Florent Meyer

Sustained delivery of siRNAs targeting viral infection by cell-degradable multilayered polyelectrolyte films.

Gene silencing by RNA interference (RNAi) has been shown to represent a recently discovered approach for the treatment of human diseases, including viral infection. A major limitation for the success of therapeutic strategies based on RNAi has been the delivery and shortlasting action of synthetic RNA. Multilayered polyelectrolyte films (MPFs), consisting of alternate layer-by-layer deposition of polycations and polyanions, have been shown to represent an original approach for the efficient delivery of DNA and proteins to target cells. Using hepatitis C virus infection (HCV) as a model, we demonstrate that siRNAs targeting the viral genome are efficiently delivered by MPFs. This delivery method resulted in a marked, dose-dependent, specific, and sustained inhibition of HCV replication and infection in hepatocyte-derived cells. Comparative analysis demonstrated that delivery of siRNAs by MPFs was more sustained and durable than siRNA delivery by standard methods, including electroporation or liposomes. The antiviral effect of siRNA-MPFs was reversed by a hyaluronidase inhibitor, suggesting that active degradation of MPFs by cellular enzymes is required for siRNA delivery. In conclusion, our results demonstrate that cell-degradable MPFs represent an efficient and simple approach for sustained siRNA delivery targeting viral infection. Moreover, this MPF-based delivery system may represent a promising previously undescribed perspective for the use of RNAi as a therapeutic strategy for human diseases.

Cyto-mechanoresponsive Polyelectrolyte Multilayer Films

Cell adhesion processes take place through mechanotransduction mechanisms where stretching of proteins results in biological responses. In this work, we present the first cyto-mechanoresponsive surface that mimics such behavior by becoming cell-adhesive through exhibition of arginine-glycine-aspartic acid (RGD) adhesion peptides under stretching. This mechanoresponsive surface is based on polyelectrolyte multilayer films built on a silicone sheet and where RGD-grafted polyelectrolytes are embedded under antifouling phosphorylcholine-grafted polyelectrolytes. The stretching of this film induces an increase in fibroblast cell viability and adhesion.

Multilayered films made from tannic acid and alkaline phosphatase with enzymatic activity and electrochemical behavior

Layer-by-layer-deposition of enzymes and polyphenols, like tannic acid may provide a reservoir of antioxidant and antibacterial molecules of controlled thickness and degradability with an additional activity due to the presence of the enzyme. The layer-by-layer deposition of films made from tannic acid and alkaline phosphatase is shown to yield an exponential growth with the number of deposited layer pairs. The films display the electrochemical behavior of tannic acid and the enzymatic activity of alkaline phosphatase. However, it is shown that only the enzyme close to the film-solution interface is active and follows the Michaelis-Menten mechanism. Similarly, only tannic acid close to the electrode-film interface can be oxidized. The enzymatic activity is almost completely lost when the multilayer film is treated with sodium periodate which oxidizes tannic acid even if the solubilized enzyme is not affected by the oxidant. This shows that the formation of covalent bonds between alkaline phosphatase and tannic acid is deleterious for its conformation and activity.

Optimizing the silanization of thermally-decomposed iron oxide nanoparticles for efficient aqueous phase transfer and MRI applications

The design of magnetic iron oxide nanoparticles (IONPs) as contrast agents for magnetic resonance imaging (MRI) requires good magnetic properties of the core but also an organic coating suitable for in vivo applications. IONPs synthesised by thermal decomposition display optimal properties due to their excellent monodispersity, controlled morphology and high crystallinity; however, their in situ coating by hydrophobic ligands make them only dispersible in nonpolar solvents. A wide range of methods was developed to coat IONPs with molecules or polymers bearing anchoring groups such as carboxylates mainly. Nonetheless, very few have dealt with silane based molecules due to difficulties (e.g., slow kinetics of reaction, NPs aggregation during reaction, non miscibility of solvents) to graft homogeneously and efficiently silanes at the surface of hydrophobic NPs. In this work, a new and versatile method was developed to graft hydrophilic silanes on the surface of hydrophobic IONPs based on the direct reaction of IONPs in miscible polar/apolar co-solvents: EtOH/CHCl3. The feasibility of this efficient process was demonstrated by using various silanes bearing amino and carboxylate end-groups. We show that this novel process allows several improvements in comparison with the few existing methods to silanize hydrophobic IONPs: (i) shorter reaction time, (ii) increased amount of processed NPs per cycle, (iii) the establishment of a silane limit stoichiometry to ensure good colloidal properties and (iv) easier implementation without the need of specific or stringent treatments, which are all key issues for scale-up aspects. IONPs grafted with aminosilanes display an excellent colloidal stability in ethanol and only in acidic aqueous solutions (pH < 5). By contrast, carboxylated silane-IONPs were shown to exhibit excellent colloidal stability in the physiological pH range (pH = 6–8). Moreover, such new silanized NPs display MRI contrast enhancement as efficient as commercially available magnetic NPs.

Performance of Pyridylthiourea-Polyethylenimine Polyplex for siRNA-Mediated Liver Cancer Therapy in Cell Monolayer, Spheroid, and Tumor Xenograft Models

Medical application of siRNAs relies on methods for delivering nucleic acids into the cytosol. Synthetic carriers, which assemble with nucleic acids into delivery systems, show promises for cancer therapy but efficiency remains to be improved. In here, the effectiveness of pyridylthiourea‐polyethylenimine (πPEI), a siRNA carrier that favors both polyplex disassembly and endosome rupture upon sensing the acidic endosomal environment, in 3 experimental models of hepatocellular cancer is tested. The πPEI‐assisted delivery of a siRNA targeting the polo‐like kinase 1 into Huh‐7 monolayer produces a 90% cell death via a demonstrated RNA interference mechanism. Incubation of polyplex with Huh‐7 spheroids leads to siRNA delivery into the superficial first cell layer and a 60% reduction in spheroid growth compared to untreated controls. Administration of polyplexes into mice bearing subcutaneous implanted Huh‐7Luc tumors results in a reduced tumor progression, similar to the one observed in the spheroid model. Altogether, these results support the in vivo use of synthetic and dedicated polymers for increasing siRNA‐mediated gene knockdown, and their clinical promise in cancer therapeutics.

Mimicking the chemistry of natural eumelanin synthesis: the KE sequence in polypeptides and in proteins allows for a specific control of nanosized functional polydopamine formation

The oxidation of dopamine and of other catecholamines leads to the formation of conformal films on the surface of all known materials and to the formation of a precipitate in solution. In some cases, it has been shown that the addition of additives in the dopamine solution, like certain surfactants or polymers, polyelectrolytes, and certain proteins, allows to get polydopamine nanoparticles of controlled size and the concomitant decrease, in an additive/dopamine dependent manner, in film formation on the surface of the reaction beaker. However, the mechanism behind this controlled oxidation and self-assembly of catecholamines is not known. In this article, it is shown that a specific diad of amino acids in proteins, namely KE, allows for specific control in the oxidation-self-assembly of dopamine to obtain polydopamine@protein core-shell nanoparticles which are biocompatible. The interactions between dopamine and the adjacent KE amino acids potentially responsible for the size control of polydopamine aggregates was investigated by molecular dynamics simulations. The obtained core-shell nanoparticles display the biological activity of the protein used to control the self-assembly of PDA. The photon to heat conversion ability of PDA is conserved in the PDA@protein particles.

Polyphenols at interfaces

Polyphenols are important molecules in living organisms, particularly in plants, where they serve as protectants against predators. They are also of fundamental importance in pharmacology for their antioxidant and antibacterial activities. Since a few years polyphenols are also used in surface functionalization mimicking the tannin deposition observed when tea or red wine are in contact with the surface of cups or glasses respectively. The interaction of polyphenols with proteins to yield colloids and of polyphenol with surfaces will be reviewed in this article to provide an overview of such particles and surface functionalization methods in modern surface science. Particular emphasis will be given to biological applications of polyphenols at interfaces.

Hybrid extracellular matrix microspheres for development of complex multicellular architectures

This study aims to develop a system for tissue engineering or wound management using the advantages of ECM components like gelatin, hyaluronic acid, fibronectin. We developed for the first time a hybrid CaCO3 microparticular system for creating a building block strategy. This promising method is easy to apply, versatile and will allow the buildup of any kind of targeted tissues.

How a grafting anchor tailors the cellular uptake and in vivo fate of dendronized iron oxide nanoparticles

Superparamagnetic spherical iron oxide nanoparticles of 10 nm diameter have been synthesized by thermal decomposition and grafted through a direct ligand exchange protocol with two dendrons bearing respectively a monophosphonic anchor (D2) or a biphosphonic tweezer (D2-2P) at their focal point. Physico-chemical characterization techniques such as dynamic light scattering (DLS), zeta potential, Fourier transform infrared spectroscopy (FTIR), transmission electron microscopy (TEM) and superconducting quantum interference device (SQUID) magnetometry were used to assess their composition, colloidal stability and magnetic properties. High-resolution magic angle spinning (HR-MAS) nuclear magnetic resonance (NMR) spectroscopy studies have been conducted to understand the organic shell composition and to determine both the grafting rate of the dendrons onto the nanoparticle surface and the influence of the remaining oleic acid originating from the synthesis protocol on the cellular uptake. Both dendronized IONPs showed moderate in vitro toxicity (MTT and LDH tests) in human cancer and primary cell lines. Furthermore, in vivo MRI studies showed high contrast enhancement as well as renal and hepatobiliary excretions and highlighted the influence of the grafting anchor (mono- versus bi-phosphonate) on the in vivo fate of dendronized magnetic iron oxides.

Composite vector formulation for multiple siRNA delivery as a host targeting antiviral in a cell culture model of hepatitis C virus (HCV) infection

Hepatitis C virus (HCV) infection is a major cause of chronic liver disease and cancer worldwide. RNA interference (RNAi)-based gene therapies have emerged recently as a promising tool to treat chronic viral infections. Indeed, small interfering RNAs (siRNAs) provide an opportunity to target host factors required for the viral life cycle. In this study, we evaluated a novel nanovector-based approach for siRNA delivery in a model of chronically infected hepatic cells. We designed original composite nanoparticles by coating the calcium phosphate core with siRNAs targeting HCV host-factors and pyridylthiourea-grafted polyethyleneimine (πPEI). Using combinations of different siRNAs, we observed an efficient and prolonged decrease of HCV replication. Moreover, we showed that the layer-by-layer technique of coating applied to our nanoparticles triggers a sequential release of siRNAs acting on different steps of the HCV life cycle. Together, our results demonstrate the efficacy of these nanoparticles for siRNA delivery and open new perspectives for antiviral therapies.