Biomedical Engineering Reference
In-Depth Information
was related to the ability of the injected DNA plasmids to enter the epithelial cells
by both endocytosis and the intrastromal pressure effect due to volume change from
injection of isotonic buffer. The expression of plasmids expressing small interfering
RNA (siRNA) against VEGF was also achieved after injecting them into the corneal
stroma of mice [31] . Intrastromal injection has been suggested mainly to treat acute
corneal diseases [29] , because the durations of gene retention and expression in the
cornea after intrastromal injection were found to be relatively short irrespective of
the rapid expression.
The mechanism for naked DNA-mediated gene transfer is not yet clear. It was
suggested that naked DNA is taken up by parenchymal cells in vivo by an active,
receptor-mediated process [32] . The hypothesis is based on results showing that intra-
vascularly administered DNA is presented to hepatocytes immediately after injection,
but DNA had reached only 5-10% of hepatocytes after 1 h of DNA injection. Also,
coinjection with excess polyanions inhibits both DNA uptake and expression, indi-
cating competitive inhibition of DNA uptake by polyanions. Applying high pressure
in intravascular injections by administering higher volumes enhances gene expres-
sion, most likely by facilitating egress of plasmid DNA out of blood vessels [32] .
Although naked DNA delivery has shown promise in smaller animals, its real
clinical benefit will be fulfilled only if reproducible significant gene expression is
achieved in bigger animals. To achieve such transfection, an external physical force
is required. Subsequent sections of this chapter describe the use of various physical
methods employing external force for enhanced DNA expression.
3.3 Electroporation
Despite some promising early results, gene therapy has not yet fulfilled expectations
because of inefficient gene transfection even after advances in the control of gene
expression and selection of therapeutic genes and expression systems. Hence, devel-
opment of a more effective and safer method of gene transfection in vivo is of utmost
importance for clinically relevant gene delivery. Naked gene delivery without any
physical force and vector has been a disappointment, with therapeutically low gene
transfection, and the use of vectors and methods employing physical force has been
employed to enhance gene expression.
EP has proved to be a successful method for drug delivery across the skin in vitro
and in vivo and has also been used as an effective in vitro gene delivery system in pro-
karyotic and eukaryotic cells. It employs physical force to import therapeutic drugs
and macromolecules, such as DNA and proteins, from extracellular compartments
into cells having transiently increased cell membrane permeability caused by short,
pulsed electric field application, temporarily disrupting the structural integrity of cell
membranes. This electric pulse is applied to the cells between the electrodes to form
small membrane pores across the cell membrane within 3 milliseconds (ms), which
may increase up to 120 nm within 20 ms [33] . These formed pores are transient in
nature, and they reseal within a few seconds to minutes, without causing any signifi-
cant damage to the cell membrane of the exposed cells. During this period of time, a
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