Elaboration of Sterically Stabilized Liposomes for S-Nitrosoglutathione Targeting to Macrophages.

S-nitrosoglutathione (GSNO) is a potential therapeutic for infectious disease treatment because of its pivotal role in macrophage-mediated inflammatory responses and host defense in addition to direct antibacterial activities. In this study, sterically stabilized cationic liposomes (SSCL) and sterically stabilized anionic liposomes (SSAL) were developed as nanocarriers for macrophage targeting. Elaborated liposomes were characterized in terms of size, zeta potential, morphology, encapsulation efficiency, in vitro drug release behavior and cytotoxicity. Their versatility in targeting monocytes/macrophages was determined by confocal laser scanning microscopy and transmission electron microscopy. Flow cytometry revealed that cellular uptake of both SSCL and SSAL was governed by several endocytic clathrin- and caveolae-dependent mechanisms. Quantitative assessments of intracellular nitric oxide demonstrated highly efficient uptake of GSNO-loaded SSCL that was twenty-fold higher than that of GSNO-free molecules. GSNO-loaded SSCL displayed strong bacteriostatic effects on Staphylococcus aureus and Pseudomonas aeruginosa, which can be involved in pulmonary infectious diseases. These results reveal the potential of liposomal GSNO as an anti-infective therapeutic due to its macrophage targeting capacity and direct antibacterial effects.

Core-shell alginate@silica microparticles encapsulating probiotics.

Lactobacillus rhamnosus GG (LGG) was encapsulated in core-shell alginate-silica microcapsules by coating the electrosprayed ionogel with a silica shell via hydrolysis/condensation of alkoxysilane precursors. The viability of encapsulated LGG highly depends on the mineralisation conditions (in aqueous or organic phases), identified as a critical step. More importantly, due to the unswelling of silica and to its mesoporosity that allows nutriment-metabolite diffusion, it was possible to avoid cell leakage and additionally insure bacterial growth inside the microcapsules. The results of this work gave a proof-of-concept for controlled bacterial proliferation in microcompartments, which have straightforward applications in oral delivery of probiotics.

Interest of designed cyclodextrin-tools in gene delivery.

Cyclodextrins (CyDs) currently displays even today the image of a natural macrocyclic compound largely dominant in the formation of inclusion complexes with small hydrophobic molecules. During the past 10years, advances in this field allowed to achieve more and more sophisticated CyDs derivatives opening a simple access in scale-up quantities to original and better CyD-based gene delivery systems. In addition, possibility to combine covalent and supramolecular approaches offers new venues for the design of tailor-made CyD-based nanovehicles to improve their transfection ability and gene transfer in cells. In this account, we describe our recent progress in the construction of a novel CyD-based G0 (generation number) core dendrimer, scalable to CyD oligomers by a strategy using protonable guanidine tethers and whose concept can be generalized for the assembly of CyD pre-coated dendrimers. The synthetic strategy based on an original Staudinger-Aza-Wittig tandem coupling reaction. We present an outline of the different analytical strategies to characterize CyD-ODN (cyclodextrin-oligodeoxynucleotide) complexes. Among them, Capillary electrophoresis (CE) was used to perfectly characterize our CyD-siRNA and CyD-DNA complexes and shown to be a very attractive method with advantages of low sample consumption, rapid analysis speed, and high efficiency that make this technology a major tool for association constant measurement. Finally, we present the different biological methods that can be used, in vitro, to study gene delivery, and more precisely ones we have performed to evaluate the capability of our original model bis-guanidinium-tetrakis-ß-cyclodextrin dendrimeric tetrapod, to deliver efficiently DNA or siRNA in eukaryotic cells.

Antiseptic properties of two calix[4]arenes derivatives on the human coronavirus 229E.

Facing the lack in specific antiviral treatment, it is necessary to develop new means of prevention. In the case of the Coronaviridae this family is now recognized as including potent human pathogens causing upper and lower respiratory tract infections as well as nosocomial ones. Within the purpose of developing new antiseptics molecules, the antiseptic virucidal activity of two calix[4]arene derivatives, the tetra-para-sulfonato-calix[4]arene (C[4]S) and the 1,3-bis(bithiazolyl)-tetra-para-sulfonato-calix[4]arene (C[4]S-BTZ) were evaluated toward the human coronavirus 229E (HCoV 229E). Comparing these results with some obtained previously with chlorhexidine and hexamidine, (i) these two calixarenes did not show any cytotoxicity contrary to chlorhexidine and hexamidine, (ii) C[4]S showed as did hexamidine, a very weak activity against HCoV 229E, and (iii) the C[4]S-BTZ showed a stronger activity than chlorhexidine, i.e. 2.7 and 1.4log10 reduction in viral titer after 5min of contact with 10⁻³mol L⁻¹ solutions of C[4]S-BTZ and chlorhexidine, respectively. Thus, the C[4]S-BTZ appeared as a promising virucidal (antiseptic) molecule.

[Time-kill curves and evolution of membrane permeability after para-guanidinoethylcalix[4]arene exposure]. / Cinétique d'action du para-guanidinoéthylcalix[4]arène, et évolution de la perméabilité membranaire.

UNLABELLED: Three problems at the moment: multidrug-resistant bacteria, healthcare-associated infections, and decrease of active antibiotics. We have an urgent need of new antibacterials, with an innovative mechanism of action, in order to avoid too quickly bacterial resistance. The first interface between bacteria and antibiotics is the bacterial cell wall. It is a very interesting target, as far as some components or motives are highly conserved between genus or species, and wall destabilization conduct rapidly to bacterial lysis. However, few methods are at our disposal to study rapidly impact of such antibacterials on the structure, composition or functions of the bacterial cell wall. The paraguanidinoethylcalix[4]arene (Cx1) is a new cationic antibacterial drug, with a broad spectrum, not toxic, active on multidrug-resistant bacteria, with a possible parietal target, but with unknown kind of activity (i.e. bactericidal or bacteriostatic). We thus developed, at the same time as the realization of the time-kill curves, a technique to stain bacteria with two dyes: SYTO9 and propidium iodide (PI), to follow the membrane permeability modifications, due to Cx1 exposure. The obtained results demonstrate, for Escherichia coli ATCC 25922, that Cx1 possesses a bactericidal activity, concentration-dependent, with a gradual achievement of membrane permeability, time- and concentration-dependent, with the presence of filamentous bacteria. IN CONCLUSION: the SYTO9-PI double staining, allows a simple and fast detection, easy to implement, of the impact of new antibacterial on the bacterial wall; and Cx1 interacts well with the bacterial wall, pulling in the end a loss of membrane integrity.

Synthesis and complexation ability of a novel bis- (guanidinium)-tetrakis-(beta-cyclodextrin) dendrimeric tetrapod as a potential gene delivery (DNA and siRNA) system. Study of cellular siRNA transfection.

The facile synthesis of a novel bis-(guanidinium)-tetrakis-(beta-cyclodextrin) tetrapod, the first example of a new host family, was described, and the ability of the cyclodextrin CyD tetrapod to form molecular association with siRNA and DNA guest molecules was demonstrated. Affinity capillary electrophoresis was used to determine the binding constant with the evaluation of the shift in the electrophoretic mobility mu of injected siRNA when various CyD tetrapod concentrations were added to the run buffer. A significant association constant (K(a) =16,000 M(-1)) was obtained with borate buffer when double-stranded siRNA was primarily opened with the help of temperature. An efficient cellular transfection of siRNA into human embryonic lung fibroblasts was observed by fluorescence microscopy.

Characterization of the human ACTH receptor gene and in vitro expression.

The coding sequence of the human ACTH receptor, cloned in 1992, contains no intron, but the presence of one intron (of about 18 kb) separating the coding exon from an upstream exon has been demonstrated. One major transcription start site was located in this first exon. Northern blot analysis of cultured human adrenocortical cells revealed several transcripts that can be partly explained by the use of different polyadenylation sites. We have isolated a 1 kb fragment of genomic DNA upstream of exon 1 and studied its basal promoter activity. The sequence of this region shows several putative CREs that could be responsible for the stimulation by ACTH of its own receptors as demonstrated on human adrenocortical cells. To functionally characterize the human ACTH receptor, we have prepared cells stably transfected with either the normal receptor or a mutant receptor. This model allows the study of both binding to ACTH and coupling to adenylate cyclase. Two naturally mutated receptors, described in patients with Familial Glucocorticoid Deficiency, have been studied. Both mutations (C251F and D107N) strongly impaired the binding of ACTH to its receptors and are then responsible for the absence of biological response to ACTH.