Anne Greet Bittermann

Cellular uptake and intracellular pathways of PLL-g-PEG-DNA nanoparticles.

Polycationic molecules form condensates with DNA and are used for gene therapy as an alternative to viral vectors. As clinical efficacy corresponds to cellular uptake, intracellular stability of the condensates, and bioavailability of the DNA, it is crucial to analyze uptake mechanisms and trafficking pathways. Here, a detailed study of uptake, stability, and localization of PLL-g-PEG-DNA nanoparticles within COS-7 cells is presented, using FACS analysis to assess the involvement of different uptake mechanisms, colocalization studies with markers indicative for different endocytotic pathways, and immunofluorescence staining to analyze colocalization with intracellular compartments. PLL-g-PEG-DNA nanoparticles were internalized in an energy-dependent manner after 2 h and accumulated in the perinuclear region after >6 h. The nanoparticles were found to be stable within the cytoplasm for at least 24 h and did not colocalize with the endosomal pathway. Nanoparticle uptake was approximately 50% inhibited by genistein, an inhibitor of the caveolae-mediated pathway. However, genistein did not inhibit gene expression, and PLL-g-PEG-DNA nanoparticles were not colocalized with caveolin-1 indicating that caveolae-mediated endocytosis is not decisive for DNA delivery. Clathrin-mediated endocytosis and macropinocytosis pathways were reduced by 17 and 24%, respectively, in the presence of the respective inhibitors. When cells were transfected in the presence of double and triple inhibitors, transfection efficiencies were increasingly reduced by 40 and 70%, respectively; however, no differences were found between the different uptake mechanisms. These findings suggest that PLL-g-PEG-DNA nanoparticles enter by several pathways and might therefore be an efficient and versatile tool to deliver therapeutic DNA.

Cyto- and hemocompatibility of a biodegradable 3D-scaffold material designed for medical applications

In this study, the polyester urethane Degrapol (DP) was explored for medical applications. Electrospun DP-fiber fleeces were characterized with regard to fiber morphology, swelling, and interconnectivity of interfiber spaces. Moreover, DP was assayed for cell proliferation and hemocompatibility being a prerequisite to any further in vivo application. It was shown that DP-fiber fleeces produced at different humidity while spinning affects interconnectivity of interfiber spaces, such that the higher the humidity the looser the resulting fiber fleeces. When the spinning target was cooled with dry ice, the resulting DP-fibers remained less fused to each other. However, permeability for fluorescent beads was not significantly increased. Fibroblast adhesion and proliferation occurred in a comparable manner on native as well as on fibronectin or collagen I adsorbed DP-fiber fleeces. On DP-surfaces fibroblasts proliferated equally well as compared with glass or PLGA surfaces or DP-surfaces adsorbed with fibronectin or collagen I. In contrast, human umbilical vein endothelial cells proliferated only after adsorption of DP-surfaces with fibronectin or collagen I, indicating that different cell types respond differently to DP-surfaces. Furthermore, hemocompatibility of DP-surfaces was found to be similar or better to PLGA or stainless steel, both medically used materials. These experiments indicate that DP-fiber fleeces or surfaces might be useful for tissue engineering.

Do lower cuff pressures reduce damage to the tracheal mucosa? A scanning electron microscopy study in neonatal pigs.

Modern high volume–low pressure (HVLP) endotracheal tubes (ETT) cuffs can seal the trachea using baseline cuff pressures (CP) lower than peak inspiratory airway pressures (PIP). The aim of the study was to determine whether this technique reduces the damage to the tracheal mucosa compared to constant CP of 20 cmH2O.

The Role of Purple Sulphur Bacteria in Carbonate Precipitation of Modern and Possibly Early Precambrian Stromatolites

Since the description of stromatolites a century ago by Kalkowsky (1908) as products of ancient microbial activity, the exact understanding of its formation remained unclear and is still matter of debate.

Analysis of Effective Interconnectivity of DegraPol-foams Designed for Negative Pressure Wound Therapy

Many wounds heal slowly and are difficult to manage. Therefore Negative Pressure Wound Therapy (NPWT) was developed where polymer foams are applied and a defined negative pressure removes wound fluid, reduces bacterial burden and increases the formation of granulation tissue. Although NPWT is used successfully, its mechanisms are not well understood. In particular, different NPWT dressings were never compared. Here a poly-ester urethane Degrapol® (DP)-foam was produced and compared with commercially available dressings (polyurethane-based and polyvinyl-alcohol-based) in terms of apparent pore sizes, swelling and effective interconnectivity of foam pores. DP-foams contain relatively small interconnected pores; PU-foams showed large pore size and interconnectivity; whereas PVA-foams displayed heterogeneous and poorly interconnected pores. PVA-foams swelled by 40 %, whereas DP- and PU-foams remained almost without swelling. Effective interconnectivity was investigated by submitting fluorescent beads of 3, 20 and 45 μm diameter through the foams. DP- and PU-foams removed 70-90 % of all beads within 4 h, independent of the bead diameter or bead pre-adsorption with serum albumin. For PVA-foams albumin pre-adsorbed beads circulated longer, where 20 % of 3 μm and 10 % of 20 μm diameter beads circulated after 96 h. The studies indicate that efficient bead perfusion does not only depend on pore size and swelling capacity, but effective interconnectivity might also depend on chemical composition of the foam itself. In addition due to the efficient sieve-effect of the foams uptake of wound components in vivo might occur only for short time suggesting other mechanisms being decisive for success of NPWT.