Biomedical Engineering Reference
In-Depth Information
100
90
80
70
60
50
40
30
20
pH changed from
7.2 TO 5.0
10
0
0 0 0 0 0 0 0 0
Time (h)
Figure 4.14
Release of DNA from a poly(ortho ester) prepared from 3,9-diethylidene-2,4,8,10-
tetraoxaspiro [5.5] undecane, TEG, 1,2-propane diol, and TEG-GL (100/35/15/45/5). Micro-
spheres, 5
μ
m, phosphate buffer (pH 7.4) or sodium acetate buffer (pH 5.0) at 37 °C.
followed using the pico green method, release was slow, corresponding to slow
polymer erosion, as shown in Figure 4.14 [27]. However, when the pH of the
buffer was changed to 5.0, the pH of endosomes, an immediate and signifi cant
acceleration of DNA release was noted. Since a lowered pH results in an increased
erosion rate, this plot provides unequivocal evidence of an erosion-controlled
DNA release.
4.4.6.1
DNA Plasmid Stability
As analyzed by gel electophoresis, DNA plasmid retained its active conformation
(supercoiled and relaxed) when released from the POE microspheres placed into
a pH 7.4 buffer and also that remained in the microspheres. However, when the
microspheres were placed into a pH 5.0 buffer, signifi cant damage to the DNA
plasmid was noted. This is consistent with the known acid sensitivity of DNA. The
DNA plasmid remaining in the microspheres dispersed in the pH 5.0 buffer
retained its active conformation.
This is a signifi cant fi nding indicating that the internal pH within the poly(ortho
ester) matrix must be above pH 5.0 and that the microspheres are able to protect
the DNA plasmid from a low pH external environment.
4.4.6.2
Microencapsulation Procedure
Microspheres were prepared by a modifi ed water - in - oil - water double emulsion,
solvent evaporation procedure. The two phases consisting of 250
L of DNA solu-
tion (250 mg of DNA) and 7 mL of methylene chloride containing 200
μ
μ
g POE were
