Katharina Achazi

A facile approach for dual-responsive prodrug nanogels based on dendritic polyglycerols with minimal leaching.

A novel pH and redox dual-responsive prodrug nanogel was prepared by an inverse nanoprecipitation method, which is mild and surfactant free, and based on a thiol-disulfide exchange reaction and thiol-Michael addition reaction. Highly biocompatible hyperbranched polyglycerol (hPG) was cross-linked with disulfide bonds, to obtain biodegradable nanogels, which could be degraded under intracellular reductive conditions. Doxorubicin (DOX) was conjugated to the biodegradable nanogel matrix via an acid-labile hydrazone linker. This is the first dual-responsive prodrug nanogel system that shows very low unspecific drug leaching, but efficient intracellular release of the payload triggered by the intracellular conditions. Two different prodrug nanogels were prepared with a size of approximately 150nm, which is big enough to take the advantage of the enhanced permeation and retention (EPR) effect in tumor tissue. Cell culture analysis by microscopy and flow cytometry revealed that the prodrug nanogels were efficiently internalized by tumor cells. Distinct release profiles of DOX were achieved by adjusting the nanogel architecture, and online detection of cytotoxicity showed that, unlike free DOX, the dual-responsive prodrug nanogels exhibited a delay in the onset of toxicity, indicating the different uptake mechanism and the need for prodrug activation to induce cell death.

The specificity of cytokinin signalling in Arabidopsis thaliana is mediated by differing ligand affinities and expression profiles of the receptors.

Arabidopsis thaliana has three membrane-located cytokinin receptors (AHK2, AHK3 and CRE1/AHK4), which are sensor histidine kinases containing a ligand-binding CHASE domain. Despite their structural similarity the role of these receptors differs in planta. Here we have explored which parameters contribute to signal specification. In a bacterial assay, the CHASE domain of AHK2 has a similar ligand binding spectrum as CRE1/AHK4. It shows the highest affinity for isopentenyladenine (iP) and trans-zeatin (tZ) with an apparent K(D) of 1.4 and 4.0 nm, respectively. Real-time PCR analysis of cytokinin primary response genes in double mutants retaining only single receptors revealed that all receptors are activated in planta by cytokinin concentrations in the low nanomolar range. However, there are differences in sensitivity towards the principal cytokinins iP and tZ. The activation of the cytokinin-sensitive P(ARR5) :GUS reporter gene in three different double mutants shows specific, but also overlapping, spatial domains of activity, which were for all receptors predominantly in the shoot apical meristems and root cap columella. AHK2 and AHK3 signal specifically in leaf parenchyma cells, AHK3 in stomata cells, and CRE1/AHK4 in the root vasculature. Promoter-swap experiments demonstrate that CRE1/AHK4 can functionally replace AHK2 but not AHK3. However, the cytoplasmic AHK3 histidine kinase (Hk) domain can be replaced by the CRE1/AHK4 Hk domain, which suggests that functionality is mediated in this case by the extracytosolic domain. Together, the data show that both differential gene expression and ligand preference contribute to specify the receptor activity.

Mussel‐Inspired Dendritic Polymers as Universal Multifunctional Coatings

A rapid and universal approach for multifunctional material coatings was developed based on a mussel-inspired dendritic polymer. This new kind of polymer mimics not only the functional groups of mussel foot proteins (mfps) but also their molecular weight and molecular structure. The large number of catechol and amine groups set the basis for heteromultivalent anchoring and crosslinking. The molecular weight reaches 10 kDa, which is similar to the most adhesive mussel foot protein mfp-5. Also, the dendritic structure exposes its functional groups on the surface like the folded proteins. As a result, a very stable coating can be prepared on virtually any type of material surface within 10 min by a simple dip-coating method, which is as fast as the formation of mussel byssal threads in nature.

Rodents as sentinels for the prevalence of tick-borne encephalitis virus.

INTRODUCTION Tick-borne encephalitis virus (TBEV) causes one of the most important flavivirus infections of the central nervous system, affecting humans in Europe and Asia. It is mainly transmitted by the bite of an infected tick and circulates among them and their vertebrate hosts. Until now, TBE risk analysis in Germany has been based on the incidence of human cases. Because of an increasing vaccination rate, this approach might be misleading, especially in regions of low virus circulation. METHOD To test the suitability of rodents as a surrogate marker for virus spread, laboratory-bred Microtus arvalis voles were experimentally infected with TBEV and analyzed over a period of 100 days by real-time (RT)-quantitative polymerase chain reaction. Further, the prevalence of TBEV in rodents trapped in Brandenburg, a rural federal state in northeastern Germany with autochthonous TBE cases, was determined and compared with that in rodents from German TBE risk areas as well as TBE nonrisk areas. RESULTS In experimentally infected M. arvalis voles, TBEV was detectable in different organs for at least 3 months and in blood for 1 month. Ten percent of all rodents investigated were positive for TBEV. However, in TBE risk areas, the infection rate was higher compared with that of areas with only single human cases or of nonrisk areas. TBEV was detected in six rodent species: Apodemus agrarius, Apodemus flavicollis, Apodemus sylvaticus, Microtus agrestis, Microtus arvalis, and Myodes glareolus. M. glareolus showed a high infection rate in all areas investigated. DISCUSSION AND CONCLUSION The infection experiments proved that TBEV can be reliably detected in infected M. arvalis voles. These voles developed a persistent TBE infection without clinical symptoms. Further, the study showed that rodents, especially M. glareolus, are promising sentinels particularly in areas of low TBEV circulation.

Fabrication of thermoresponsive nanogels by thermo-nanoprecipitation and in situ encapsulation of bioactives

A synthetic method for thermoresponsive, glycerol based nanogels has been developed. The nanogels were synthesized by nanoprecipitation of the orthogonally functionalized macromonomers and their gelation in water. The crosslinking points were generated by strain promoted azide–alkyne cycloaddition which enabled the in situ encapsulation of Doxorubicin HCl. The mild and surfactant free reaction conditions make these nanogels ideal candidates for biomedical applications.

pH-responsive dendritic core-multishell nanocarriers.

In this paper we describe novel pH-responsive core-multishell (CMS) nanocarrier (pH-CMS), obtained by introducing an aromatic imine linker between the shell and the core. At a pH of 5 and lower the used imine linker was rapidly cleaved as demonstrated by NMR studies. The CMS nanocarriers were loaded with the dye Nile red (NR) and the anticancer drug doxorubicin (DOX), respectively. The transport capacities were determined using UV/Vis spectroscopy, and the sizes of the loaded and unloaded CMS nanocarriers were investigated using dynamic light scattering (DLS). We could show that CMS nanocarriers efficiently transported NR in supramolecular aggregates, while DOX was transported in a unimolecular fashion. After cellular uptake the DOX-loaded pH-responsive nanocarriers showed higher toxicities than the stable CMS nanocarriers. This is due to a more efficient DOX release caused by the cleavage of the pH-labile imine bond at lower pH within the intracellular compartments.

Surface-Independent Hierarchical Coatings with Superamphiphobic Properties

Facile approaches for the fabrication of substrate independent superamphiphobic surfaces that can repel both water and organic liquids have been limited. The design of such super-repellent surfaces is still a major challenge of surface chemistry and physics. Herein, we describe a simple and efficient dip-coating approach for the fabrication of highly hierarchical surface coatings with superamphiphobic properties for a broad range of materials based on a mussel-inspired dendritic polymer (MI-dPG). The MI-dPG coating process provides a precise roughness control, and the construction of highly hierarchical structures was achieved either directly by pH-controlled aggregation or in combination with nanoparticles (NP). Moreover, the fabrication of coatings with a thickness and roughness gradient was possible via simple adjustment of the depth of the coating solution. Subsequent postmodification of these highly hierarchical structures with fluorinated molecules yielded a surface with superamphiphobic properties that successfully prevented the wetting of liquids with a low surface tension down to about 30 mN/m. The generated superamphiphobic coatings exhibit impressive repellency to water, surfactant containing solutions, and biological liquids, such as human serum, and are flexible on soft substrates.

Engineering thermoresponsive polyether-based nanogels for temperature dependent skin penetration

Highly biocompatible thermoresponsive nanogels (tNGs) based on oligo ethylene glycol (OEG) as thermoresponsive unit and dendritic polyglycerol (dPG) as cross-linker, were precisely engineered in terms of size and volume phase transition temperature (VPTT). Preliminary uptake studies into human skin were realized to show the temperature-dependent internalization behavior of these systems.

Multivalent anchored and crosslinked hyperbranched polyglycerol monolayers as antifouling coating for titanium oxide surfaces

A set of new catecholic monolayer coatings was developed to improve the antifouling performance of TiO2 surfaces. To solve the problem of the weak charge-transfer interaction between a single catechol anchor and TiO2, multiple catechol groups were combined with hyperbranched polyglycerol (hPG) which is a distinct dendritic scaffold that exposes its multivalent anchor groups on the surface. Thus, multivalent catecholic hPGs can be easily prepared for surface modification. The immobilization of the compounds was monitored by quartz crystal microbalance with dissipation monitoring. Surface properties of the coatings were analyzed by water contact angle, X-ray photoelectron spectroscopy, and atomic force microscopy. The antifouling ability and stability were investigated by protein adsorption and cell adhesion. By increasing the number of catechol groups on the hPG scaffold, the stability and surface coverage could be significantly enhanced. Moreover, the inner-layer crosslinking of the coatings by grafting and initiating vinyl groups clearly improved their long-term stability. As a result, hPG with a catecholic functional degree of 10% (hPG-Cat10) and hPG with both catecholic and vinylic functional degree of 5% (hPG-Cat5-V5) were identified as the best catecholic hPGs to prepare bioinert and stable monolayer coatings on TiO2.

Enzymatically cross-linked hyperbranched polyglycerol hydrogels as scaffolds for living cells.

Although several strategies are now available to enzymatically cross-link linear polymers to hydrogels for biomedical use, little progress has been reported on the use of dendritic polymers for the same purpose. Herein, we demonstrate that horseradish peroxidase (HRP) successfully catalyzes the oxidative cross-linking of a hyperbranched polyglycerol (hPG) functionalized with phenol groups to hydrogels. The tunable cross-linking results in adjustable hydrogel properties. Because the obtained materials are cytocompatible, they have great potential for encapsulating living cells for regenerative therapy. The gel formation can be triggered by glucose and controlled well under various environmental conditions.