(490g) Development of Encapsulated siRNA Nanoparticles for Targeted Delivery

RNA interference (RNAi) is rapidly gaining importance as a set of tools to control gene expression and has the potential to develop into new classes of drugs for therapy. The full potential of these new compounds has been stunted by the lack of appropriate delivery mechanisms. In this project, we have developed nanoparticles of small inhibitory RNA (siRNA) using the biodegradable polymer poly(lactic acid) (PLA) and the copolymer PLA/poly(ethylene glycol) (PLA/PEG). Supercritical carbon dioxide (SC-CO2) was used as an anti-solvent and the drug/polymer solutions were sprayed into the supercritical fluid (SCF) to form particles by rapid precipitation. The high compressibility of SCF allows for tunable solvent properties and tight process control that enable optimization of particle size and particle size distribution. Due to the unique properties of SC-CO2, no purification or drying steps are needed, and the process is compatible with bioactive compounds, such as proteins and nucleic acids.

Initially, unmodified siRNA targeting the mRNA encoding mutant N171K keratin 6a (K6a: this mutation results in the skin disorder pachyonychia congenita) was processed using the SCF process yielding siRNA nanoparticles in the sub-100 nm range. The resulting siRNA particles were analyzed by scanning electron microscopy and dynamic light scattering for particle size distribution. A functional cell culture assay was performed to ensure biological activity of the siRNA after particle formation. In this assay the K6a cDNA was fused to a luciferase reporter, resulting in a bicistronic expression vector that allowed rapid detection of the reporter construct and activity of the siRNA nanoparticles. The siRNA mediated reduction of the keratin producing gene was correlated with light emission in live cell assays, and this revealed that siRNA activity was preserved after the SCF process.

Subsequently, the siRNA particles were encapsulated using PLA or PLA/PEG. The release of active siRNA was shown to be highly dependent on polymer concentration, polymer molecular weight, and SCF process conditions (such as flow rate, temperature and pressure). Encapsulated siRNA particles formed particles in the 200-500 nm range which could be controlled by process conditions. SCF processes offer new approaches for the formation of biologically active nanoparticles that may lead to effective tools for controlled delivery of siRNA