PAMAM versus PEI complexation for siRNA delivery: interaction with model lipid membranes and cellular uptake.

PURPOSE: Cationic polymers have many advantages as vectors for mediated cellular entry and delivery of siRNA. However, toxicity related to their cationic charge has compromised clinical use. It is hypothesized that the siRNA-vector complex composition and properties can be controlled to optimize therapeutic performance. Here we investigate siRNA complexes with branched polyethylenimine (bPEI) versus generation 4 polyamidoamine dendrimers (PAMAM) on interactions with immobilized lipid membranes, and cellular uptake and toxicity. METHODS: A model siRNA was complexed with either PAMAM or bPEI, and their size and zeta-potential characterized. Interaction of the complexes and parent polymers with lipid bilayers was investigated using atomic force microscopy and correlated with the uptake and toxicity in HeLa cells. RESULTS: PAMAM and its siRNA complexes formed circular shaped micron-sized holes in lipid bilayers, while bPEI formed nanoscale holes. Flow cytometry and fluorescence microscopy demonstrated PAMAM-siRNA complexes to have a higher cellular uptake than bPEI-siRNA complexes. bPEI-siRNA complexes did not impact on viability, however PAMAM-siRNA complexes demonstrated increasing cell toxicity as N/P ratio increased. PAMAM-siRNA complexes accumulated around the cell nucleus, while PEI-siRNA complexes were located closer to the cell wall. CONCLUSION: Complexation of PAMAM dendrimer or bPEI with siRNA modified physicochemical properties of the parent polymer, however it did not impact on the mechanism of interaction with model lipid bilayers or how the polymer/siRNA complex interacted and was internalized by HeLa cells. Interaction of siRNA polymer complexes with cells is related to the action of the parent polymer.

Interfacial analysis of siRNA complexes with poly-ethylenimine (PEI) or PAMAM dendrimers in gene delivery.

Solution and interfacial analysis has been employed to gain insight into the complexation of siRNA using either G4 PAMAM dendrimers or 25kDa branched poly-ethylenimine (bPEI). The size, charge and shape/structure of the complexing agents were probed using atomic force microscopy (AFM), circular dichroism spectrometry (CD), dynamic light scattering (DLS), and gel electrophoresis (GE). The binding capability of these cationic polymers to the siRNA molecule, subsequently controls the surface/adsorption behaviour of the complexes to a negatively charged surface. G4 PAMAM dendrimers bind to the major groove of the siRNA structure, while bPEI binds to both major and minor groove. PAMAM-siRNA complexes form a thin uniform surface film with adsorption of monomeric particles, whilst bPEI-siRNA complexes adsorb as particles in random orientations at low bPEI concentration and form network structures across the surface at high charge ratio. This is due to their ability to bind to both regions within siRNA. This new understanding of the interfacial behaviour of siRNA complexes correlates with observations of cellular transfection and can be used in the design of optimal transfection agents.