uptake, intracellular distribution and cellular responses

Silver nanoparticles (SNP) are among the most commercialized nanoparticles worldwide. They can be found in many diverse products, mostly because of their antibacterial properties. Despite its widespread use only little data on possible adverse health effects exist. It is difficult to compare biological data from different studies due to the great variety in sizes, coatings or shapes of the particles. Here, we applied a novel synthesis approach to obtain SNP, which are covalently stabilized by a small peptide. This enables a tight control of both size and shape. We applied these SNP in two different sizes of 20 or 40 nm (Ag20Pep and Ag40Pep) and analyzed responses of THP-1-derived human macrophages. Similar gold nanoparticles with the same coating (Au20Pep) were used for comparison and found to be non-toxic. We assessed the cytotoxicity of particles and confirmed their cellular uptake via transmission electron microscopy and confocal Raman microscopy. Importantly a majority of the SNP could be detected as individual particles spread throughout the cells. Furthermore we studied several types of oxidative stress related responses such as induction of heme oxygenase I or formation of protein carbonyls. In summary, our data demonstrate that even low doses of SNP exerted adverse effects in human macrophages

Tandem fusion of hepatitis B core antigen allows assembly of virus-like particles in bacteria and plants with enhanced capacity to accommodate foreign proteins

The core protein of the hepatitis B virus, HBcAg, assembles into highly immunogenic viruslike particles (HBc VLPs) when expressed in a variety of heterologous systems. Specifically, the major insertion region (MIR) on the HBcAg protein allows the insertion of foreign sequences, which are then exposed on the tips of surface spike structures on the outside of the assembled particle. Here, we present a novel strategy which aids the display of whole proteins on the surface of HBc particles. This strategy, named tandem core, is based on the production of the HBcAg dimer as a single polypeptide chain by tandem fusion of two HBcAg open reading frames. This allows the insertion of large heterologous sequences in only one of the two MIRs in each spike, without compromising VLP formation. We present the use of tandem core technology in both plant and bacterial expression systems. The results show that tandem core particles can be produced with unmodified MIRs, or with one MIR in each tandem dimer modified to contain the entire sequence of GFP or of a camelid nanobody. Both inserted proteins are correctly folded and the nanobody fused to the surface of the tandem core particle (which we name tandibody) retains the ability to bind to its cognate antigen. This technology paves the way for the display of natively folded proteins on the surface of HBc particles either through direct fusion or through non-covalent attachment via a nanobody

Highly specific gene silencing by artificial microRNAs in the unicellular alga Chlamydomonas reinhardtii

MicroRNAs (miRNAs) are small RNAs, 21 to 22 nucleotides long, with important regulatory roles. They are processed from longer RNA molecules with imperfectly matched foldback regions and they function in modulating the stability and translation of mRNA. Recently, we and others have demonstrated that the unicellular alga Chlamydomonas reinhardtii, like diverse multicellular organisms, contains miRNAs. These RNAs resemble the miRNAs of land plants in that they direct site-specific cleavage of target mRNA with miRNA-complementary motifs and, presumably, act as regulatory molecules in growth and development. Utilizing these findings we have developed a novel artificial miRNA system based on ligation of DNA oligonucleotides that can be used for specific high-throughput gene silencing in green algae

A novel type of silver nanoparticles and their advantages in toxicity testing in cell culture systems

Silver nanoparticles (SNPs) are among the most commercialized nanoparticles worldwide. Often SNP are used because of their antibacterial properties. Besides that they possess unique optic and catalytic features, making them highly interesting for the creation of novel and advanced functional materials. Despite its widespread use only little data exist in terms of possible adverse effects of SNP on human health. Conventional synthesis routes usually yield products of varying quality and property. It thus may become puzzling to compare biological data from different studies due to the great variety in sizes, coatings or shapes of the particles applied. Here, we applied a novel synthesis approach to obtain SNP of well-defined colloidal and structural properties. Being stabilized by a covalently linked small peptide, these particles are nicely homogenous, with narrow size distribution, and form monodisperse suspensions in aqueous solutions. We applied these peptide-coated SNP in two different sizes of 20 or 40 nm (Ag20Pep and Ag40Pep) and analyzed responses of THP-1-derived human macrophages while being exposed against these particles. Gold nanoparticles of similar size and coating (Au20Pep) were used for comparison. The cytotoxicity of particles was assessed by WST-1 and LDH assays, and the uptake into the cells was confirmed via transmission electron microscopy. In summary, our data demonstrate that this novel type of SNP is well suited to serve as model system for nanoparticles to be tested in toxicological studies in vitro

Influence of Phosphonium Alkyl Substituents on the Rheological and Thermal Properties of Phosphonium-PAA-Based Supramolecular Polymeric Assemblies

A noncovalent synthetic strategy to supramolecular polymeric assemblies, including network structures, is described by the complexation of various phosphonium monocations and dications with the multianion, poly(acrylic acid). The alkyl chains surrounding the phosphonium cation were systematically varied from butyl, hexyl, to octyl in order to probe the effect of sterics and ion pairing on the resulting macroscopic properties of the assemblies. The supramolecular assemblies were characterized by TGA, DSC, oscillatory rheometry, steady-state flow rheometry, and SAXS. The rheological and thermal properties, as well as the flow activation energies, are highly dependent on the alkyl chain length. All of the supramolecular assemblies have glass transition temperatures lower than room temperature and range from 8 °C to below −40 °C. Di-ButC10PAA has the shortest alkyl chain length and affords the highest glass transition temperature. Correspondingly, it shows the largest viscosity and storage and loss moduli. For example, its viscosity is 3 orders of magnitude greater than di-OctC10PAA. In creep-recovery experiments, di-ButC10PAA shows the highest percent of strain recovery after the stress is removed, followed by di-HexC10PAA and di-OctC10PAA. The rheological and thermal properties of monoIL-PAA assemblies show similar alkyl chain length dependence, but the magnitude is significantly less because of the lack of cross-linking. A reversibility test of the supramolecular networks demonstrates that the ionic network material can fully reassemble within a short time period after disruption of the network due to heat or shear without sacrificing the mechanical properties

Polyelectrolyte--Surfactant Complexes as Thermoreversible Organogelators

Poly(N,N-dimethyl-n-octadecylammonium p-styrenesulfonate) (PSS-DMODA) polymers were prepared and investigated as organogelators for low-polarity aromatic solvents. Gels were prepared by heating polymer solutions (2.5–20% w/v polymer) at elevated temperature and then cooling in an ice bath. Gelation was confirmed by the formation of self-supporting samples that did not flow when inverted 180°. Measurement of the gel transition temperature by inversion testing showed a dependence on the concentration of the polymer, the molecular weight of the polymer, and the gelled solvent. Cavitation rheology measurements on a subset of the gels demonstrated that they were viscoelastic solids. Scanning electron microscopy measurements of freeze-dried xerogels and polarized optical microscopy measurements showed the formation of network structures and birefringent samples, respectively. Aging studies showed syneresis of the gels especially at low concentration and temperature. Gelation was interpreted using a model for reversibly associating polymers. The gelation was attributed to the clustering of the ionic groups to form a physically cross-linked network that restricts the motion of the chains. These polyelectrolyte–surfactant complexes should be a useful class of organogelators as a number of characteristics of the polymer (molecular weight, ionic groups, side-chain length) can be independently varied to tune the properties of the resultant organogels