Biomedical Engineering Reference
In-Depth Information
activating gut immunity in Japan. Recent work has revealed that the
immunostimulatory nature of b-glucans is related to the effector functions of
leukocytes as well as the inflammatory processes. Recently, dectin-1 was
identified as a major receptor involved in the recognition of b-glucans.
38,39
Dectin-1 was originally found as a dendritic cell (DC)-specific receptor and
thus it was named as ''dendritic-cell-associated C-type lectin-1''. However, it
was revealed that dectin-1 is expressed by many other antigen presenting cells
(APCs), including macrophages, monocytes, neutrophils, and a subset of T
cells.
40
The APC binding ability of b-1,3-glucans implies that the glucans can
specifically deliver the bound oligonucleotides to APCs.
We are investigating the fundamental properties of this novel polysacchar-
ide/polynucleotide complex as well as developing a new method for DNA
delivery using this complex. The present chapter reviews the characterization
of
d
n
4
y
3
n
g
|
0
the
complex,
and
its
application
to
delivery
of
CpG
and
antisense
oligonucleotides to the APCs in vitro and in vivo.
8.2
Characterization of the SPG/DNA Complex
8.2.1 Preparation of the SPG/DNA Complex
SPG (M
w
5 1.5 6 10
5
as the single chain, determined using gel-permeation
chromatography coupled to multi-angle light scattering analysis) was dissolved
in 0.25 N NaOH
aq
for more than 2 days to completely dissociate the triple
helix to single chains. All DNA samples were purified with high-performance
liquid chromatography. In this work, we used not only a phosphodiester but
also a phosphorothioate backbone. Among various nucleotide analogs,
phosphorothioate, in which one oxygen atom of the phosphodiester moiety
is replaced by sulfur, have improved nuclease resistance and cellular uptake.
6,41
The SPG solution, ODN (dA
60
) in water, and phosphate buffer solution
(330 mM NaH
2
PO
4
,pH5 4.7) were mixed. After mixing, the pH was
controlled around 7 and the mixture was stored at 4 uC overnight.
8.2.2 Solution Properties and Characterization
Figure 8.2A shows size exclusion chromatography (SEC) chromatograms for
triple-helix SPG (tSPG), dA
60
/SPG, and dA
60
detected with a light scattering
(LS) photodiode at 2h 5 90u (upper left), the refractive index (RI)
refractometer (middle left), and the UV spectrometer at l 5 260 nm (bottom
right).
42
The weight-averaged molar mass was determined for each fraction
and plotted against the elution time (upper right). Since tSPG has no
absorption at 260 nm, whereas dA has, UV chromatograms were only
observed for dA
60
/SPG and dA
60
. For the main peaks of dA
60
/SPG in the RI
and UV chromatograms, the peak-top positions and their shapes were very
similar but not identical: the RI peak was eluted more forward (i.e., higher
molecular weight) than the UV peak and showed tailing to the low molar-mass
