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.

Polyelectrolyte multilayer-mediated gene delivery for semaphorin signaling pathway control.

The capability of multilayered polyelectrolyte films (MPFs) to control the sequential expression of two genes encoding cell receptors involved in a common cell signalling activity is shown, while achieving a fully functional signal transduction. As a functional model system representative of a cell signalling process that proceeds in a top-down manner, cell collapse induced by semaphorin 3A (Sema3A) was chosen as the target. Polyelectrolyte multilayers were sequentially functionalized with two plasmids encoding Neuropilin-1 (NRP-1) and Plexin-A1 (Plx-A1), respectively, acting as co-receptors for Sema3A. By using hyaluronan and chitosan as structural components for the incorporation of plasmid DNA layers onto precursor films made of poly-allylamine hydrochloride and poly-sodium-4-styrenesulfonate, the polyelectrolyte system is established; this systems is capable of delivering both plasmids to Cos-1 cells in a manner that permits control over the timing and the respective order in which the two plasmid DNA constructs are expressed. Importantly, it was observed that, following Sema3A stimulation, COS-1 cells co-expressing Plx-A1 and NRP-1 display a collapse phenotype, which is determined by the multilayer build-up scheme, and that the expression products of both transgenes embedded in MPFs are temporally functional over several days while acting their role of co-receptors for Sema3A.

Relevance of bi-functionalized polyelectrolyte multilayers for cell transfection.

In an effort to develop new biomaterial coatings, it was shown that polyelectrolyte multilayers constitute a very powerful tool to render surfaces biologically active. The challenge is to multi-functionalize surfaces in a controlled way. We show here, for the first time, that it is possible to functionalize multilayer films simultaneously with two molecules acting in totally different ways on cells, namely plasmid DNA (pDNA), pre-complexed with poly(ethyleneimine) (PEI), and a peptide molecule, NDPMSH. This peptide, grafted to poly(L-glutamic acid) (PGA) was used as a signal molecule for melanoma cells B16-F1 and for its ability to enhance gene delivery in a receptor-independent manner. The PGA-NDPMSH chains are embedded in poly-(allylamine hydrochloride)/poly-(sodium 4-styrene sulfonate) multilayers and the pDNA-PEI complexes are deposited on top of the films. It is shown that melanoma cells (B16-F1) are efficiently transfected after 24h of contact with functionalized films. When brought in contact with Huh-7 cells that do not express the peptide receptors, these films trigger significantly the transfection rate, showing that it is possible to enhance the transfection process by incorporating specific peptides into multilayer films. Moreover, transfected cells sorted by flow cytometry produce melanin, demonstrating both activation via the peptide signaling pathway and cell transfection.

Filamentous condensation of DNA induced by pegylated poly-L-lysine and transfection efficiency.

In this paper we propose a detailed analysis of structural and morphological properties of two poly-L-lysine (PLL)-based transfection formulations, PLL/DNA and pegylated PLL (PLL-g-PEG)/DNA, by means of atomic force microscopy (AFM) and transmission electron microscopy (TEM). Comparing PLL-g-PEG/DNA with PLL/DNA polyplexes, we demonstrate that, due to the presence of PEG, the particles differ not only in size, shape, and crystalline structure, but also in transfection efficiency. While PLL condensates DNA in large agglomerates, PLL grafted with polyethylene glycol 2000 can condensate DNA in long filaments with diameters of some nanometers (6-20 nm). These structures are dependent on the grafting ratio and are more efficient than compacted ones, showing that DNA uptake and processing by cell is directly related to physicochemical properties of the polyplexes.

Polyplex-embedding in polyelectrolyte multilayers for gene delivery.

In this work, incorporation of plasmid DNA, pre-complexed with PEI, into polyelectrolyte multilayers has been studied to further develop platforms for local gene delivery. Polyplex embedding in synthetic and naturally degradable architectures was efficient for transfection of human hepato-cellular carcinoma cells.

Control of drug accessibility on functional polyelectrolyte multilayer films.

A surface coating based on polylysine/hyaluronic acid multilayers was designed and acted as a reservoir for an antiproliferative agent, paclitaxel (Taxol). Absolutely no chemical modification of polyelectrolytes or of the drug was needed and the final architecture was obtained in an extremely simple way using the layer-by-layer method. The paclitaxel dose available for human colonic adenocarcinoma cells HT29 seeded on the films could be finely tuned. Moreover, the accessibility of the drugs was controlled by adding on the top of the drug reservoir a capping made of synthetic polyelectrolyte multilayers. This capping was also required to allow adhesion of HT29 cells. Paclitaxel activity was maintained after embedding in the polyelectrolyte multilayers and cellular viability could be reduced by about 80% 96 h after seeding. The strategy described in this paper could be valuable for various other drug/cell systems.

Immunogold detection of types I and II chondrocyte collagen fibrils: an in situ atomic force microscopic investigation.

Chondrocyte tissue engineering is a major challenge in the field of cartilage repair. The phenotype of chondrocytes consists of cartilage specific proteoglycan and type II collagen. During serial passages, chondrocytes dedifferentiate into cells, presenting a fibroblast-like phenotype consisting predominately of type I collagen synthesis. Observation of native collagen fibers could be visualized by atomic force microscope. Here, we developed an original and useful atomic force microscopy-based immunogold technique allowing biochemical distinction between types I and II collagen fibers. Imaging of 40-nm gold particles staining collagen fibers was performed in tapping mode. Rat 1 fibroblasts and human chondrosarcoma cells were used as positive models for types I and II collagen, respectively. As demonstrated by our data, primary rat chondrocytes adhering for 48 h on a glass substrate synthesize type II collagen native fibers. This technique allows analyses of local areas of the extracellular matrix of fixed cells, providing complementary data about cartilage phenotype. This simple approach could be of major interest for the biologist community in routine laboratory investigations, to localize in situ, macromolecules of the extracellular matrix.

Crystal structure of the V-region of Streptococcus mutans antigen I/II at 2.4 A resolution suggests a sugar preformed binding site.

Antigens I/II are large multifunctional adhesins from oral viridans streptococci that exert immunomodulatory effects on human cells and play important roles in inflammatory disorders. Among them, Streptococcus mutans plays a major role in the initiation of dental caries. The structure of the V-region (SrV+, residues 464-840) of the antigen I/II of S. mutans has been determined using the multiwavelength anomalous diffraction phasing technique with seleno-methionine-substituted recombinant protein and subsequently refined at 2.4 A resolution. The crystal structure of SrV+ revealed a lectin-like fold that displays a putative preformed carbohydrate-binding site stabilized by a metal ion. Inhibition of this binding site may confer to humans a protection against dental caries and dissemination of the bacteria to extra-oral sites involved in life-threatening inflammatory diseases. This crystal structure constitutes a first step in understanding the structure-function relationship of antigens I/II and may help in delineating new preventive or therapeutic strategies against colonization of the host by oral streptococci.

Effect of functionalization of multilayered polyelectrolyte films on motoneuron growth.

We studied in vitro cell-substrate interaction of motoneurons with functionalized polylectrolyte films. Thin polylectrolyte films were built on glass by alternating polycations, poly(ethylene-imine) PEI, poly(L-lysine) PLL, or poly(allylamine hydrochloride) PAH, and polyanions, poly(sodium-4-styrenesulfonate) PSS or poly(L-glutamic acid) (PGA). These architectures were functionalized with Brain Derived Neurotrophic Factor (BDNF) or Semaphorin 3A (Sema3A). We used Optical Waveguide Lightmode Spectroscopy (OWLS) and Atomic Force Microscopy (AFM) to characterize the architectures. The viability of motoneurons was estimated by the acid phosphatase method, and morphometrical measures were performed to analyse the influence of different architectures on cell morphology. Motoneurons appeared to adhere and spread on all the architectures tested and preferentially on PSS ending films. The viability of motoneurons on polyelectrolyte multilayers was higher compared to polyelectrolyte monolayers. BDNF and Sema3A embedded in the films remained active and thereby create functionalized nanofilms.

Polyelectrolyte multilayers functionalized by a synthetic analogue of an anti-inflammatory peptide, alpha-MSH, for coating a tracheal prosthesis.

Polyelectrolyte multilayer films made of poly (L-lysine) (PLL) and poly (L-glutamic acid) (PGA) have been functionalized by covalent binding of a synthetic analogue of the anti-inflammatory peptide, alpha-melanocyte-stimulating hormone (alpha-MSH) to PGA to create biologically active coatings for tracheal prostheses. The morphology and in vivo stability of the films were investigated by atomic force microscopy and confocal laser scanning microscopy, respectively. For the in vivo evaluation, 87 rats were implanted and examined for a period superior to 3 months. Histological analysis, performed 1 month after implantation, showed a fibroblast colonization of the periprosthetic side and a respiratory epithelium type on the endoluminal side of the implant for all the polyelectrolyte coatings tested. However, for prostheses modified by PGA ending multilayer films, a more regular and less obstructive cell layer was observed on the endoluminal side compared to those modified by PLL ending films. Systemic anti-inflammatory IL-10 production was only detected in rats implanted with prostheses functionalized by alpha-MSH, demonstrating, in vivo, the anti-inflammatory activity of the embedded peptide into multilayer architectures.

Streptococcal antigen I/II binds to extracellular proteins through intermolecular beta-sheets.

One of the functions associated with the oral streptococcal surface protein I/II is to bind to human extracellular matrix molecules or blood components, which could act as opportunistic ligands in pathological circumstances. In order to understand the relative specificity of the binding repertoire of this bacterial adhesin, we examined by infrared measurements the mode of binding of the protein I/II from Streptococcus mutans OMZ175 (I/IIf) to fibronectin and fibrinogen. This approach revealed the beta-structure forming capacity of I/IIf upon interaction with both proteins. The forming of intermolecular beta-structures may provide a non-selective way of interaction between I/IIf and its possible targets.

Templated assembly of albumin-based nanoparticles for simultaneous gene silencing and magnetic resonance imaging.

In this article, we address the design of innovative human serum albumin (HSA)-based nanoparticles loaded with silencing RNA and grafted with gadolinium complexes having average sizes ranging from ca. 50 to 150 nm according to the siRNA/HSA composition. The non-covalent siRNA/HSA assembly is formed on isobutyramide-modified mesoporous silica and the self-supported HSA-based nanoparticles are obtained following the silica template dissolution. These original protein particles provide simultaneous magnetic resonance imaging contrast enhancement and cellular in vitro gene silencing.

Potent calcium phosphate nanoparticle surface coating for in vitro and in vivo siRNA delivery: a step toward multifunctional nanovectors.

The present study describes hybrid nanoparticles, built by alternate deposition of siRNA and modified polyethyleneimine (tyrosine-grafted PEI or tyrosine/galactose-grafted PEI) on calcium phosphate nanoparticles. These "easy to produce" nanoparticles (NPs) present an efficient gene silencing effect demonstrated in vitro in a luciferase expressing cell culture model and in vivo in a tumour xenograft mouse model. The luciferase gene silencing percentage reached up to 95% in vitro with biocompatible doses of siRNA. Interestingly, we show by SPECT imaging of radiolabeled particles that without modifying the size, stability and in vitro efficiency, the grafting of a sugar moiety on PEI can modify the in vivo biodistribution of the particles. The proof of concept that galactose-grafting on PEI could change biodistribution without changing the gene silencing efficiency makes them versatile tools for specific delivery of small interfering RNA. As they have been designed so far, biodistribution is mainly located in the liver and thus these innovative nanoparticles open a realistic and feasible strategy for siRNA delivery into the liver in vivo.

Antibacterial protection of suture material by chlorhexidine-functionalized polyelectrolyte multilayer films.

The formation of bacterial biofilms on the surface of implanted materials is a critical factor that may lead to chronic microbial infection and tissue necrosis. In the present study we analysed the stability of polyelectrolyte multilayer (ML) films on suture materials and the antibacterial effect obtained with chlorhexidine (CHX)-functionalized films built on different types of suture materials such as silk, polyester and copolymer of glycolide and L: -lactide. The comparison of Escherichia coli culture on glass coverslips and glass coverslips with ML and CHX showed at 24 h an inhibition of the bacterial relative luminescence (40.68%, P < 0.5) and at 48 h (99.46%, P < 0.001). In another way, simple soaking of suture material overnight in CHX digluconate 20% without polyelectrolyte films did not at all protect sutures from bacterial colonization but CHX-functionalized polyelectrolyte films, made from poly-L: -glutamic acid and poly-L: -lysine, inhibited Escherichia coli proliferation.

Characterization of PLL-g-PEG-DNA nanoparticles for the delivery of therapeutic DNA.

Local and controlled DNA release is a critical issue in current gene therapy. As viral gene delivery systems are associated with severe security problems, nonviral gene delivery vehicles were developed. Here, DNA-nanoparticles using grafted copolymers of PLL and PEG to increase their biocompatibility and stealth properties were systematically studied. Ten different PLL-based polymers with no, low, and high PEG grafting and PEG molecular weights as well as different PLL backbone lengths were complexed with plasmids containing 3200 to 10,100 base pairs. Stable complexes were formed and selected for cytotoxicity and transfection efficiency. Predominantly, PLL-g-PEG-DNA nanoparticles grafted with 4 or 5% PEG moieties of 5 kDa transfected 40% COS-7 cells without reduction of cell viability when formed at N/P ratios between 0.1 and 12.5. The molecular weight of PLL did not significantly affect transfection efficiency or cytotoxicity indicating that a specific cationic charge-density-to-PEG-ratio is important for efficient transfection and low cytotoxicity. The PLL-g-PEG-DNA nanoparticles were spherical with a diameter of approximately 100 nm and did not aggregate over 2 weeks. Moreover, they protected included plasmid DNA against serum components and DNase I digestion. Therefore, such storage stable and versatile PLL-g-PEG-DNA nanoparticles might be useful to deliver differently sized therapeutic DNA for in vivo applications.

Transfection ability and intracellular DNA pathway of nanostructured gene-delivery systems.

Considerable efforts have been devoted to the design of structured materials with functional properties. Polyelectrolyte multilayer films are now a well-established nanostructured concept with numerous potential applications, in particular as biomaterial coatings. This technique allows the preparation of nanostructured architectures exhibiting specific properties for cell-activation control and local drug delivery. In this study, we used a multilayered system made of poly-(l-lysine)/hyaluronic acid (PLL/HA) as a reservoir for active DNA complexes with nonviral gene-delivery vectors, PLL, beta-cyclodextrin (CD), and PLL-CD. When embedded into the multilayered films, the transfection efficiencies of the DNA complexes and the cell viability were improved. The highest transfection efficiency was obtained with the PLL-CD/plasmid DNA (pDNA) complexes. We found that this high transfection efficiency was related to an efficient internalization of the complexes in the cell cytoplasm and selected nuclei domains through a nonendocytotic pathway. For the first time, we report the intracellular pathway of the pDNA in complexes incorporated into the multilayered system.

Polyelectrolyte multilayer films modulate cytoskeletal organization in chondrosarcoma cells.

The aim of this study was to evaluate polyelectrolyte multilayer films as interfaces for implants. Polyelectrolyte multilayers were built up with different terminating layers by alternate deposition of oppositely charged polyelectrolytes on which chondrosarcoma (HCS-2/8) cells were grown in the presence of serum. Films formed by an increasing number of layers were investigated. The terminating layer was made of one of the following polyelectrolytes: poly-sodium-4-styrenesulfonate (PSS), poly-L-glutamic acid (PGA), poly-allylamine hydrochloride (PAH), or poly(L-lysine) (PLL). Cell viability, inflammatory response, adherence, and cytoskeletal organization were studied. Induction of interleukin-8 (IL-8) secretion was detected on PAH and PLL ending polyelectrolyte films. Early cellular adherence was enhanced with PGA, PAH, PLL, and, to a lower extent, PSS terminating layers. Adherence was independent of the number of layers constituting the films. The presence of actin filaments and vinculin focal adhesion spots was observed on PSS or PAH ending films. They were respectively partially and totally absent on PGA and PLL terminating multilayer architectures. For PLL ending films, vinculin and actin organization was clearly dependent on the number of deposited layers. The results of this study suggest that PSS ending multilayered films constitute a good interfacial micro-environment at the material surface for HCS-2/8 cells.

Viability, adhesion, and bone phenotype of osteoblast-like cells on polyelectrolyte multilayer films.

The aim of this study was to develop new biocompatible coatings for bone implants by the alternating deposition of oppositely charged polyelectrolytes. Polyelectrolyte films were built up with different terminating layers on which SaOS-2 osteoblast-like cells and human periodontal ligament (PDL) cells were grown. The terminating layer was made of one of the following polyelectrolytes: poly(ethylene imine) (PEI), poly(sodium 4-styrenesulfonate) (PSS), poly(allylamine hydrochloride) (PAH), poly(L-glutamic acid) (PGA), or poly(L-lysine) (PLL). Cell adherence, viability, stability of osteoblast phenotype, and inflammatory response were studied. Adherence and viability were good on all terminating layers except the PEI-terminating layer, which was cytotoxic. Maintenance of osteoblast phenotype marker expression was observed on PSS- and PGA-terminating films for both cell types, whereas downregulation, associated with the induction of Interleukin-8 (IL-8) secretion, was detected on PEI and PAH for both cell types and on PLL for PDL cells. These results suggested a good biocompatibility of PSS- and PGA-ending films for PDL cells and of PSS-, PGA-, and PLL-terminating films for SaOS-2 cells. As a result, polyelectrolyte multilayer films could emerge as new alternatives for implant coatings.

3-D surface charges modulate protrusive and contractile contacts of chondrosarcoma cells.

Up to now, most of the studies addressing the critical roles played by protrusive and contractile cell-matrix contacts in cell adhesion, guidance, migration, matrix assembly, and activation of signaling molecules have been performed on two-dimensional surfaces. Here, we analysed the organization of chondrosarcoma cell contacts in a new three-dimensional environment made of titanium beads. Surface charges were modified by deposition of polyelectrolyte multilayer films built up by alternated polycations poly-(L-lysine) or poly(allylamine hydrochloride) and polyanions poly-(L-glutamic acid) or poly(sodium 4-styrenesulfonate). Negatively charged 3-D titanium surfaces amplified the occurrence and length of cell protrusions. These protrusions had pseudopod characteristics extended to 200 microm in length, growing off the substratum to distant beads. Pseudopod formation is inhibited by the exocytosis inhibitor concanamycin A and is triggered by a secreted factor. Chondrosarcoma cells adhering on uncoated or on negatively charged surfaces contained discrete focal spots of vinculin and actin cables. In cells plated onto these surfaces, phosphorylation of p44/42 MAPK/ERK was twofold increased. In contrast, no cytoskeletal vinculin and actin organization was observed when the surface was positively charged. These data suggest that chondrosarcoma cells adapt a more stable adhesion on uncoated or negatively charged surfaces. This point may be critical in tissue engineering strategies designed for cartilage repair.