Polyplexes between siRNA and poly(ethylene imine) (PEI) derivatives are promising nonviral carriers for siRNA. The polyplex stability is of critical importance for efficient siRNA delivery to the cytoplasm. Here, we investigate the effect of PEGylation at a constant ratio ( approximately 50%) on the biophysical properties of the polyplexes. Particle size, zeta potential, and stability against heparin as well as RNase digestion and reporter gene knockdown under in vitro conditions of different siRNA polyplexes were characterized. Stability and size of siRNA polyplexes were clearly influenced by PEI-PEG structure, and high degrees of substitution such as PEI(25k)-g-PEG(550)(30) resulted in large (300-400 nm), diffuse complexes (AFM) which showed condensation behavior only at high N/P ratios. All other polyplexes and the PEI control showed similar sizes (150 nm) and compact structures in AFM, with complete condensation reached at N/P ratio of 3. Stability of siRNA polyplexes against heparin displacement and RNase digestion could be modified by PEGylation. Protection against RNase digestion was highest for PEI(25k)-g-PEG(5k)(4) and PEI(25k)-g-PEG(20k)(1), while siRNA/PEI provided insufficient protection. In knockdown experiments using NIH/3T3 fibroblasts stably expressing beta-galactosidase, it was shown that PEG chain length had a significant influence on biological activity of siRNA. Polyplexes with siRNA containing PEI(25k)-g-PEG(5k)(4) and PEI(25k)-g-PEG(20k)(1) yielded similar efficiencies of ca. 70% knockdown as lipofectamine controls. Confocal microscopy demonstrated enhanced cellular uptake of siRNA into cytosol by polyplexes formation with PEI copolymers. In conclusion, both the chain length and graft density of PEG were found to strongly influence siRNA condensation and stability and hence affect the knockdown efficiency of PEI-PEG/siRNA polyplexes.

Over the past 10 years, the interest in intranasal drug delivery in pharmaceutical R&D has increased. This review article summarises information on intranasal administration for local and systemic delivery, as well as for CNS indications. Nasal delivery offers many advantages over standard systemic delivery systems, such as its non-invasive character, a fast onset of action and in many cases reduced side effects due to a more targeted delivery. There are still formulation limitations and toxicological aspects to be optimised. Intranasal drug delivery in the field of drug development is an interesting delivery route for the treatment of neurological disorders. Systemic approaches often fail to efficiently supply the CNS with drugs. This review paper describes the anatomical, histological and physiological basis and summarises currently approved drugs for administration via intranasal delivery. Further, the review focuses on toxicological considerations of intranasally applied compounds and discusses formulation aspects that need to be considered for drug development. Graphical abstract

This study describes the physicobiological characterization of PEI- and PEG-PEI polyplexes containing partially 2'-OMe modified 25/27mer dicer substrate siRNAs (DsiRNAs) and their in vivo behavior regarding biodistribution and systemic bioavailability after pulmonary application as well as their ability to knock down gene expression in the lung. Biophysical characterization included circular dichroism of siRNA in polyplexes, condensation efficiency of polymers and in vitro stability. After in vivo application, biodistribution and kinetics of radiolabeled polyplexes were quantified and recorded over time in three-dimensional SPECT images and by end point scintillation counting. The influence on lung tissue and on the humoral and cellular immunosystem was investigated, and finally knockdown of endogenous gene expression in the lung was determined qualitatively. While all of the polymers used in our study were proven to effectively condense siRNA, stability of the complexes depended on the PEG grafting degree. Interestingly, PEI 25 kDa, which showed the least interaction with mucin or surfactant in vitro, performed poorly in vivo. Our nuclear imaging approach enabled us to follow biodistribution of the instilled nanocarriers over time and indicated that PEGylated nanocarriers are more suitable for lung application. While moderate proinflammatory effects were attributed to PEI25k-PEG(2k)(10) nanocarriers, none of the treatments caused histological abnormalities. Our preliminary in vivo knockdown experiment suggests that PEG-PEI/siRNA complexes are promising nanomedicines for pulmonary siRNA delivery. These results encouraged us to further investigate possible adverse effects and to quantify in vivo gene silencing in the lung after intratracheal instillation of PEG-PEI/siRNA complexes.

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