Microfluidic self-assembly of folate-targeted monomolecular siRNA-lipid nanoparticles

Non-viral delivery of nucleic acids for therapies based on RNA interference requires a rational design and optimal self-assembly strategies. Nucleic acid particles need to be small, stable and functional in terms of selective cell uptake and controlled release of encapsulated nucleic acids. Here we report on small (∼38 nm) monomolecular nucleic acid/lipid particles (mNALPs) that contain single molecules of short double-stranded oligonucleotides covered by a tight, highly curved lipid bilayer. The particles consist of DOPE, DOTAP, DOPC and DSPE-PEG(2000) and are assembled with 21 bp double-stranded DNA or small interfering RNA by solvent exchange on a hydrodynamic-focusing microfluidic chip. In comparison to vortex mixing by hand this method increases the encapsulation efficiency by 20%, and yields particles with a narrower size distribution, negligible aggregate formation and high stability in blood plasma and serum. Modification of mNALPs with folate-conjugated PEG-lipids results in specific binding and uptake by epithelial carcinoma KB cells overexpressing folate receptors. Binding is significantly reduced by competitive inhibition using free folate and is not observed with non-targeted mNALPs, revealing high specificity. The functionalized mNALPs show gene silencing in the presence of chloroquine, an endosome-destabilizing agent. Together, the robust self-assembly of small-sized mNALPs with their high stability and receptor-specific cell uptake demonstrate that the tight, PEG-grafted lipid-bilayer encapsulation may offer a promising approach towards the delivery of short double-stranded oligonucleotides.