Delivery to Intracellular Targets by Nanosized Particles - Intracellular Delivery: Fundamentals and Applications - page 86

A positively charged polysaccharide, chitosan, has also been used to complex
nucleic acids. Gene silencing in H1299 human lung carcinoma cells has been
achieved (Liu et al. 2007 ). Cyclodextrins, molecular cages of glucose units, are not
positively charged but can be coupled to a cationic polymer to form effective sys-
tems for complexing nucleic acids. When modified with PEG and a targeting moi-
ety (again transferrin) these particles show gene knock-down in vitro and inhibition
of the EWS-Fli-1 gene in Ewing's sarcoma in vivo (Bartlett and Davis 2007 ;
Hu-Lieskovan et al. 2005 ).
Micellar systems have also been used to deliver nucleic acids to intracellular
compartments. Examples are PEG-poly (aspartic acid) copolymers (Kakizawa et al.
2004 ) which can exert a “proton sponge” effect in endosomes, lactosylated PEG
which can form complexes with poly (lysine) and AS-ODNs and allow targeting to
galactose receptors on liver cells (Oishi et al. 2005 ; Oishi et al. 2007 ) and PEG-
PMMA block copolymers (Kakizawa et al. 2006 ) which can be used to produce
hybrid organic-inorganic nanoparticles.
The adsorption of nucleic acids onto preformed nanoparticles requires a posi-
tively charged surface. The first systems described used cationic surfactant on the
surface of poly (alkylcyanoacrylates) (PACA) (Fattal et al. 1998 ) or poly (lactide-
co-glycolide) (Singh et al. 2003 ; Oster et al. 2005 ). AS-ODN adsorbed onto PACA
nanoparticles are protected from nucleases (Lambert et al. 1998 ) and can be deliv-
ered intracellularly (Chavany et al. 1994 ). These formulations were found to be able
to inhibit the proliferation of mutated Ha-ras-transformed cells proliferation and
reduce tumorigenicity in nude mice (Schwab et al. 1994 ). More recently, cationic
nanoparticles made up of a biodegradable core of poly (isobutylcyanoacrylate) sur-
rounded by a shell of chitosan were prepared by de Martimprey et al. ( 2008 ).
A siRNA directed against the Ret/PTC1 junction oncogene was adsorbed onto
these particles and was able to silence the gene in papillary thyroid carcinoma cells
and exert an anti-tumor effect in mice.
PLGA-based nanoparticles have also been modified to provide a cationic surface.
Notably, Nafee et al. ( 2007 ) coated them with chitosan and were able increase intra-
cellular uptake of AS-ODN into A549 lung cells. Cationic nanoparticles have also
been prepared by co-polymerization of methylmethacrylate and aminoalkyl-
methacrylate monomers (Zobel et al. 2000 ). These nanoparticles can also promote
intracellular accumulation of AS-ODN (Tondelli et al. 1998 ; Zobel et al. 2000 ).
Despite the fact that adsorption onto the nanoparticle surface has produced some
results with the intracellular delivery of nucleic acids, better protection from nucle-
ases would be obtained if the nucleic acids were incorporated within the interior of
the particles. However, the hydrophilic character of the nucleic acids is not compat-
ible with the hydrophobic polymers; Thus, Lambert et al. ( 2000 ) developed a
method of producing nanocapsules (NC) with an aqueous core and a PIBCA shell
which could encapsulate hydrophilic molecules such as nucleic acids. These nano-
capsules were able to protect AS-ODN from nuclease degradation and showed
evidence of intracellular uptake by their ability to transfect cells, including vascular
smooth muscle cells, which are notoriously difficult to transfect (Toub et al. 2005 ).
The same system was also able to deliver siRNA to cells (Toub et al. 2006 ).
Fluorescent labeling showed a punctate pattern of siRNA within the cells, suggesting