Dokument

Summary:
In this thesis, toxicity of PEI-based non-viral vector systems for siRNA application into the lungs was comprehensively described and analyzed in vitro as well as in vivo. Chapter 1 introduced in basic information about the lung anatomy and physiology and general considerations for pulmonary application as well as gave an overview of the two major groups of non-viral vector systems for pulmonary application and highlighted their impact in nanomedicine and nanotoxicology. The search for more predictive toxicity tools for (polymeric) non-viral vector systems is still of great concern in the community and was pointed out in this chapter. Chapter 2 described the toxicicological and immunomoldulatory effects of two different PEI-based nanocarriers for siRNA delivery in different murine lung cells. Two different PEI nanocarriers (branched vs. linear, and low vs. high molecular weight PEI) were evaluated regarding standard toxicity endpoints, but also immunomodulatory effects caused by the pure polymers and their respective polyplexes with siRNA. The results pointed out, that epithelial cells were much more sensitive in response to such polymers and the polyplexes appeared to be less toxic than the pure polymers. In addition, the immunomodulatory effects of such polymeric non-viral vector systems should be further investigated for their underlying mechanism. Chapter 3 hypothesized that poly(ethylene glycol) (PEG) reduces the cytotoxicity of high molecular weight, branched PEI25 kDa and investigated the cell-compatibility and cytotoxicity of a panel of different PEI-PEG polymers in vitro. This in vitro study highlighted the inflammatory potential of such PEI-PEG polymers which seemed to be higher when cytotoxicity was extremely reduced. Hypothesizing that inflammatory and oxidative stress response play an important role when using PEI-based nanocarriers, especially for pulmonary application, in Chapter 4 a toxicity and stress pathway focused gene expression profiling was described for selected PEI-PEG polymers. This gene array clearly stressed the inflammatory potential of the modified PEI-PEG polymers with reduced apoptotic signalling pathways, but increasing inflammatory and oxidative stress response, in contrast to PEI25 kDa. Due to the higher proinflammatory potential and elevated oxidative stress parameters, the question of genotoxicity was addressed in Chapter 5. The mutant frequency of selected PEI-based nanocarriers was investigated by using a transgenic lung epithelial cell culture in vitro model, but was regarded to be less and PEI-based nanocarriers were not mutagenic in such an in vitro model. After toxicity analysis in vitro two main questions raised (i) what kind of effects would be induced by the polymers or their polyplexes in vivo when directly administered to the lungs and (ii) could we find any in vitro/ in vivo correlation for biomarkers indicating toxicity, inflammation and/or oxidative stress? Chapter 6 focused on the in vivo toxicity, inflammatory, and oxidative stress response of selected PEI-based nanocarriers for siRNA in mice after intratracheal instillation and tried to answer the two upcoming questions from the in vitro studies. Almost all modified PEI-based nanocarriers showed very high acute inflammation, but with different resolving kinetics. Hydrophobic modification of low molecular weight PEI and highly hydrophilic PEGylated PEI-based nanocarriers seemed to be well tolerable in contrast to moderate hydrophilic PEGylated and fatty-acid modified PEI-based polymers which showed very high and sustained inflammation in the lungs. In contrast to safety issues (which represent the main part of this thesis) in chapter 7 the in vivo efficacy and the cell–type specific targeting was reported of PEI-based nanocarriers, same carriers selected as in Chapter 6, for pulmonary siRNA delivery. Surprisingly, the highly inflammatory PEI-based nanocarriers yielded high knock down effects, but only the fatty acid modified PEI-based nanocarrier, seemed to avoid off-target effects. Leucocytes were targeted to some extent, but seemed not to be the main targeted cell type in the lung after PEI-based nanocarriers application for siRNA delivery. Thus, for clinical trials the polymers should be carefully optimized and evaluated for cytotoxicity, high acute inflammatory and oxidative stress response and their in vivo performance of siRNA delivery. Development of polymers with reduced cytotoxicity and negligible off-target effects, but high in vivo efficacy represents one of the biggest challenges for the next decades before entry to clinics. In addition, optimized in vitro models for predictive toxicity are still needed.

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41. Olivia M. Merkel*, Andrea Beyerle*, Benedikt M. Beckmann, Roland K. Hartmann, Tobias Stoeger, and Thomas Kissel Off-target effects in non-viral siRNA delivery – A study on the effect of polymer genomics under in vitro cell culture conditions Submitted to Molecular Therapy
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45. Andrea Beyerle, Holger Schulz , Thomas Kissel, Tobias Stoeger Nanohale -Cytotoxic and proinflammatory evaluation of various PEI particles on lung target cells Oral Presentation, 49. DGPB Tagung, Lübeck, April 2008 9.5 Curriculum vitae Persönliche Daten: Name: Beyerle, Christiane Andrea Barbara Beruf: Apothekerin Geburtsdatum: 10.09.1979
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48. Andrea Beyerle, Andrea Braun, Atrayee Banerjee, Nuran Ercal, Oliver Eickelberg, Thomas Kissel, Tobias Stoeger Side-effects and their clinical relevance of Poly(ethylene imine)-based siRNA nanocarriers for pulmonary application in mice Submitted to European Respiratory Journal (ERJ-00161-2010)
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