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
oligonucleotides 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 nanovesicle was determined to be ∼2500 using fluorescence correlation spectroscopic analyses with fluorescently labeled SSOs. Furthermore, the nanovesicle stably encapsulated
ribonuclease H (
RNase H) as an effector
enzyme at ∼10 per nanovesicle through simple vortex-mixing with preformed nanovesicles. Ultimately, the
RNase H-encapsulated nanovesicle efficiently delivered SSOs with
RNase H into cultured
cancer cells, thereby eliciting the significantly higher gene knockdown compared with empty nanovesicles (without
RNase H) or a mixture of nanovesicles with
RNase H without encapsulation. These results demonstrate the great potential of noncovalently stabilized nanovesicles for the codelivery of two varying bio-macromolecule payloads for ensuring their cooperative
biological activity.