Non-viral Gene Therapy - Intracellular Delivery: Fundamentals and Applications - page 668

To achieve an enhanced anticancer therapy, a combination of therapeutics of
proapoptotic peptide D-(KLAKLAK) (2) and Bcl-2 antisense ODN G3139 was
simultaneously delivered using cationic liposomes by Torchilin and coworkers (Ko
et al. 2009a ). For the preparation of these multifunctional liposomes, the cationic
peptide D-(KLAKLAK) (2) was first compacted with the anionic ODN G3139 to
form negatively charged peptide/ODN complexes, which were then incorporated
into DOTAP/DOPE cationic liposomes (CL). In vitro treatment of mouse mela-
noma B16(F10) with CL containing D-(KLAKLAK)(2)/G3139 showed signifi-
cantly enhanced antitumor efficacy, mediated by stimulated induction of apoptotic
(caspase 3/7) activity, when compared to CL loaded with G3139 alone. Intratumoral
injection of this dual-delivery formulation in 1,316(F10) mice xenograft also
resulted in suppressed tumor growth associated with enhanced apoptotic activity.
Accordingly, the simultaneous delivery of proapoptotic peptide D-(KLAKLAK)(2)
and antisense ODN G3139 via cationic liposomes could give rise to enhanced
apoptotic/antitumor efficacy, which may provide a promising tool for cancer
treatment.
In addition, multifunctional nanoparticles that combine gene delivery with the
ability to cross tissue and membrane barriers can function as ideal non-viral vectors
for gene therapy. One of the first indications of the potential of multifunctional
platforms for gene delivery is the work performed by Pardridge and coworkers
(Zhang et al. 2003 ). With the aim to transfer exogenous genes to the entire retina,
83-14 MAb antibody was conjugated to the PEGylated liposomes. After systemic
administration to rhesus monkeys, these 'stealthy' immunoliposomes were targeted
across the blood-retinal barrier, resulting in specific gene expression in the eye by
using the opsin promoter. On the other hand, targeting tumor cells or ischemic
myocardium by liposomes has been achieved via conjugating TAT peptide and/or
anti-myosin antibody by Torchilin's group (Kale and Torchilin 2007 ; Gupta et al.
2007 ; Ko et al. 2009b ).
Harashima and coworkers have created a multifunctional envelope-type nano
device (MEND) as a non-viral gene delivery system (Kogure et al. 2008 ). The ideal
MEND consists of a condensed DNA core and a lipid envelope structure equipped
with the various functionalities such as longevity and targetability. The condensa-
tion of DNA by cationic polymers (such as PLL) allows protection of DNA from
DNase, size control and improvement in packaging efficiency. To control topology,
polyplexes are incorporated into lipid envelopes by hydration of a lipid film with
subsequent sonication such that the DNA core and lipid envelope exist as separate
structures, rather than a disordered mixture. The packaging mechanism was based
on electrostatic interactions between DNA, polycations and lipids. As an example,
pDNA was first condensed with a polycation such as PLL by vortexing. The kinetic
control of this process is important to control the size and charge of the resulting
polyplexes. In the case of PLL, small (around 100 nm) and positively charged
(around 30 mV) PLL/DNA complexes were prepared at a N/P ratio of 2.4. The lipid
film containing a negatively charged lipid such as cholesteryl hemisuccinate was
hydrated in an aqueous solution containing PLL/DNA polyplexes. The packaging
of the PLL/DNA nanoparticles into a lipid bilayer was achieved by sonication.