Gene Therapy in Bone Regeneration: A Summary of Delivery Approaches for Effective Therapies - Intracellular Delivery: Fundamentals and Applications - page 831

causes membrane destabilization at low pH permitting endocytic escape. While
penton base complexes have been shown to follow multiple endocytic routes (as
with native adenovirus), it is thought that the complexes that contribute to nuclear
delivery follow a clathrin-mediated endocytic route (Rentsendorj et al. 2006 ). Also
similar to native adenovirus, penton base proteins seem to be capable of utilizing
microtubules and dynein to traffic to the perinuclear region, potentially making
them useful for not only vesicular escape but for increasing nuclear uptake as well
(Rentsendorj et al. 2006 ). The RGD peptide sequences found in integrin-binding
adenoviral proteins have been also incorporated into synthetic carriers; however,
this approach met with mixed success. Linear RGD modification of PEI was shown
to be beneficial for transgene expression in a melanoma cell line, which appeared
to be due to increased complex attachment to the cell surface (Kunath et al. 2003 ).
However, our own group found that modifying PEI (branched, 25 kDa) with RGD
did not increase polymer binding to bone marrow stromal cells (Clements et al.
2006 ). Attaching RGD to non-cationic polymers (i.e., polymers unable to interact
with anionic cell surface), on the other hand, were shown to be beneficial in
increasing nucleic acid delivery (Xiong et al. 2010 ). As much as the ligand, the
polymeric carrier used for complex formation seems to be critical for the ultimate
success of this strategy.
Unlike peptide-mediated specific uptake mechanisms, it is possible to take a
non-specific approach to enhance cellular crossing of the complexes by imparting
a hydrophobic character to the carriers. This could be achieved by lipid substitution
(Incani et al. 2009 ). Modifying cationic polymers, such as PEI (Neamnark et al.
2009 ) and PLL (Abbasi et al. 2007 ), with various lipids resulted in increased trans-
fection efficiency, in parallel with increased uptake of polymer/plasmid
complexes.

3.3.2 Nuclear Import

While many carriers can successfully achieve cytoplasmic localization, nuclear
targeting remains a major rate-limiting step. This is especially true in cells with low
mitotic activity where carriers face the barrier of an intact nuclear envelope. Entry
into the nucleus is regulated by NPC and, although small segments of DNA (1 Kb)
can enter the nucleus unassisted (Hagstrom et al. 1997 ), DNA larger than 2 Kb
mostly remains in the cytoplasm. The NPC is generally regarded to be permeable
to molecules up to 39 nm (Pante and Kann 2002 ). Considering that the plasmid
DNAs for typical therapeutic genes are several Kbs in size and form complexes
>100 nm in size with carriers, passage through the NPC is a major challenge.
Movement from the cytoplasm to the nucleus can be a passive diffusion process, or
an active process based on the properties of the plasmid carrier. Some polymeric
carriers, such as PEI and PLL, appear to have the intrinsic affinity to nucleus
(Farrell et al. 2007 ), which lipid-based systems lack (Pollard et al. 1998 ). Cationic
lipids appear to be dependent on cell mitosis, and dissolution of the nuclear mem-
brane, for efficient nuclear delivery of plasmid DNA and transgene expression