Biomedical Engineering Reference
In-Depth Information
membrane-disruption agents. PEI-mediated gene delivery has been achieved in numerous
in vitro
and
in vivo
models [100].
A disadvantage of the use of PEI as a transfection agent is its nonbiodegradable nature.
Toxicity has also been associated with the excessive positive charges on the polymer [98].
Shielding the PEI/DNA complexes with PEG, similar to liposomes, does significantly reduce
toxicity [101]. Further targeting of the system can be achieved by the addition of ligands
either with or without the PEG shield [102, 103].
Polyamidoamine cascade polymers, or Starburst dendrimers, are another highly posi-
tively charged polymer. They are spherical and highly branched (see Figure 11.3), with
varying degrees of branching forming the different generations of dendrimer that are com-
mercially available. Like PEI, their high amine density enables neutralization of the acid
pH within the endosomal vesicle, allowing DNA to escape degradation. They have been
shown to be effective as a transfection agent, without the need for additional endosomolytic
agents [104].
11.2.5 Assessing the physical properties of a non-viral vector
Physical characterization of the non-viral vector is an important initial step in the develop-
ment of non-viral gene delivery systems. The size and surface charge of the vector/DNA
particle are both crucial parameters, influencing the passage of the DNA through the cell
and its ultimate expression. Preliminary experiments to explore these characteristics are
relatively easy and highly worthwhile in order to enhance future understanding and aid
optimization of the delivery system.
Simple mixing of the polycationic delivery agent and the DNA results in electrostatic
binding, condensation and charge neutralization of the DNA to form particles (see Proto-
cols 11.5 and 11.6). Visualization of neutralization can be demonstrated through a DNA
retardation assay (Figure 11.4a). Inhibition of electrophoretic mobility indicates when suf-
ficient vector has been added to the DNA to neutralize charges. Particle formation and
subsequent size and charge can be more readily assessed using a technique called dynamic
light scattering (DLS), alternatively called photon correlation spectroscopy. This measures
the scattering of laser light upon a population of particles, resulting in values of average
particle diameter (
Z
-average) and average net surface charge of a particle (zeta potential).
It also establishes some level of homogeneity of the particle population (polydispersity). It
is not, however, quantitative for particle numbers or relative numbers within a heteroge-
neous population. Figure 11.4b shows the size profile for a peptide-DNA complex, giving
its average diameter (
Z
-average) and an indication of the level of homogeneity within the
sample of particles (polydispersity, range 0-1).
The cation-to-anion ratio (also calculated in terms of the N : P ratio, i.e. amine to phos-
phate) within the complex is an important consideration, with a slightly positive net charge
being the optimal for delivery to cell lines
in vitro
. DLS is capable of measuring the net
surface charge of a complex, which unquestionably plays a key role in initial binding to the
cell surface. The zeta potential reading is a value in millivolts depicting the movement of
the particles within an electric field, giving a positive or negative value depending on its
speed and direction of movement. Figure 11.4c shows the net surface charge of the same
peptide-DNA complex prepared in different solutions. The surface charge has been found
to be highly influenced by the presence of salts within the buffer.
