Challenges and Opportunities in Gene Delivery - Challenges in Delivery of Therapeutic Genomics and Proteomics - page 59

words, DNA-polycation complex internalization followed by subsequent transgene
expression, is likely to occur through the multistage process of the binding of DNA-
polycation complex to the cell surface, followed by entry into cell cytoplasm and the
nucleus. The binding of DNA-polycation complex with cell surface proteoglycans
results in the two-dimensional aggregation of these cell surface proteins, which acts
as a strong stimulus for complex internalization by cell endocytosis. The extent of
internalization of this complex in the cell, and the subsequent transgene expression
and cytotoxicity of the cells caused by the complex, generally run parallel to one
another. A general association between cytotoxicity and transfection suggests essen-
tial membrane damage for successful DNA entry into the cytoplasm [113] . However,
for successful and clinically beneficial transfection, one should be able to correctly
balance sufficient DNA entry into cytoplasm without causing excessive and nonre-
versible cell damage.
The process of endocytosis observed on the cell surface may be either nonclathrin
mediated, as in the case of endocytosis of low-density lipoprotein [114] , or adsorp-
tion mediated with subsequent internalization process, as in the case of cationic lip-
ids and polymers [115] . The internalization of the DNA-polycation complex has also
been shown to be mediated through clathrin-coated pits and has been widely reported
[116] . The cellular internalization by DNA-polycation complex in the cell lines
has also been found to occur by phagocytosis [117] . The phagocytosis of the DNA-
polycation complex by caveolae-mediated internalization has also been observed and
has been demonstrated by enhanced folate receptor-mediated cell uptake in tumor
cell lines by using folate-attached complexes that are internalized through folate
receptors richly present in the caveolae [118] .
The presence of some overexpressed receptors on particular cell types allows pref-
erential targeting by enhanced cellular internalization of the ligand-attached complexes
and delivery systems through receptor-mediated endocytosis after the ligand-receptor
interaction. These ligands can be small molecules (e.g., folate, galactose) or peptides
and proteins (e.g., transferrin and antibodies), and they are internalized through their
specific receptors overexpressed on the cell surface. A large number of systems have
been studied involving transferrin for tumor cells targeting [34,84] and galactose for
hepatocyte targeting [102] . Binding of V and 5 integrins of the cell surface to
the tripeptide Arg-Gly-Asp (RGD) sequence attached to the transfection complex
enhances transfection in vitro by promoting cell internalization following integrin
binding [87] .
Macropinocytosis is another internalization process observed during uptake of
nonviral gene delivery vectors because of its ability to internalize larger structures
such as bacterium [119] . However, in comparison to phagocytosis, macropinocytosis
is a rare event and has been observed in fewer mammalian cell lines [119] .
Currently, the relative influence of these various internalization pathways on cell
uptake is not completely understood. However, it can be said that, in different cell
types, different mechanisms operate simultaneously but to a different extent in inter-
nalizing DNA-polycation complex.