Biomedical Engineering Reference
In-Depth Information
(b)
(a)
60
%
in
class
40
20
Zav 723nm
Polydisp. 0.2
1:0
1:0.1 1:0.2
1:0.5
1:1
1:2
1:3
10
100
1000
Diameter (nm)
(d)
(c)
40
30
Zeta
Potential
(mV)
20
10
0
−
10
5% Dextrose
5% Dextrose
PBS
10 mM tris
Figure 11.4
Physical characterization of vector-DNA complexes. (a)
DNA retardation assay
.
The (Lys)
16
-molossin peptide [91] was added to pGL3 plasmid DNA, containing the luciferase
reporter gene, in PBS at the various w/w ratios indicated and loaded on to an 0.8% agarose gel
after 30 min (loaded at arrow). Electrophoretic mobility is reduced as the vector concentration
increases. Charge neutralization occurs at a peptide/DNA w/w ratio of
∼
1:1. (b)
Size analysis
of peptide-DNA particles by DLS
. (Lys)
16
-molossin peptide mixed with pGL3 plasmid DNA at a
ratio of 3 : 1(w/w), at 10
μ
g/ml DNA, in PBS, analyzed at 30 min on a zetasizer 3000HS (Malvern
Instruments, Malvern, UK). Average diameter (Zav) of the particles is 723 nm with a polydispersity
of 0.2 (range 0-1) indicating a relatively homogeneous population. (c)
Zeta potential analysis of
peptide-DNA particles by DLS
. Peptide-DNA complexes prepared as for (b) but in PBS, 5% dextrose
or 5% dextrose 10 m
M
Tris solution. Net surface charge measured on the zetasizer 3000HS at
30 min. In the presence of salt the charge is substantially reduced. (d)
Macropinocytosis
of peptide-DNA complexes viewed by transmission electron microscopy
. (Lys)
16
-molossin-DNA
particles prepared as in (b) but using biotin-labeled peptide and conjugated to 10 nm streptavidin
gold, were transfected into HUH7 hepatoma cells. After a 4 h incubation the cells were fixed and
processed. Two particles are visualized, both with a halo of gold beads. One inside the cell is
surrounded by a vesicle and the other is entering through the cell membrane. The bar is 500 nm.
Liposomes themselves have also been used together with other non-viral vectors, such as the
arginine-glycine-aspartate (RGD)-containing peptides [75-77] and some viruses, including
adenovirus [78] and the hemaglutinating virus of Japan [79], to improve DNA delivery.
Some cell toxicity and acute inflammation reactions have been reported in both cell and
animal models [80], as well as following clinical trials [81], which currently pose limitations
on the success of lipid-mediated gene therapy in the clinic.
