Noncovalent stabilization of vesicular polyion complexes with chemically modified/single-stranded oligonucleotides and PEG-b-guanidinylated polypeptides for intracavity encapsulation of effector enzymes aimed at cooperative gene knockdown.

For the simultaneous delivery of antisense oligonucleotides and their effector enzymes into cells, nanosized vesicular polyion complexes (PICs) were fabricated from oppositely charged polyion pairs of oligonudeotides and poly(ethylene glycol) (PEG)-b-polypeptides. First, the polyion component structures were carefully designed to facilitate a multimolecular (or secondary) association of unit PICs for noncovalent (or chemical cross-linking-free) stabilization of vesicular PICs. Chemically modified, single-stranded oligonucleotides (SSOs) dramatically stabilized the multimolecular associates under physiological conditions, compared to control SSOs without chemical modifications and duplex oligonucleotides. In addition, a high degree of guanidino groups in the polypeptide segment was also crucial for the high stability of multimolecular associates. Dynamic light scattering and transmission electron microscopy revealed the stabilized multimolecular associates to have a 100 nm sized vesicular architecture with a narrow size distribution. The loading number of SSOs per nanoveside was determined to be similar to 2500 using fluorescence correlation spectroscopic analyses with fluorescently labeled SSOs. Furthermore, the nanovesicle stably encapsulated ribonudease H (RNase H) as an effector enzyme at similar to 10 per nanovesicle through simple vortexmixing with preformed nanovesides. Ultimately, the RNase H-encapsulated nanoveside efficiently delivered SSOs with RNase H into cultured cancer cells, thereby eliciting the significantly higher gene knockdown compared with empty nanovesides (without RNase H) or a mixture of nanovesicles with RNase H without encapsulation. These results demonstrate the great potential of noncovalently stabilized nanovesides for the codelivery of two varying bio-macromolecule payloads for ensuring their cooperative biological activity.