Insight into the relationship between the cell culture model, cell trafficking and siRNA silencing efficiency.

Despite research efforts, cell uptake processes determining siRNA silencing efficiency remain unclear. Here, we examine the relationship between in vitro cell culture models, cellular trafficking and siRNA silencing efficiency to provide a mechanistic insight on siRNA delivery system design. Model siRNA-polyplexes, based on chitosan as a 'classical' condensing agent, were applied to a panel of lung epithelial cell lines, H1299, A549 and Calu-3 and cell internalization levels, trafficking pathways and gene silencing assessed on exposure to pharmacological inhibitors. The data reveal striking differences in the internalization behaviour and gene silencing efficiency in the tested cell lines, despite their common lung epithelial origins. The model system's silencing was lower where clathrin internalization pathway predominated in Calu-3, relative to silencing in H1299 cells where a non-clathrin internalization appears dominant. Increased silencing on endosomal disruption was apparent in Calu-3 cells, but absent when cellular internalization was not predominantly clathrin-mediated in A549 cells. This highlights that identifying cell trafficking pathways before incorporation of functional components to siRNA delivery systems (e.g. endosomolytic compounds) is crucial. The study hence stresses the importance of selection of appropriate cell culture model, relevant to in vivo target, to assess the gene silencing efficiency and decide which functionalities the 'stratified siRNA silencing vector' requires.

Water-soluble substituted chitosan derivatives as technology platform for inhalation delivery of siRNA.

Despite research efforts full potential of siRNA-based therapeutics has not yet been fully realized due to a need for suitable, effective delivery formulations. Here, we examine a potential of a new class of water-soluble chitosans as siRNA platform for pulmonary delivery. The system is based on piperazine-substituted chitosans, a material designed to integrate established, safe application of chitosan for mucosal administration with novel properties: the piperazine-substituted chitosans are freely water-soluble at physiological pH, possess low cytotoxicity (no significant reduction in cell viability up to 0.1 mg/ml), and provide efficient incorporation of siRNA into sub-300 nm colloidal complexes (at relatively low polymer/siRNA ratio of 5:1). In vitro, the complexes achieved silencing of a model gene at a level of 40-80%, when tested in a panel of lung epithelial cells. Considering the formulation 'developability', there were no significant changes in the complexes' size and integrity on aerosolisation by microsprayer (PenCentury™) device. Following intratracheal aerolisation, the complexes deposited throughout the lung, although relatively inhomogeneously, as judged from IVIS imaging of the isolated mouse lung (visualizing DY647-siRNA). In vivo data illustrate absence of adverse effects on repeated administration of complexes and significant tumor reduction in atopical lung cancer model in mice. Altogether, the data illustrates potential of substituted chitosan derivatives to be utilized as a safe system for inhalation delivery of siRNA.

Stereochemically inactive lone pairs in phosphorus(III) compounds: the characterisation of some derivatives with the 2,5-(CF3)2C6H3 (Ar) substituent and their complexation behaviour towards Pt(II) species.

Some new phosphorus(III) derivatives Ar(2)PX (X = Br, Cl, F or H), ArPX(2) (X = Br or Cl), Ar(3)P and Ar(t)BuPCl, with the 2,5-bis(trifluoromethyl)phenyl (Ar) substituent on phosphorus, have been prepared, and characterised by (31)P and (19)F NMR solution-state spectroscopy. The complexing ability of Ar(2)PX, Ar(3)P and Ar(t)BuPCl towards the dimeric platinum(II) complexes [PtY(µ-Y)(PEt(3))](2) (Y = Cl or Br, the latter for X = Br only) has also been investigated. Single-crystal X-ray diffraction studies at low temperature have been carried out for Ar(3)P, Ar(2)PCl and the hydrolysis or oxidation products Ar(2)P(H)OH and Ar(2)P(O)OH. The structures of Ar(3)P and Ar(2)PCl are particularly interesting as in each compound the geometry around P is approximately octahedral. In Ar(3)P there are three short contacts to fluorine as well as the three bonded C atoms for both of the independent molecules in the unit cell. For Ar(2)PCl there are two short P-F contacts, and the octahedron is completed by a weak P-P interaction to a neighbouring molecule. In both instances the lone pair on the P(III) centre appears to be stereochemically inactive, and does not play a significant role in the structure.