Highly Stable RNA Capture by Dense Cationic Polymer Brushes for the Design of Cytocompatible, Serum-Stable SiRNA Delivery Vectors.

The high density of polymer brushes confers to these coatings unique physicochemical properties, in particular for the regulation of biomolecular interaction and the design of highly selective coatings for biosensors and protein patterning. Here, we show that high density poly(dimethylaminoethyl methacrylate) cationic polymer brushes enable the stable uptake of high levels of oligonucleotides. This is proposed to result from the high degree of crowding and associated increase in entropic driving force for the binding of polyelectrolytes such as nucleic acid molecules. We further demonstrate the ease with which such coatings allow the design of highly structured nanomaterials for siRNA delivery using block copolymer-brush-based nanoparticles that allow the protection of oligonucleotides by a protein-resistant outer block. In particular, these nanomaterials display a high serum stability and low cytotoxicity while retaining excellent knock down efficiencies. Polymer brush-based nanomaterials therefore appear particularly attractive for the rational design of a new generation of high performance theranostics and RNA delivery probes.

Solution Conformation of Polymer Brushes Determines Their Interactions with DNA and Transfection Efficiency.

Polymer brush-functionalized nanomaterials offer interesting features for the design of gene delivery vectors as their physicochemical and structural properties can be designed independently of the chemistry, size and shape of the nanomaterial core. However, little is known of the parameters regulating the adsorption and infiltration of DNA molecules at the surface of positively charged polymer brushes, despite the importance of such processes for gene delivery. Here we investigate the role of the molecular environment (e.g., pH, type of buffer, concentration) on the interactions between plasmid DNA and positively charged poly(dimethylaminoethyl methacrylate) (PDMAEMA) brushes using a combination of light scattering, electrophoretic light scattering, in situ ellipsometry, and surface plasmon resonance. We show that the conformation of swollen PDMAEMA brushes is modulated by the surrounding buffer and that this impacts strongly on the ability of such brushes and nanomaterials based on these coatings to complex DNA molecules. In turn, the levels of transfection efficiency measured correlate with changes in brush conformation and DNA binding. Therefore, this work demonstrates the importance of molecular design of polymer brushes to control DNA complexation and release in order to optimize the performance of polymer brush-functionalized nanomaterials for gene delivery applications.

In Vitro Antibacterial Activity of Curcumin-Polymyxin B Combinations against Multidrug-Resistant Bacteria Associated with Traumatic Wound Infections.

Bacterial infections resulting from nonsurgical traumatic wounds can be life threatening, especially those caused by multidrug-resistant (MDR) bacteria with limited therapeutic options. The antimicrobial activity of polymyxin B (1) and curcumin (2) alone and in combination was determined versus MDR bacterial isolates associated with traumatic wound infections. Cytotoxicity assays for 1 and 2 were undertaken in keratinocyte cell lines. Minimum inhibitory concentrations of 1 were significantly reduced in the presence of 2 (3- to 10-fold reduction), with synergy observed. Time-kill assays showed the combinations produced bactericidal activity. Cytotoxicity assays indicate the toxicity of 2 was reduced in the presence of 1.

In Vitro Activity of Epigallocatechin Gallate and Quercetin Alone and in Combination versus Clinical Isolates of Methicillin-Resistant Staphylococcus aureus.

Topical infections can become life threatening in immunocompromised patients. However, fewer treatments are available as multi-drug-resistant bacteria become more common. The natural compounds epigallocatechin gallate (1) and quercetin (2) alone and in combination were tested as potential antimicrobial clinical therapies. Strong antimicrobial activity was produced by 1 alone against methicillin-resistant Staphylococcus aureus, and activity was significantly increased in the presence of 2. A synergistic interaction was observed between the two compounds. Kill kinetics indicate the combination is bactericidal over 24 h.

Nucleocytoplasmic shuttling: a common theme in mechanotransduction.

Cells sense their mechanical and physical environment through diverse mechanisms, and these interactions specify a wide range of responses including growth, survival, migration and differentiation. Although much work has focused on dissecting the adhesive and structural components of the cell responsible for transducing external mechanical forces into biochemical signalling cascades, only recently have studies begun to examine how mechanical signals are transmitted to the nucleus and activate specific gene expression programmes. One necessary step in these processes is the transport of signalling molecules from the cytoplasm to the nucleus. The SRF (serum-response factor) and YAP (Yes-associated protein)/TAZ (transcriptional co-activator with PDZ-binding motif) pathways are known mediators of this process in multiple cell types, including mesenchymal stem cells, keratinocytes, mammary epithelial cells and smooth muscle cells. In addition, recent evidence suggests a potential role for ß-catenin and Smad signalling in mechanotransduction, but further mechanistic studies are needed to prove this hypothesis. As a model system, the epidermis of the skin is one tissue in which nucleocytoplasmic shuttling mediates cellular mechanosensing and is essential for tissue development, homoeostasis and repair. We propose that nuclear translocation is a common element of mechanotransduction conserved across multiple cell types and tissues.

Nuclear actin modulates cell motility via transcriptional regulation of adhesive and cytoskeletal genes.

The actin cytoskeleton is a classic biomechanical mediator of cell migration. While it is known that actin also shuttles in and out of the nucleus, its functions within this compartment remain poorly understood. In this study, we investigated how nuclear actin regulates keratinocyte gene expression and cell behavior. Gene expression profiling of normal HaCaT keratinocytes compared to HaCaTs over-expressing wild-type ß-actin or ß-actin tagged with a nuclear localization sequence (NLS-actin), identified multiple adhesive and cytoskeletal genes, such as MYL9, ITGB1, and VCL, which were significantly down-regulated in keratinocytes with high levels of nuclear actin. In addition, genes associated with transcriptional regulation and apoptosis were up-regulated in cells over expressing NLS-actin. Functionally, accumulation of actin in the nucleus altered cytoskeletal and focal adhesion organization and inhibited cell motility. Exclusion of endogenous actin from the nucleus by knocking down Importin 9 reversed this phenotype and enhanced cell migration. Based on these findings, we conclude that the level of actin in the nucleus is a transcriptional regulator for tuning keratinocyte migration.

Nanoparticulate zinc oxide as a coating material for orthopedic and dental implants.

Orthopedic and dental implants are prone to infection. In this study, we describe a novel system using zinc oxide nanoparticles (nZnO) as a coating material to inhibit bacterial adhesion and promote osteoblast growth. Electrohydrodynamic atomisation (EHDA) was employed to deposit mixtures of nZnO and nanohydroxyapatite (nHA) onto the surface of glass substrates. Nano-coated substrates were exposed to Staphylococcus aureus suspended in buffered saline or bovine serum to determine antimicrobial activity. Our results indicate that 100% nZnO and 75% nZnO/25% nHA composite-coated substrates have significant antimicrobial activity. Furthermore, osteoblast function was explored by exposing cells to nZnO. UMR-106 cells exposed to nZnO supernatants showed minimal toxicity. Similarly, MG-63 cells cultured on nZnO substrates did not show release of TNF-α and IL-6 cytokines. These results were reinforced by both proliferation and differentiation studies which revealed that a substrate coated with exclusively nZnO is more efficient than composite surface coatings. Finally, electron and light microscopy, together with immunofluorescence staining, revealed that all cell types tested, including human mesenchymal cell (hMSC), were able to maintain normal cell morphology when adhered onto the surface of the nano-coated substrates. Collectively, these findings indicate that nZnO can, on its own, provide an optimal coating for future bone implants that are both antimicrobial and biocompatible.