Andrea Beyerle

Toxicity pathway focused gene expression profiling of PEI-based polymers for pulmonary applications.

Polyethylene imine (PEI) based polycations, successfully used for gene therapy or RNA interference in vitro as well as in vivo, have been shown to cause well-known adverse side effects, especially high cytotoxicity. Therefore, various modifications have been developed to improve safety and efficiency of these nonviral vector systems, but profound knowledge about the underlying mechanisms responsible for the high cytotoxicity of PEI is still missing. In this in vitro study, we focused on stress and toxicity pathways triggered by PEI-based vector systems to be used for pulmonary application and two well-known lung toxic particles: fine crystalline silica (CS) and nanosized ZnO (NZO). The cytotoxicity profiles of all stressors were investigated in alveolar epithelial-like type II cells (LA4) to define concentrations with matching toxicity levels (cell viability >60% and LDH release <10%) for subsequent qRT-PCR-based gene array analysis. Within the first 6 h pathway analysis revealed for CS an extrinsic apoptotic signaling (TNF pathway) in contrast to the intrinsic apoptotic pathway (mitochondrial signaling) which was induced by PEI 25 kDa after 24 h treatment. The following causative chain of events seems conceivable: reactive oxygen species derived from particle surface toxicity triggers TNF signaling in the case of CS, whereby endosomal swelling and rupture upon endocytotic PEI 25 kDa uptake causes intracellular stress and mitochondrial alterations, finally leading to apoptotic cell death at higher doses. PEG modification most notably reduced the cytotoxicity of PEI 25 kDa but increased proinflammatory signaling on mRNA and even protein level. Hence in view of the lung as a sensitive target organ this inflammatory stimulation might cause unwanted side effects related to respiratory and cardiovascular disorders. Thus further optimization of the PEI-based vector systems is still needed for pulmonary application.

PEGylation affects cytotoxicity and cell-compatibility of poly(ethylene imine) for lung application: structure-function relationships.

Poly(ethylene imine) (PEI) has widely been used as non-viral gene carrier due to its capability to form stable complexes by electrostatic interactions with nucleic acids. To reduce cytotoxicity of PEI, several studies have addressed modified PEIs such as block or graft copolymers containing cationic and hydrophilic non-ionic components. Copolymers of PEI and hydrophilic poly(ethylene glycol) (PEG) with various molecular weights and graft densities were shown to exhibit decreased cytotoxicity and potential for DNA and siRNA delivery. In this study, we evaluated the cytotoxicity and cell-compatibility of different PEGylated PEI polymers in two murine lung cell lines. We found that the degree of PEGylation correlated with both cytotoxicity and oxidative stress, but not with proinflammatory effects. AB type copolymers with long PEG blocks caused high membrane damage and significantly decreased the metabolic activity of lung cells. In addition, they significantly increased the release of two lipid mediators such as 8-isoprostanes (8-IP) and prostaglandin E(2) (PGE(2)) in a dose-dependent manner. In contrast, the cytokine profiles which indicated high levels of acute-phase cytokines such as TNF-alpha, IL-6, and G-CSF did not follow any clear structure-function relationship. In conclusion, we found that modification of PEI 25 kDa with high degree of PEGylation and low PEG chain length reduced cytotoxic and oxidative stress response in lung cells, while the proinflammatory potential remained unaffected. A degree of substitution in the range of 10 to 30 and PEG-chain lengths up to 2000 Da seem to be beneficial and merit further investigations.

Comparative in vivo study of poly(ethylene imine)/siRNA complexes for pulmonary delivery in mice

Pulmonary siRNA delivery offers a new way to treat various lung diseases. Poly(ethylene imines) (PEIs) are promising cationic nanocarriers and various modifications are still under investigations to improve their cytotoxicity and efficacy for siRNA delivery. In this study, we analyzed two different types of PEI-based nanocomplexes in mice after intratracheal administration regarding their toxicity and efficacy in the lungs. Ubiquitously enhanced green fluorescent protein (EGFP) expressing transgenic and BALB/c mice were intratracheally instilled with 35μg siRNA complexed with the different types of PEI nanocarriers. Lung toxicity and inflammation were investigated after 24h, 3d and 7d treatment and knockdown of EGFP expression was analyzed by flow cytometry and fluorescence microscopy five days post instillation. Three different polyplexes caused more than 60% knockdown of EGFP expression, but only the fatty acid modified low molecular weight PEI 8.3kDa (C16-C18-EO25)1.4 specifically reduced EGFP expression in CD45+ leucocytes (25±12%) and CD11b-/CD11c+ lung macrophages (36±14%). Hydrophobic and hydrophilic PEG modifications on PEI caused severe inflammatory response and elevated levels of IgM in broncho-alveolar fluid (BALF). Thus, the PEG modification reduced cytotoxicity, but elevated the immune response and proinflammatory effects. Further investigations of the proinflammatory and immunomodulatory effects of the PEI-modified carriers are necessary to clarify the highly unspecific knockdown effects in the lung in more detail. Nevertheless, the more hydrophobic modification of PEI based non-viral vector system appeared to be a promising approach for improved siRNA therapeutics offering successful pulmonary siRNA delivery.

Inflammatory responses to pulmonary application of PEI-based siRNA nanocarriers in mice.

Polymeric non-viral vector systems for pulmonary application of siRNA are promising carriers, but have failed to enter clinical trials because of safety and efficiency problems. Therefore, improving their transfection efficiency, as well as their toxicological profile, is the subject of intensive research efforts. Six different poly(ethylene imine) (PEI)-based nanocarriers, with hydrophilic and hydrophobic PEG modifications, were toxicologically evaluated for pulmonary application in mice. Nanocarriers were intratracheally instilled to determine their toxicological profile, with particular focus on the inflammatory response in the lungs. Nanocarriers from both groups caused an acute inflammatory response in the lungs, albeit with different resolution kinetics and cytotoxicity. Hydrophobic modifications caused a severe inflammatory response with increased epithelial barrier permeability, accompanied by an acute antioxidant response. Hydrophilic modifications, with high PEG-grafting degrees, induced less proinflammatory effects without depleting macrophages and disrupting the epithelial/endothelial barrier in the lungs, while showing only a minor oxidative stress response. For pulmonary applications, local proinflammatory effects should be optimized by further development of nanocarriers with highly grafted PEG-PEI-based carriers or Jeffamine-modified hydrophobic PEI modifications.

Poly(ethylene imine) nanocarriers do not induce mutations nor oxidative DNA damage in vitro in MutaMouse FE1 cells.

Genotoxicity information on polymers used for gene delivery is scant, but of great concern, especially when developing polymeric nanocarriers as nonviral vector systems for cancer treatment. The genotoxicity of some engineered nanomaterials, e.g., metal oxides like ZnO, TiO₂, and CuO but also carbon based materials like carbon black or nanotubes, has commonly been related to oxidative stress, and subsequent inflammation. Recent studies of poly(ethylene imine) (PEI)-based polymers, important nonviral vector systems for pDNA and siRNA, might raise concerns because of their toxic effects dominated by cellular oxidative stress and inflammatory responses, similar to the mentioned effects of engineered nanoparticles. In this study, we employed a FE1-MutaMouse lung epithelial cell line based mutation assay to determine the genotoxicity of three PEI-based polymers and nanosized zinc oxide particles (NZO), all of which have previously been shown to trigger oxidative stress and inflammation. In addition, oxidative DNA damage (8-OH-dG) in FE1 cells was assessed by ELISA. The well-known carcinogen benzo[a]pyrene (B[a]P) was used as positive control. FE1 lung epithelial cells were exposed for eight sequential 72 h incubations, and reporter-gene mutation frequency or 8-OH-dG formation was determined to assess mutagenicity and oxidative DNA damage, respectively. No cytotoxic effects were detected at the exposure levels examined, which are representative of PEI concentrations normally used in in vitro transfection studies. In contrast to B[a]P, neither PEI-polymers nor NZO showed any significant mutagenic activity or oxidative DNA damage in the exposed cells, although PEI-based polymers have been shown to generate significant levels of cellular stress and inflammatory responses. We suggest that the lack of any detectable mutagenic/genotoxic activity of the PEI-based polymers studied here is a crucial step toward a safe use of such nanocarriers in clinical trials.

Screening strategy to avoid toxicological hazards of inhaled nanoparticles for drug delivery: The use of a-quartz and nano zinc oxide particles as benchmark

Nanotechnology is a broad, revolutionary field with promising advantages for new medicine. In this context the rapid development and improvement of so called nanocarriers is of high pharmaceutical interest and some devices are already on the market. In our project we aim to develop well characterized nanoscaled drug delivery systems for an inhalative application. To this end, we focus on the most adverse side-effects within the lung, the cytotoxic and the proinflammatory responses to these nanoparticles (NPs). Before performing any animal experiments, we start with an in vitro screening for analyzing the cytotoxic and proinflammatory effects of the investigated particles on two murine lung target cell lines, the alveolar epithelial like typ II cell line (LA4) and the alveolar macrophage cell line (MH-S). Three different endpoints were estimated, (i) cellular metabolic activity, determined by the WST-1 assay, (ii) membrane integrity, by detection of LDH release and hemolytic activity, and (iii) secretion of inflammatory mediators. To analyze the relative particle toxicity we choose two reference particles as benchmarks, (i) fine a-quartz, and (ii) ultrafine ZnO particles. The investigation of dose-response and kinetics of proinflammatory and toxic effects caused to the named cell lines provide an insight to a close evaluation of our cell based screening strategy. oc-quartz is well known for its inflammatory and toxic potential caused by inhalation, and nanosized ZnO particles - used in a broad field of nanotechnology like electronics, but also cosmetics and pharmaceuticals - is to a high degree cytotoxic and proinflammatory in vitro. Preliminary experiments indicated not only particle and cell specific inflammatory responses, but also different susceptibilities of the cell types being exposed to our benchmark particles regarding their size and surface activities. Exposure to the μm-sized a-quartz particles affected the viability of epithelia cells less than that of macrophages, pointing to the impact of particle uptake by phagocytosis. In contrast, the nanosized ZnO particles caused much stronger decrease in cell viability and higher levels of LDH in the macrophage cell line compared to epithelial cells, even though the hemolytic activity was much higher for the a-quartz particles than for the nanosized ZnO. For the proinflammatory effects, we observed a clear dose-dependent release of acute phase cytokines (TNF-α, IL-6, G-CSF> CXCL10>CCL2) for both alveolar cell lines after Min-U-Sil exposure. After ZnO treatment the cytokine responses were negligible compare to control cells. In conclusion, our data attach value to the use of different cell types to detect different pathways of toxicity generated by different particle properties. Therefore, we will establish both lung target cell lines for an in vitro screening to analyze proinflammatory and cytotoxicity effects of nanocarriers. The implementation of the two reference particles facilitate the validated classification of the cytotoxic responses caused by the NPs investigated.

Toxicity of Polymeric-Based Non-Viral Vector Systems for Pulmonary siRNA Application

Nanomedicine has the potential of clinical benefit by combination of engineering technologies and materials (Schatzlein, 2006). Development of nanometre scaled therapeutics which provides new and improved properties by specifically targeting the site of action and causing low level of side effects would be a big challenge to treat patients with severe and live-threatening diseases like cancer. Gene therapy provides a new way to treat patients and a lot of effort is made to improve the clinical benefit. But current gene therapy is still experimental and has not proven success in the clinics. Nevertheless there is a need for new approaches to treat „undruggable“ disease sites and there are some clinical trials ongoing which using RNA inference (RNAi) as therapeutic mechanism (Table 1).