Challenges and Opportunities in Gene Delivery - Challenges in Delivery of Therapeutic Genomics and Proteomics - page 58

2.3.2.6 Cell Surface Receptor Interaction and Cellular Uptake of the
DNA Delivery System by Target Cells Through Endocytosis
2.3.2.6.1 Interaction of DNA-Polycation Complex with the
Cell Surface
The interaction of the DNA and DNA-polycation complexes to the cell surface is either
mediated by an ionic charge-based interaction or by ligand-receptor interactions. The
mammalian cell membrane is covered by the heparin sulfate proteoglycans (HSPGs)
having a strong negative charge surface. The proteoglycans are characteristic of the cell
surface and are involved in various cell processes such as cell differentiation, adhesion,
and migration [93,94] . The proteoglycans are also known to be involved in virus inter-
nalization, including HIV-1 [95] , HSV-2 [96] , AAV-2 [97] , and adenovirus [98] . The
composition of HSPGs includes a protein core with one or more attached glycosami-
noglycan moieties. Proteoglycans have been found to exist either as integral membrane
proteins, membrane-associated glycosylphosphatidylinositol (GPI)-linked proteins, or
as extracellular proteins closely associated with the cell surface.
The DNA itself has a strong negative charge, and its interaction with the cell sur-
face has been reported to be very low because of the relatively high negative charge
density of both the DNA and the cell surface. Positively charged polycations are
known to increase the cellular internalization of DNA by neutralizing the negative
charge of DNA, with the charge ratio of the complex modulating the extent of this
contact. These positively charged DNA-polycation complexes are known to interact
favorably with negatively charged cell surface HSPGs and hence help in their cellular
internalization process. The cellular interactions of the lipid-DNA complex are also
dependent on the colloidal stability of these delivery systems. The stable complexes
in colloidal range are shown to be more associated with cell surface than the aggre-
gates [99] . Improper expression of HSPGs on the cell surface, either by enzymatic
removal or metabolic inhibition of their biosynthesis, has resulted in a decreased
amount of DNA-polycation complex interaction with cells, with a subsequent reduc-
tion in transgene expression in vitro [100] .
The advancements in technology and knowledge have helped researchers to
increase the amount of DNA interacting with the cell surface and enhance the
cellular targeting. As detailed earlier, conjugation of various targeting ligands to
cationic lipids or polymers tends to increase the degree of cellular association and
internalization of DNA both in vitro [101-103] and in vivo [104-107] . Also, cova-
lent conjugation of targeting ligands directly to DNA has also been proposed [108] .
The use of physical methods to concentrate complexes near the cell surface has also
been reported. Use of poly(ethylene glycol) (PEG) [109] , colloidal silica [110] , and
centrifugation of the DNA delivery systems in cell culture plates in vitro [111,112]
for DNA concentration near cell surface have all resulted in improvements in gene
delivery in vitro .
2.3.2.6.2 Intracellular Receptor-Mediated Cell Uptake
Considering the electrostatic interaction of the negatively charged cell surface and
the positively charged DNA-polycation complex, the process of transfection, in other