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Layer-by-layer assembled siRNA-nanoparticles as tool to identify strategies for highly efficient siRNA delivery into cells

Subject Area Pharmacy
Term from 2010 to 2014
Project identifier Deutsche Forschungsgemeinschaft (DFG) - Project number 183111963
 
Final Report Year 2014

Final Report Abstract

Small interfering RNA (siRNA) holds great promise for the treatment of various diseases. A decisive step towards its broad clinical application is the development of siRNA delivery systems. However, because these delivery systems are usually characterized by the existence of different particle subspecies, an optimization is hard to achieve. Hence, we proposed Layer-by-Layer (LbL) coated nanoparticles as a tool to investigate how the efficacy of siRNA delivery can be improved. The LbL approach involves the sequential adsorption of oppositely charged polyelectrolytes on a surface using siRNA as polyanion in combination with any suitable polycation. The big advantage of LbL-coated particles is their high uniformity, which allows to fine-tune one property of the nanoparticles without changing other parameters at the same time. The first goal was to identify polyelectrolytes that provide for a sufficient stability of the delivery system in the extracellular environment accompanied by a sufficient release at the target place. We developed a Foerster Resonance Energy Transfer (FRET)-based approach to correlate the LbL film structure and the release of macromolecules. For example, the polycation poly(allylamine hydrochloride) (PAH), in combination with poly(styrene sulfonate) (PSS) - which was used as surrogate for siRNA - showed little promise for the delivery of charged macromolecules; chain indenting most likely inhibited swelling of the multilayer, thereby impeding release of these larger drugs. In contrast, the polycation poly(ethylene imine) (PEI) in combination with PSS seemed to be a promising candidate for the delivery of charged macromolecules because the film structure is driven more by stacking and rearranging than interpenetration. Hence, layer-layer cohesion is weaker, enabling water molecules to more easily expand the distance between layers. Without the development of the FRET-based approach, these results would only have been possible with extensive trial and error and the use of methods that are not widely accessible. To complete this part of the project, we are currently transferring the results from flat surfaces to nanoparticles using siRNA as a polyanion. The second goal was to determine physicochemical properties of LbL-coated nanoparticles for an efficient cellular uptake and siRNA-silencing efficacy. In a first step, we investigated the size optimum using nanoparticles cores of 20, 30, 50 and 80 nm that were coated in a LbL approach. Although the smaller particles had a significantly lower loading capacity for nucleic acids, they overcompensated this by the higher uptake and consequently also yielded a significantly higher number of therapeutic nucleic acid molecules per cell. In numbers, the efficacy of nucleic acid delivery using 80 nm core particles was only 2% of the 20 nm ones. This was the first study that involved drug-loaded nanoparticles for the determination of the size optimum and that paid attention to the size increase of nanoparticles after incubation in serum-containing culture medium. In a second step, we investigated the impact of using various polyelectrolytes on the LbL formation and on the interaction with the biological system. A major finding was that the surface topography of the LbL-coated nanoparticles is decisive for the adsorption of serum proteins and later the association with cells. Thus, a pronounced surface roughness led due to a larger surface contact area to a massive protein adsorption and consequently to a reduced cell association. It was surprising that the trilayered LbL film involving the polycation PEI for the assembly, led to a pronounced surface roughness. The third goal was to track the integrity of siRNA during the delivery process. To this end, we developed a siRNA-sensor that is based on an energy transfer between two fluorescent dyes, which were introduced as base surrogates. The principle of the siRNA-sensor was a red fluorescence emission of the double-stranded siRNA and a green signal as soon as strand dissociation occurred. We applied the sensor as a favorable tool to monitor siRNA integrity in living cells in real time. In contrast to other available sensors, it had a remarkable dynamic range and showed an excellent contrast in the biological environment. We also demonstrated that the siRNA-sensor, depending on the position of the base surrogates, maintained its gene-silencing efficacy. Furthermore, we developed new energy transfer pair that (i) are “clickable” to DNA and RNA which facilitates the synthetic access significantly, (ii) show improved photostability which is an important prerequisite for long-term fluorescence cell imaging and additional readout by electron microscopy, and (iii) exhibit excellent emission color contrasts and brightness. The new energy transfer systems must now be transferred to RNA and evaluated in the context of this project, mainly for trafficking siRNA delivery. With respect to the tremendous synthetic work, this final part of the project is still elusive and needs to be performed in the future. In conclusion, our results helped to clarify existing limitations for siRNA delivery such as the size and surface properties of nanoparticles. This information will help to design new and efficient materials for siRNA delivery.

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