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O. Mykhaylyk, D. Vlaskou, Ch. Plank, New magnetic nanoparticle formulations for DNA and siRNA delivery, 13th Annual Congress of the European Society of Gene Therapy, Oct. 29 - Nov. 1, 2005, Prague, Czech Republic
A set of the core/shell type magnetic nanomaterials (MNs) was synthesized using precipitation of the iron hydroxides from aqueous salt solution and transformation into magnetite in an oxygen free atmosphere. Nanocrystals were stabilized by spontaneous adsorption of the hydrophobically modified dextrans or polyethylene imines (PEI) combined with fluorinated surfactants. The NMs are easily incorporated into lipid microbubbles together with a plasmid. The resulting microbubbles are small, uniform and stable. The materials are highly efficient in pBLuc adsorption and magnetofection in 3T3 cells. Positively charged NMs demonstrate high self-activity in the plasmid adsorption and delivery, whereas other materials are efficient in combination with positively charged enhancer (Metafectene (Mf) and PEI tested). When combined with Mf, in the low DNA concentration range (1 to 75 ng DNA/ml) positively and slightly negatively charged NMs are 10-fold more effective than optimal Mf concentrations. NM with relatively high negative Z-potential (-57 mV) in combination with Mf demonstrates a 5-15-fold increase in a transfection efficiency in the range of 15 to 1250 ng pBluc/ml compared to the optimal Mf concentration. The nanomaterials are highly efficient in siRNA magnetofection in HeLa cells stably transduced with GFP- and Luciferase-genes. Positively charged NM demonstrates high self-activity in siRNA transfection without any enhancer (66% inhibition of the GFP-expression, 8 nM siRNA, 72 h). Formulations of NMs with PEI are highly efficient in siRNA magnetofection (84% GFP-expression inhibition at 8 nM siRNA, 72 h). The combined use of the INF-7 fusion peptide and NMs improves siRNA magnetofection results. Combinations of NMs with Metafectene are more efficient for DNA delivery than those with PEI, whereas for siRNA delivery, formulations of nanoparticles with PEI are much more efficient. Inhomogeneous steady state magnetic field (Nd-Fe-B magnet) is a catalyst of the DNA transfection with synthesized magnetic nanomaterials.
C. M. Schmidt, S. Huth, O. Mykhaylyk, C. Plank, C. Rudolph and J. Rosenecker. 2006. Efficient downregulation of EnaC activity by synthetic siRNAs. Molecular Therapy. 13 (Suppl. 1): p267: 691.
The amiloride-sensitive epithelial sodium channel (ENaC) is assumed to play a major role in the pathogenesis of chronic lung disease in cystic fibrosis patients. Its natural regulation by the cystic fibrosis transmembrane conductance regulator (CFTR) appears to be compromised, based on the impaired function of CFTR. The missing inhibition of the channel results in increased absorption of sodium ions and fluid across airway epithelia leading to the depletion of the airway surface liquid (ASL) layer and to the depression of mucociliar clearance. Several observations suggest that a downregulation of ENaC restores the periciliar liquid layer, thereby rehydrating the mucus and improving ciliary clearance in the lung. Therefore, we attempt to specifically downregulate ENaC expression by RNA interference.
ENaC is a tetrameric ion channel localized in the apical membrane of epithelia. It is comprised of three subunits alpha, beta and gamma. Alpha ENaC itself is the pore forming subunit. To study the downregulation of the subunits by RNAi we designed ENaC-specific synthetic and plasmid based siRNA sequences. To analyze RNAi for ENaC expression we used the murine renal cortical collecting duct cell line M-1. This cell line is well characterized for ENaC expression; in addition we detected a strong amiloride dependent signal in a fluorescence based assay capable of measuring changes in the cells membrane potential.
To identify optimal siRNA transfection conditions for M-1 cells we used a GFP expressing M-1 cell line. The highest GFP knockdown was achieved when using HiPerFect (Qiagen) in combination with CombiMag (OZ Biosciences). After 72h a GFP knockdown to 40% was detected by fluorescence measurements.
To identify alpha ENaC knockdown we analyzed six different siRNA sequences designed by conventional design programs. In addition we included a sequence tested by Li and Folkesson against rat ENaC adapted to the murine sequence. These sequences were transfected in M-1 epithelial cells in 6-well format; the knockdown is analyzed on mRNA and protein level via quantitative real-time PCR and Western blotting. The most efficient ENaC knockdown resulting in 70% reduction was achieved with the modified sequence published by Li and Folkesson 2005. These results suggest that ENaC activity can be inhibited with synthetic siRNAs which might lead to a functional effect on the sodium transport.
S. Huth, C. Schmidt, C. Rudolph and J. Rosenecker. 2006. Analysis of the stability and functionality of siRNA after nebulization of siRNA polyplexes. Molecular Therapy. 13 (Suppl. 1): p272: 703.
Aerosol application of siRNA is a promising therapeutical approach to reach and silence genes which are leading to diseases of the lung, e. g. Cystic fibrosis (CF). Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) are leading to a missing down regulation of the epithelial sodium channel (ENaC) which results in an increased absorption of sodium ions and fluid across the airway epithelia leading to the depletion of the perciliary liquid layer and to the depression of mucus clearance. Therefore silencing ENaC by RNA interference might help to treat this disease. The integrity of the siRNA nebulized is a precondition for this new approach.
In this work, we studied the properties of siRNA polyplexes after the nebulization process. In particular, we analyzed its stability and functionality before and after nebulization.
The physical stability of siRNA was studied by polyacrylamide gel electrophoresis. Its functionality was analyzed in vitro by determining the efficiency of silencing by flow cytometry using cells stably expressing GFP and siRNA against GFP. We investigated the siRNA properties of complexes based on different non-viral vectors e.g. polyethylenimine (PEI) and Oligofectamine™.
Our data shows that complexed siRNA unlike to naked siRNA is not harmed by the nebulization process. After the nebulization process the complexes are still able to down regulate the GFP expression.
In conclusion, aerosol application is an appropriate method for siRNA delivery.
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