Tuneable Time Delay in the Burst Release from Oxidation-Sensitive Polymersomes Made by PISA.

Reactive polymersomes represent a versatile artificial cargo carrier system that can facilitate an immediate release in response to a specific stimulus. The herein presented oxidation-sensitive polymersomes feature a time-delayed release mechanism in an oxidative environment, which can be precisely adjusted by either tuning the membrane thickness or partial pre-oxidation. These polymeric vesicles are conveniently prepared by PISA allowing the straightforward and effective in situ encapsulation of cargo molecules, as shown for dyes and enzymes. Kinetic studies revealed a critical degree of oxidation causing the destabilization of the membrane, while no release of the cargo is observed beforehand. The encapsulation of glucose oxidase directly transforms these polymersomes into glucose-sensitive vesicles, as small molecules including sugars can passively penetrate their membrane. Considering the ease of preparation, these polymersomes represent a versatile platform for the confinement and burst release of cargo molecules after a precisely adjustable time span in the presence of specific triggers, such as H2 O2 or glucose.

Polysaccharide valproates: Structure - property relationships in solution.

Polysaccharides are promising macromolecular platforms for use in the life sciences. Here, bioactive cellulose, pullulan, and dextran valproates are characterized hydrodynamically by sedimentation velocity and thermodynamically by sedimentation equilibrium analytical ultracentrifugation. Using sedimentation-diffusion analysis of sedimentation velocity experiments by numerical solution of the Lamm equation enabled the calculation of sedimentation and diffusion coefficients, and consequently molar masses. Sedimentation equilibrium experiments were then also used to determine the average molar masses. The corresponding set of data, with independently performed self-diffusion measurements by nuclear magnetic resonance spectroscopy, and together with size exclusion chromatography molar masses by coupling to refractive index-, viscometric-, and multi-angle laser light scattering detection, were subsequently correlated to each other by the hydrodynamic invariant and sedimentation parameter. We assess statistically most relevant average values of the molar masses of these polysaccharide valproates with relevant macromolecular conformational characteristics.

Hydrodynamic Analysis Resolves the Pharmaceutically-Relevant Absolute Molar Mass and Solution Properties of Synthetic Poly(ethylene glycol)s Created by Varying Initiation Sites.

The solution behavior originating from molecular characteristics of synthetic macromolecules plays a pivotal role in many areas, in particular the life sciences. This situation necessitates the use of complementary hydrodynamic analytical methods as the only means for a complete structural understanding of any macromolecule in solution. To this end, we present a combined hydrodynamic approach for studying in-house prepared, low dispersity poly(ethylene glycols)s (PEGs), also known as poly(ethylene oxide)s (PEOs) depending on the classification used, synthesized from varying initiation sites by the living anionic ring opening polymerization. The series of linear PEGs in the molar mass range of only a few thousand to 50 000 g mol-1 have been studied in detail via viscometry and sedimentation-diffusion analysis by analytical ultracentrifugation. The obtained estimations for intrinsic viscosity, diffusion coefficients, and sedimentation coefficients of the macromolecules in the solution-based analysis clearly showed self-consistency of the followed hydrodynamic approach. This self-consistency is underpinned by appropriate and physically sound values of hydrodynamic invariants, indicating adequate values of derived absolute molar masses. The classical scaling relations of Kuhn-Mark-Houwink-Sakurada of all molar-mass dependent hydrodynamic estimates show linear trends, allowing for interrelation of all parametric macromolecular characteristics. Differences among these are ascribed to the observation of α-end and chain-length dependent solvation of the macromolecules, identified from viscometric studies. This important information allows for analytical tracing of variations of scaling relationships and a physically sound estimation of hydrodynamic characteristics. The demonstrated self-sufficient methodology paves an important way for a complete structural understanding and potential replacement of pharmaceutically relevant PEGs by alternative macromolecules offering a suite of similar or tractably distinct physicochemical properties.

Characterization of poly(2-oxazoline) homo- and copolymers by liquid chromatography at critical conditions.

In this study liquid chromatography at critical conditions for poly(2-ethyl-2-oxazoline)s (PEtOx) has been performed for the first time in order to analyze functional PEtOx homopolymers and block copolymers. Besides the verification of the critical point of adsorption with two series of ester end group functionalized PEtOx homopolymers, to evaluate the effect of both the chain length dependence and the end group polarity, using a cyano column with a solvent combination of 2-propanol and water, also two-dimensional liquid chromatography (2D-LC) has been applied for a poly(2-oxazoline) block copolymer. The combined characterization techniques provided further information about the polymerization procedure with regard to the formation of side-products by separation of the block copolymer from the corresponding homopolymer impurities. In addition, hyphenation of LCCC with MALDI-TOF MS and ESI-Q-TOF tandem mass spectrometry verified the obtained results.

π-Conjugated Donor and Donor-Acceptor Metallo-Polymers.

Two zinc(II)- and two ruthenium(II) containing π-conjugated metallo-polymers were synthesized and characterized in detail. We could prove by SEC, analytical ultracentrifugation (AUC) and viscosimetry the ruthenium(II) metallo-polymers to be high molar mass materials (M(fs) = 20 000 g · mol(-1) Ru1-2; M(fs) = 34 000 g · mol(-1) Ru1) exhibiting intrinsic viscosities of up to [η] = 192 · cm(3) · g(-1) . Applying spin-coating we produced homogeneous films of the polymers and could, subsequently, investigate the photophysical properties in the solid state. Introducing the Ru(II) metallo-polymers mixed with PCBM[60] as photoactive layer in bulk-heterojunction solar cells resulted in very low efficiencies due to morphology problems.

Growth and percolation of metal nanostructures in electrode gaps leading to conductive paths for electrical DNA analysis.

Metal nanostructures are promising novel labels for microarray-based biomolecular detection. Additional silver deposition on the surface-bound labels strongly enhances the sensitivity of the system and can lead to continuous metal areas, which enable an electrical readout especially for simple and robust point-of-care analyses. In this paper, atomic force microscopy (AFM) was used to study different routes of metal deposition on labelled DNA-DNA duplexes in electrode gaps. Besides the well-established metal-induced silver enhancement, a recently introduced enzymatic silver deposition was applied and proved highly specific. The in situ characterization was especially focused on the nanostructure percolation-the moment at which the nanoparticulate film becomes continuous and electrically conducting. The formation of conducting paths, continuous from one electrode to the other, was followed by complementary electrical measurements. Thereby, a percolation threshold was determined for the surface coverage with metal structures, i.e. the required metallized area to achieve conductance. Complementary graphic simulations of the growth process and graphic 'conductance measurements' were developed and proved suitable to model the metal deposition and electrical detection. This may help to design electrode arrays and identify optimum enhancement parameters (required seed concentration and shell growth) as well as draw quantitative conclusions on the existing label (i.e. analyte) concentration.

Chip-based molecular diagnostics using metal nanoparticles.

BACKGROUND: Chip-based bioanalytical methods represent a promising approach for a highly parallel and robust analysis with minimal sample volumes. Key process parameters that can be decisive for certain applications are determined by the detection scheme utilized. OBJECTIVE: This review addresses typical requirements of chip-based detection systems, especially for the emerging field of point-of-care diagnostics that make possible field detection with less-trained personnel, robust assays as well as low instrumentation costs. METHODS: The use of metal nanoparticles as labels represents a promising approach. They exhibit a high stability in signal and new detection schemes that would allow for robustness and low-cost readout. RESULTS/CONCLUSION: First examples of this kind have been established and are in the market, and more are in the development pipeline.

A parallel approach for subwavelength molecular surgery using gene-specific positioned metal nanoparticles as laser light antennas.

An optical technique for the parallel manipulation of nanoscale structures with molecular resolution is presented. Bioconjugated metal nanoparticles are thereby positioned at the location of interest, such as, e.g., certain DNA sequences along metaphase chromosomes, prior to pulsed laser light irradiation of the whole sample. The nanoparticles are designed to absorb the introduced energy highly efficiently, in that way acting as nanoantenna. As result of the interaction, structural changes of the sample with subwavelength dimensions and nanoscale precision are observed at the location of the particles. The process leading to the nanolocalized destruction is caused by particle ablation as well as thermal damage of the surrounding material.

The gut fermentation product butyrate, a chemopreventive agent, suppresses glutathione S-transferase theta (hGSTT1) and cell growth more in human colon adenoma (LT97) than tumor (HT29) cells.

PURPOSE: The gut fermentation product of dietary fiber, butyrate, inhibits growth of HT29, an established tumor cell line. It also induces detoxifying enzymes belonging to the glutathione S-transferase family (GSTs), namely hGSTM2, hGSTP1, hGSTA4, but not of hGSTT1 . Here we investigated kinetics of effects in HT29 and compared sensitivities with preneoplastic LT97 colon adenoma cells, to assess mechanisms of colon cancer chemoprevention in two stages of cell transformation. METHODS: We determined cell growth after butyrate treatment by quantifying DNA, GST expression by Northern/Western Blotting or biochemical analysis and butyrate consumption by measuring the residual concentrations in the cell culture supernatants. Stability of GST-theta (hGSTT1) mRNA was assessed in HT29 cells after inhibition of transcription with actinomycin D. RESULTS: LT97 adenoma cells consumed twofold more butyrate and were more sensitive to growth inhibition than HT29 (EC(50)1.9 mM and 4.0 mM, respectively). Butyrate did not induce GSTs, but instead reduced hGSTT1 in LT97 and HT29. CONCLUSIONS: Butyrate has suppressing-agent activities in human colon cells by inhibiting two survival factors, namely hGSTT1 and cell growth, with LT97 more sensitive than HT29. These findings indicate that butyrate formation in the gut lumen of humans could be protective by reducing survival of transformed colon cells.

Optimization of gold nanoparticle-based DNA detection for microarrays.

DNA microarrays are promising tools for fast and highly parallel DNA detection by means of fluorescence or gold nanoparticle labeling. However, substrate modification with silanes (as a prerequisite for capture DNA binding) often leads to inhomogeneous surfaces and/or nonspecific binding of the labeled DNA. We examined both different substrate cleaning and activating protocols and also different blocking strategies for optimizing the procedures, especially those for nanoparticle labeling. Contact angle measurements as well as fluorescence microscopy, atomic force microscopy (AFM), and a flatbed scanner were used to analyze the multiple-step process. Although the examined different cleaning and activating protocols resulted in considerably different contact angles, meaning different substrate wettability, silanization led to similar hydrophobic surfaces which could be revealed as smooth surfaces of about 2-4 nm roughness. The two examined silanes (3-glycidoxypropyltrimethoxysilane (GOPS) and 3-aminopropyltriethoxysilane (APTES)) differed in their DNA binding homogeneity, maximum signal intensities, and sensitivity. Nonspecific gold binding on APTES/PDC surfaces could be blocked by treatment in 3% bovine serum albumin (BSA).

Human adenoma cells are highly susceptible to the genotoxic action of 4-hydroxy-2-nonenal.

Oxidative stress and resulting lipid peroxidation are important risk factors for dietary-associated colon cancer. To get a better understanding of the underlying molecular mechanisms, we need to characterise the risk potential of the key compounds, which cause DNA damage in cancer-relevant genes and especially in human target cells. Here, we investigated the genotoxic effects of 4-hydroxy-2-nonenal (HNE) and hydrogen peroxide (H(2)O(2)) in human colon cells (LT97). LT97 is a recently established cell line from a differentiated microadenoma and represents cells from frequent preneoplastic lesions of the colon. The genomic characterisation of LT97 was performed with 24-colour FISH. Genotoxicity was determined with single cell microgelelectrophoresis (Comet assay). Comet FISH was used to study the sensitivity of TP53-a crucial target gene for the transition of adenoma to carcinoma-towards HNE. Expression of glutathione S-transferases (GST), which deactivates HNE, was determined as GST activity and GSTP1 protein levels. LT97 cells were compared to primary human colon cells and to a differentiated clone of HT29. Karyotyping revealed that the LT97 cell line had a stable karyotype with only two clones, each containing a translocation t(7;17) and one aberrant chromosome 1. The Comet assay experiments showed that both HNE and H(2)O(2) were clearly genotoxic in the different human colon cells. HNE was more genotoxic in LT97 than in HT29clone19A and primary human colon cells. After HNE incubation, TP53 migrated more efficiently into the comet tail than the global DNA, which suggests a higher susceptibility of the TP53 gene to HNE. GST expression was significantly lower in LT97 than in HT29clone19A cells, which could explain the higher genotoxicity of HNE in the colon adenoma cells. In conclusion, the LT97 is a relevant model for studying genotoxicity of colon cancer risk factors since colon adenoma are common preneoplastic lesions occurring in advanced age.