Biomedical Engineering Reference
In-Depth Information
Okino
et al
. [20] used EP for
in vivo
delivery of chemotherapeutic agents to solid tumors.
Since then EP has been used in humans and animals for the delivery of drugs and genes. EP
is now frequently used for plasmid DNA gene transfer as an alternative to delivery with viral
vectors or lipid-soluble products. Heller and Heller [21] have listed the numerous reports
of using EP to deliver many types of transgenes genes into multiple cell types and organs
to test their efficacy in reducing cancer and immune reactions. EP is an efficient method of
increasing gene expression several fold over direct injection of naked DNA (plasmids).
EP is not a Trojan horse but a bombardment to breach the cell wall. The mechanism
of EP delivery is straightforward. Electrodes use voltage or current in pulses to make the
membrane of cells semi-permeable by briefly opening pores in the lipid membrane of the
cell without doing permanent damage. Molecules that are usually excluded from entering
the cell (hydrophilic, non-lipid-soluble or anionic molecules) can physically pass through
the pores. The challenge is to find the right parameters for different genes, cell types and
purposes. The voltage (volts), current (amps), pulse width (short or long), pulse interval
(seconds), pulse shape (square, bipolar, exponential), pulse number (single, multiple, trains)
and frequency of stimulation can all affect the outcome. Long low-voltage pulses yield high
expression in muscle but not in cancer cells. To avoid direct current damage, high field
strength pulses (
>
700 V/cm) are usually mixed with short pulses (microseconds) and low
field strength (
<
700V/cm) by long pulses (9ms). Several systems are sold commercially.
If in doubt, refer to the numerous papers which have reported enhanced DNA delivery each
with their own set of parameters. For a review, see Heller and Heller [21].
12.1.2.3 Liposomes
DNA is negatively charged. Cationic liposomes bind to DNA and form a liposome-DNA
complex, which fuses with lipid membranes of the cell and allows DNA to enter. Many
purified cationic liposomes are commercially available and are widely used (e.g. Lipofec-
tamine, DOPE, DOTAP). They are best for
in vitro
delivery into cells, but are used for
in
vivo
delivery, although there has to be a check for complement activation or toxicity.
12.1.2.4 Dendrimers
Dendrimers have the potential for highly efficient gene delivery [22]. They are a class of
synthetic spherical polymers which are highly branched. They can be made to almost any
size and are stable and soluble in water. They have a positive charge on the surface to bind
to lipid cell membranes and carry negatively charged DNA on the inside. They enhance the
delivery of DNA and RNA, single- and double-stranded. They have been shown to enhance
the delivery of plasmids-mediated gene transfer in the heart [23]. A DNA : dendrimer ratio of
1 : 20 was used to show the uptake and expression of
β
-galactosidase in mouse hearts [23].
12.1.3 Viral delivery
Gene modification for cell transplantation has mainly focused on transferring genes or
recombinant genes in cassettes by viral delivery for high transfection efficiency and high
expression level that can be long lasting. Several reviews and topics have adequately covered
the pros and cons of different viruses as vectors [24] and so these will only be briefly
summarized here. The main issues to consider are: (i) whether the cells to be transfected
are dividing or non-dividing; (ii) the carrying capacity of the virus - that is, whether the
