Non-viral Gene Therapy - Intracellular Delivery: Fundamentals and Applications - page 624

conjugated PEG (PEG-Ada) via host-guest interactions between b-CD units and Ada
groups, the pDNA-PEI-CD polyplexes clearly mediated transgene expression in
tissues like liver, kidney and lung of immune-compromised female nude mice bearing
subcutaneous PC3 tumors (Pun et al. 2004a ). In addition to formulations for systemic
administration, a recent study revealed that polyplexes formed by pDNA and PEI-CD
could be used for constructing multilayer structures which were able to mediate in
situ gene transfection (Hu et al. 2010 ). Besides the reduction in toxicity, modification
using CDs can endow the resulting polyplexes with flexible modulability via
inclusion complex formation. For instance, in vitro and in vivo stability, prolonged
bloodstream circulation as well as targeting capability can be achieved using this
strategy, which have been well demonstrated by Davis'group (Davis 2009 ).
For non-viral delivery vectors, the limited endosomolytic capacity is one of the
important factors responsible for their relative low transfection efficiency compared
with viral systems. Covalently linking melittin to PEI25 could significantly increase the
intracytoplasmic release of DNA, due to the membrane lytic activity of melittin (Ogris
et al. 2001 ). Compared with PEI25, the transfection efficiency of this conjugate was
strongly increased within a broad range of cell lines (such as different tumor cells, primary
hepatocytes, and human umbilical vein endothelial cells). It was found that melittin not
only could increase the endosomal release of DNA-loaded polyplexes, but also could
enhance their intranuclear transport. The non-immunogenic all-(D)-melittin, a stereoi-
somer of natural all-(L)-melittin, is also effective to mediate enhanced gene expression
similar to the natural one (Boeckle et al. 2005 ). All-(D)-melittin conjugates of PEI
displayed up to 160-fold higher luciferase activity than unmodified PEI. The site of
melittin linkage strongly influenced the membrane-destabilizing activities of both
conjugates and polyplexes. C-terminus conjugated PEI (C-mel-PEI) is highly lytic at
neutral pH, and therefore increased doses of C-mel-PEI polyplexes elicited high toxicity.
In contrast, N-terminus conjugated polycation (N-mel-PEI) was less lytic at physiological
pH but retained higher lytic activity than C-mel-PEI at endosomal pH. This apparently
implies that N-terminus conjugation of melittin is preferred for a better transfection.
To overcome the inherent lytic activity of melittin at neutral pH that may provoke high
cytotoxicity, acidic modification of melittin was implemented by replacing neutral
glutamines with glutamic acid residues. Through this protocol, the lytic activity of
C-mel-PEI at endosomal pH was greatly improved (Boeckle et al. 2006 ).

3.2.6 Modification Using Hydrophobic and Amphiphilic Segments

Hydrophobic modification of cationic polymers has been demonstrated to be a useful
strategy to developing new gene vectors with improved delivery characteristics (Incani
et al. 2010 ). Since lipids are the main component of cell membrane, incorporation of
hydrophobic moieties may result in additional hydrophobic interaction between poly-
plexes and cell membranes, which in turn facilitates the delivery of genetic payload
into cells. In addition, the amphiphilicity of hydrophobically modified polycations
possess the self-assembly potential for constructing complex structures with advanced
functions. Table 6 lists the frequently used hydrophobic moieties.