stabilized Au NPs and Au NPs capped with dihydrolipoic acid (Au@DHLA). Both the gold content of Au NPs and the concentration of NPs (using NP diameter measured by transmission electron microscopy) have been calculated.

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.

Role of gold nanoparticles capping density on stability and surface reactivity to design drug delivery platforms.

Five-nanometer sized gold nanoparticles (Au NPs) stabilized with citrate ions have been reacted with various amounts of dihydrolipoic acid (DHLA) (×28, ×56, ×140, ×222, relative to Au NPs). Ligand exchange between citrate and the dithiol resulted in DHLA-capped Au NPs, whose degree of inertia was found to be related to the density of capping. The results revealed the importance of DHLA coating density to enhance the colloidal stability and modulate the reactivity toward free radicals and proteins of biological relevance. Thus, Au NPs capped with the highest amount of DHLA were found to be the ones that were, first, the most resistant to environmental changes, then characterized by the lowest residual catalytic reactivity of their metallic core, and finally the lowest interacting with proteins through nonspecific adsorption. The physicochemical properties conferred to Au NPs prepared with the ×222 excess should be valuable for further pharmaceutical development of nanoparticle platforms.

Endothelial γ-Glutamyltransferase Contributes to the Vasorelaxant Effect of S-Nitrosoglutathione in Rat Aorta

S-nitrosoglutathione (GSNO) involved in storage and transport of nitric oxide (•NO) plays an important role in vascular homeostasis. Breakdown of GSNO can be catalyzed by γ-glutamyltransferase (GGT). We investigated whether vascular GGT influences the vasorelaxant effect of GSNO in isolated rat aorta. Histochemical localization of GGT and measurement of its activity were performed by using chromogenic substrates in sections and in aorta homogenates, respectively. The role of GGT in GSNO metabolism was evaluated by measuring GSNO consumption rate (absorbance decay at 334 nm), •NO release was visualized and quantified with the fluorescent probe 4,5-diaminofluorescein diacetate. The vasorelaxant effect of GSNO was assayed using isolated rat aortic rings (in the presence or absence of endothelium). The role of GGT was assessed by stimulating enzyme activity with cosubstrate glycylglycine, as well as using two independent inhibitors, competitive serine borate complex and non-competitive acivicin. Specific GGT activity was histochemically localized in the endothelium. Consumption of GSNO and release of free •NO decreased and increased in presence of serine borate complex and glycylglycine, respectively. In vasorelaxation experiments with endothelium-intact aorta, the half maximal effective concentration of GSNO (EC50 = 3.2±0.5.10−7 M) increased in the presence of the two distinct GGT inhibitors, serine borate complex (1.6±0.2.10−6 M) and acivicin (8.3±0.6.10−7 M), while it decreased with glycylglycine (4.7±0.9.10−8 M). In endothelium-denuded aorta, EC50 for GSNO alone increased to 2.3±0.3.10−6 M, with no change in the presence of serine borate complex. These data demonstrate the important role of endothelial GGT activity in mediating the vasorelaxant effect of GSNO in rat aorta under physiological conditions. Because therapeutic treatments based on GSNO are presently under development, this endothelium-dependent mechanism involved in the vascular effects of GSNO should be taken into account in a pharmacological perspective.

Structure and Stability Studies of Pharmacologically Relevant S-Nitrosothiols: A Theoretical Approach

Nowadays, S-nitrosothiols (RSNOs) represent a promising class of nitric oxide (NO) donors that could be successfully used as drugs to compensate the decrease of NO production that usually arises in conjunction with cardiovascular diseases. Nevertheless, notwithstanding their pharmacological interest, the structure-stability relationship in RSNOs is still unclear, and this issue, together with the mechanism of NO donation in the physiological medium, deserves further investigation. As a first step forward in this direction, in this paper, the overall stability and structural preference of two pharmacologically relevant S-nitrosothiol molecules were studied in detail by means of computational strategies. In particular, performing calculations in implicit solvent (water) on the S-nitroso-N-acetylpenicillamine and the S-nitroso-N-acetylcysteine and analyzing the noncovalent interactions networks of their most stable conformers, we observed that the structure and the stability of these molecules can be directly related to the formation of stabilizing hydrogen-bond and chalcogen-chalcogen intramolecular interactions. The obtained results represent the starting point for further investigations to be conducted also on larger RSNOs to shed further light on the role played by intra- and intermolecular interactions and by solvation effects in stabilizing this class of molecules. The obtained insights will be hopefully helpful to design new RSNO-based drugs characterized by an enhanced pharmacological potency.

Are in situ formulations the keys for the therapeutic future of S-nitrosothiols?

S-nitrosoglutathione (GSNO) and S-nitroso-N-acetylpenicillamine (SNAP) were formulated into in situ forming implants (ISI) and microparticles (ISM) using PLGA and either N-methyl-2-pyrrolidone (NMP) or triacetin. Physicochemical characterization was carried out, including the study of matrix structure and degradation. A strong correlation between drug hydrophobicity and the in vitro release profiles was observed: whatever the formulation, GSNO and SNAP were completely released after ca. 1 day and 1 week, respectively. Then, selected formulations (i.e., SNAP-loaded NMP formulations) demonstrated the ability to sustain the vasodilation effect of SNAP, as shown by monitoring the arterial pressure (telemetry) of Wistar rats after subcutaneous injection. Both ISI and ISM injections resulted in a 3-fold extended decrease in pulse arterial pressure compared with the unloaded drug, without significant decrease in the mean arterial pressure. Hence, the results emphasize the suitability of these formulations as drug delivery systems for S-nitrosothiols, widening their therapeutic potential.