Polymeric Micelles for siRNA Delivery - RNA Interference from Biology to Therapeutics - page 169

Hydrophobic forces were also utilized to stabilize the micelles by Kim et al. who
reported the development of hydrophobic polycations by using PAsp(DET) (poly{ N -
[ N -(2-aminoethyl)-2-aminoethyl]aspartamide}) as the backbone polycation and
stearoyl groups as a hydrophobic moiety to facilitate core formation [ 64 ] . To opti-
mize the interaction between polycations and siRNA, stearoyl PAsp(DET) with dif-
ferent substitution degrees were synthesized and characterized for siRNA complex
stability and RNAi activity in cultured cells. The stearoyl introduction onto
PAsp(DET) side chains led to the complex stabilization via hydrophobic interaction
and micelles presented low cytotoxicity. Additionally, the PAsp(DET) polycations
backbone contributed to the excellent endosomal escape of siRNA complexes,
resulting in an improved RNAi activity in vitro. The in vivo application of micelles
comprising stearoyl PEG-SS-PAsp(DET) complexed with siRNA presented longer
stability in the blood as verified by intravital real-time confocal laser scanning
microscopy [ 65 ]. The extended circulation time of PEGylated micelles that were
hydrophobically stabilized allowed effective passive accumulation in solid tumors.
The significant in vivo gene silencing through the use of this smart siRNA complex
after systemic administration strongly indicates successful siRNA delivery.
Inorganic/organic interactions can facilitate the formation of nanoparticles and
the encapsulation of siRNA. The precipitation of inorganic salts apparently has the
electrostatic force as the main driving force. When mixed in an aqueous environ-
ment, inorganic compounds precipitate to form insoluble particles, which soon
aggregate with each other. In this case, physical barriers surrounding the salt are
needed to avoid further aggregation and crystal growth. The use of additives to pro-
vide colloidal stability through steric repulsion of the precipitates includes block
copolymers and other molecules.
Interestingly, negatively charged nucleic acids are entrapped in inorganic nano-
particles during wet precipitation. Inorganic/organic hybrid nanoparticles carrying
siRNA hold great promise for clinical application since they are easily prepared,
water dispersible, and stable in biological environments. In addition, due to physi-
ological stability, hybridizing inorganic nanoparticles with organic compounds such
as polymers as the physical barrier to aggregation offers a potential alternative for
effective nanocarriers [ 66- 68 ]. This hypothesis was realized through the use of
PEG-polyanion as the physical barrier to calcium phosphate nanoparticles aggrega-
tion in the report of the first calcium phosphate hybrid system [ 51 ] . Kakizawa et al.
obtained stable PEGylated hybrid nanoparticles as a low cytotoxic nanocarrier of
antisense DNA.
The first application of hybrid nanoparticles for siRNA delivery that involved
calcium phosphate (CaP) nanoparticles coated with poly(ethylene glycol)-block-
poly(aspartic acid) (PEG-P(Asp)) shows promising results [ 69 ] . Through a simple
mixing of separate solutions containing calcium, phosphate, siRNA, and PEG-
P(Asp), nanoparticles were formed having a size smaller than 100 nm. While the
loading capacity of siRNA reached almost 100% under optimal conditions, the par-
ticle size could be regulated to some extent by adjusting the PEG-P(Asp) concentra-
tion. Eventually, the intracellular environment with appreciably lowered calcium
ion concentration compared to the exterior allowed the release of the incorporated