Non-viral Gene Therapy - Intracellular Delivery: Fundamentals and Applications - page 632

a targeting ligand can be introduced onto the PEG shell of assemblies containing
gene segments, by using a transferrin-PEG-Ada conjugate (Bartlett and Davis
2007 ). In vivo transfection experiments in both murine models and non-human
primates showed that the ligand functionalized nano-assemblies can efficiently
deliver the gene payload to tumor tissues, and perform their therapeutic efficacy
after intravenous injections (Pun et al. 2004b ; Hu-Lieskovan et al. 2005 ; Heidel
et al. 2007 ). These studies also confirmed the safety of these non-viral nanoparti-
cles for systemic administrations.
Taking advantage of the host-guest interactions between b-CD units and Ada
cationic derivatives, Burckbuchler et al. were able to construct a pseudo-polycation
functioning as a vector for gene delivery (Burckbuchler et al. 2008 ). Gel retarda-
tion, z-potential and surface enhanced Raman spectroscopy (SERS) experiments
demonstrated the formation of polyplex assemblies by host-guest polycations and
pDNA encoding a luciferase gene. In addition, gel retardation and small angle
neutron scattering (SANS) measurements suggested the stability of assembled
polyplexes was mainly related to the chemical structure of guest molecules.
The polyplexes based on the polycation of Ada3 and Ada4 were relatively stable
under higher NaCl concentrations. In vitro transfection was performed on HepG2
and KEK293 cells. The optimized polyplexes based on Ada4 exhibited a transfec-
tion efficiency with the same order as that of DOTAP, a commercialized transfec-
tion agent. Nevertheless, the former transfection was carried out in the presence of
the fusogenic peptide JTS-1.
CDs based polypseudorotaxanes (CDPRs) are a family of polymers with CDs
threading onto the polymer chains. Since their dependent discoveries by several
groups (Ogata et al. 1976 ; Harada and Kamachi 1990 ; Wenz and Keller 1992 ), there
have been overwhelming interests on the developments of functional polymer
materials and delivery systems based on CDPRs (Harada et al. 2006 ; Huang and
Gibson 2005 ; Araki and Ito 2007 ; Loethen et al. 2007 ; Li and Loh 2008 ; Li 2009 ).
Nevertheless, the applications of CDPR polymers for gene delivery emerged as a
new direction more recently. Yui et al. synthesized a biocleavable polyrotaxane
(Ooya et al. 2006 ), in which dimethylaminoethyl-modified a-CDs were threaded
onto a PEG chain capped with benzyloxycarbonyl tyrosine via disulfide linkages.
AFM observation together with gel electrophoresis and x-potential measurements
confirmed the formation of polyplexes as well as the successful condensation of
pDNA. The cleavage of disulfide linkages under reducible conditions in cytosolic
milieu facilitates the de-condensation and intracellular trafficking of pDNA cargo.
In vitro transfection revealed that the assembled polyplexes can rapidly escape from
endosome and deliver pDNA to the nucleus. On the other hand, Li et al. designed
cationic CDPRs with multiple oligoethylenimine-grafted CDs threaded on a PEO-
PPO-PEO chain (Fig. 2 ) (Li et al. 2006 ). These cationic polyrotaxanes with different
oligoethylenimines can efficiently condense pDNA into nanoparticles, which was
confirmed by agarose gel electrophoresis, particle size, and x-potential measure-
ments. Importantly, these supramolecular polycations are less toxic that PEI25 for
cultured L929 and HEK 293 cells. Transfection studies using luciferase as a marker
gene in HEK293 showed that the efficiency of assembled polyplexes was even