State transition identification in multivariate time series (STIMTS) applied to rotational jump trajectories from single molecules.

Time resolved data from single molecule experiments often suffer from contamination with noise due to a low signal level. Identifying a proper model to describe the data thus requires an approach with sufficient model parameters without misinterpreting the noise as relevant data. Here, we report on a generalized data evaluation process to extract states with piecewise constant signal level from simultaneously recorded multivariate data, typical for multichannel single molecule experiments. The method employs the minimum description length principle to avoid overfitting the data by using an objective function, which is based on a tradeoff between fitting accuracy and model complexity. We validate our method with synthetic data from Monte Carlo simulations modeling fluorescence resonance energy transfer and rotational jumps, respectively. The method is applied to quantify rotational jump dynamics of single terrylene diimide (TDI) molecules deposited on a solid substrate. Depending on the substitution pattern of the TDI molecules and the chosen substrate materials, we find significant differences in time scale and geometry of molecular reorientation. From an additional application of our state transition identification in multivariate time series approach, a significant correlation between shifts of emission spectra and the occurrence of rotational jumps was found.

A novel method for the assessment of targeted PEI-based nanoparticle binding based on a static surface plasmon resonance system.

The delivery of nucleic acids is a major hurdle in gene therapy or therapeutic gene knockdown, and the development of intelligent and safe nanoparticles as carrier systems is thus under intense investigation. The introduction of ligands for their targeted delivery is of major interest. Here, we describe a novel approach for the analysis of the binding properties of antibody-functionalized nanoparticles, using surface plasmon resonance (SPR) in a static cuvette system. By chemical coupling of the Epidermal Growth Factor Receptor (EGFR)-specific antibody cetuximab to poly(ethylene imine) (PEI) via a PEG-spacer and subsequent DNA or siRNA complexation, we generated targeted nanoplexes with low surface charge. Antibody-mediated uptake into EGFR overexpressing cells was observed. SPR measurements with use of a novel, protein A-based sandwich system for the immobilization of the target receptor in its correct steric orientation allowed the analysis of the specific PEI-PEG-cetuximab binding to EGFR and the determination of binding affinities. Importantly, our cuvette-based SPR assay system was also suitable for the monitoring of ligand-mediated nanoparticle binding, without convection or shear stress. We conclude that our SPR sandwich system allows the precise analysis of the binding of ligand-functionalized nanoparticles in real-time, and we thus establish SPR for the in vitro evaluation of ligand modifications for generating targeted nanoparticles.

Polyelectrolyte complexes of DNA and linear PEI: formation, composition and properties.

In the present study, the complexation between linear 13.4 kDa poly(ethylene imine) (LPEI) and plasmid DNA was investigated. Analytical ultracentrifugation (AUC) was used for size and molar mass determination. Additionally, the morphology was studied by scanning force microscopy. The polyplex formation was investigated in a wide range of PEI nitrogen to DNA phosphate ratios (N/P). At N/P ratios below 1, the PEI/DNA complex formation is characterized by an incomplete DNA condensation and the formation of the primary DNA/PEI complexes. The merging of the initially formed polyplexes occurs at N/P ~2, resulting in the formation of polyplexes with much larger size and high aggregation rate. Stable and uniform polyplexes were formed at N/P > 10, with average sizes of the polyplexes of about 170 ± 65 nm. The content of uncomplexed PEI chains in the polyplex dispersion was estimated at four different N/P ratios, 6.2, 11.6, 28.6, and 57.8, by combining preparative centrifugation with a copper complex assay and by sedimentation velocity analysis as an alternative method. It is demonstrated that virtually all added PEI binds to the DNA at N/P < 2.5; further addition of PEI results in the appearance of a large amount of free PEI in solution. Nevertheless, PEI is able to bind in the whole range of N/P ratios tested. According to the data collected by sedimentation velocity analysis and scanning force microscopy, the single PEI/DNA complexes are composed on average of 8 to 32 single condensed DNA plasmids and 70 ± 25 PEI molecules.

Rethinking the impact of the protonable amine density on cationic polymers for gene delivery: A comparative study of partially hydrolyzed poly(2-ethyl-2-oxazoline)s and linear poly(ethylene imine)s.

To gain a more profound insight into the impact of the number and the density of protonable amines on the performance of polycations as non-viral vectors, a series of linear poly(ethylene imine)s (LPEIs) with different numbers of ethylene imine (EI) units was compared to partially hydrolyzed (21 to 86%, 20 kDa) poly(2-ethyl-2-oxazoline)s (PHPEtOxs) with a corresponding number of EI units but with varying densities. PHPEtOx polyplexes demonstrated lower transfection efficiencies than the corresponding LPEIs although having the same number of EI units as LPEI, exhibiting smaller or comparable polyplex diameters, similar zeta potentials, and similar or even preferred cyto- and hemocompatibility profiles. The lower efficiency was found to be related to a lower DNA binding capacity and less efficient protection of plasmid DNA against enzymatic degradation. The direct comparison of both types of polymers revealed that the density of charges within the polymer backbone seems to be more important than the total number of EI units. In conclusion, the reduction of the EI density to produce more biocompatible polyplexes must be critically examined, since the presence of high numbers of EI next to each other seems to have a dramatically higher impact on the transfection efficiency than on the in vitro toxicity.

Poly(ethyleneimines) in dermal applications: biocompatibility and antimicrobial effects.

Cationic polyamines, such as poly(ethyleneimines) (PEIs), may recommend themselves for antimicrobial applications as they can interact with microbial membranes resulting in their disruption. The purpose of the study was the assessment of biocompatibility and antibacterial activity of PEIs with different architectures (branched (b) and linear (l)) and molar masses (0.8-750 kDa). lPEI and bPEI exhibited a strong antibacterial activity against Staphylococcus aureus and Escherichia coli with a more pronounced effect on the Gram-positive bacteria. lPEIs further demonstrated a higher antibacterial efficacy compared to bPEIs but no significant differences between 5 and 25 kDa were observed. In accordance, antibacterial activity of bPEI did not specifically depend on molar mass. Only slightly lower minimal inhibitory concentrations (MIC) were observed at 5 kDa (S. aureus) and 25 kDa (E. coli) in the tests. As PEIs are compelling candidates for use in antimicrobial treatment, two basic aspects have to be investigated: treatment effectiveness and safety. PEIs clearly induced molecular weight dependent cytotoxic effects in vitro. PEIs with low molecular weight (0.8 and 5 kDa) exhibited higher biocompatibility. Nonetheless, the results confirmed a low genotoxic potential of lPEI and bPEIs. In conclusion, 2.5 kDa-lPEI and 0.8 kDa-bPEI can be recommended for use as antimicrobial polymers in dermal applications due to their high biocompatibility with concomitant antibacterial efficacy.

Poly(2-ethyl-2-oxazoline) as alternative for the stealth polymer poly(ethylene glycol): comparison of in vitro cytotoxicity and hemocompatibility.

Limitations of PEG in drug delivery have been reported from clinical trials. PEtOx (0.4-40 kDa) as alternative is synthesized by a living, microwave-assisted polymerization, and is directly compared to PEG of similar molar mass regarding cytotoxicity and hemocompatibility. In short-term treatments, both types of polymers are well tolerated even at high concentrations. Moderate concentration and molar mass dependent cytotoxic effects occurred only after long-term incubation at concentrations higher than therapeutic doses. PEtOx possesses not only an easy route of synthesis and beneficial physicochemical characteristics such as low viscosity and high stability, which are advantageous over PEG, but additionally in vitro toxicology comparable to PEG.