Chemistry Reference
In-Depth Information
ecules was found in the presence of oleyl amine even after 15 days, three main
steps on the release profi le can be identifi ed when DNA is confi ned within the
pure nonionic columnar system. During the fi rst 3 days, no release is detected,
and this can be attributed to the lag time needed for DNA molecules to diffuse
out of the H
II
cylinders. In the range of 4-9 days the release of DNA from the
LLC system into the excess water begins, progressing at a relatively high rate,
as indicated by the sharp slope observed in Fig. 8.12. After 9 days the release
of DNA slowed down signifi cantly and reached a plateau. At this point a sig-
nifi cant amount of DNA, approximately 65% of the initial content, has already
been released by the nonionic H
II
mesophase. These results are consistent with
the conclusions based on SAXS and ATR-FTIR analyses stating that hydro-
gen bonds between DNA, water, and GMO head-groups are responsible for
the DNA confi nement within the water channels, in the case of the nonionic
hexagonal mesophase, which allows transport of DNA into the excess water.
In contrast, the presence of cationic oleyl amine in the ionic hexagonal meso-
phase leads to such a strong electrostatic confi nement of the DNA at the
water-lipid interface that release into the excess water is prevented: This for-
mulation is then more reliable for permanent encapsulation and protection of
the DNA rather than a controlled release.
In order to gain insight on the correlations between the release loads and
the molecular conformation of the DNA molecules released, single-molecule
atomic force microscopy (AFM) was used to provide the needed information
on DNA contour length distributions (Fig. 8.13). Figure 8.13a depicts the DNA
contour length distribution prior to the release experiment. As can be seen,
several DNA fragments are generated upon the enzymatic treatment and can
be divided into three main lengths: relatively small fragments (
100 nm
contour length), medium size DNA strands (130-160 nm), and large macro-
molecules (
=
190 to 200 nm). The contour length distribution of DNA after its
release from the nonionic system was examined on the third, fourth, and the
fi fteenth days of release, corresponding to the lag time, the sharp slope, and
plateau regions as identifi ed in the release profi le (Figs. 8.13 b - 8.13 d).
During the lag time, on the third day of the release experiment, only very
short-length DNA fragments with contour lengths below 40 nm could be
observed by AFM analysis (Fig. 8.13b). These very short strands were too small
and in too low concentration to be detected by the spectrophotometer, result-
ing in the fl at absorption signal. The following day (day 4), in correspondence
with the sharp increase in the diffusion as measured by the UV-visible absorp-
tion, the medium-size DNA strands, mainly 40-100 nm in contour length,
started to appear, coexisting with the very short DNA fragments (Fig. 8.13c).
Finally, after 15 days, when the release profi le has reached a plateau, the
longest fragments, 190-200 nm in contour length, also appeared in the AFM
analysis of the excess water (Fig. 8.13d). Interestingly, after 15 days, but only
after this time, the contour length distribution of the DNA released matches
closely the initial distribution, indicating that release is nearly entirely com-
pleted (Fig. 8.13d).
∼
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