2.11 Model Drug Release
PEG-DGE-1:0.25, PEG-DGE-1:0.5, BS3-1:0.25, and BS3-1:0.5 crosslinked
microgels loaded with Texas Red-dextran were prepared by adding the dextran
(15 wt% of dextran with respect to elastin) to the aqueous phase in the inverse
emulsion in which the microgels were synthesized, under the same conditions as the
blank samples. In order to determine the amount of Texas Red-dextran taken up by
the micro-conetworks, the extracted Texas Red-dextran in the separated water was
quantified by measuring the absorbance at 596 nm and using a standard calibration
curve. The difference from the feed was taken as the amount loaded to the microgels.
To study the release of loaded dextran at 20 and 40
C, the microgels were taken up in
10 mL PBS buffer at pH 5 by shaking the cuvettes at 100 rpm. After leaving for 0, 1,
5, 10, 15, 25, 36, 48 or 72 h, the microgels were centrifuged. The release was
monitored by measuring the extinction of the supernatants. The sedimented
microgels were resuspended in fresh buffer solution. All studies were performed
in triplicate and the cumulative release was normalized to the original uptake:
Encapsulation efficiency% ¼ D t =D 0
ð
ÞÂ100,
ð4Þ
where D t is the actual amount of the dextran in the microgel at time t and D 0 is the
initial amount of dextran in the microgel.
3 Results and Discussion
Solubilized native elastin with a molecular weight range of 10–60 kDa was
dispersed in an inverse miniemulsion and crosslinked by reaction of PEG-DGE or
BS3 with the ε-amino groups of lysines in the sequence of the elastin peptide
fragments (Scheme 1).
PEG-DGE crosslinked microgels can be regarded as a conetwork of the hydrophilic PEGs and the thermoresponsive elastin. In contrast, the linking groups of the
BS3 crosslinked microgels are hydrophobic and are expected to promote precipitation above the coacervation temperature. The crosslinking density was varied by
adding different amounts of the telechelic linkers. In Table 1, the amount of linker
is given as a molar ratio of elastin and linker based on the molecular weight of
M n ¼ 60,000. Ratios of 4–0.5 crosslinker per elastin molecule correspond to
relatively low crosslinking degrees because of the lower molecular weight fraction
in the elastin sample. It must be noted, however, that the microgels were fractionated by separation of very loosely crosslinked and very small particles during the
isolation/sedimentation procedure. Thus, the crosslinker ratios can only be taken as
an indication of the relative variation in crosslinking density. Figure 1 depicts the
most densely crosslinked microgels (elastin to crosslinker ratio of 0.5) by scanning
electron microscopy (SEM) cryo-electron micrographs as quenched from ambient
422
S. Singh et al.
PEG-DGE-1:0.25, PEG-DGE-1:0.5, BS3-1:0.25, and BS3-1:0.5 crosslinked
microgels loaded with Texas Red-dextran were prepared by adding the dextran
(15 wt% of dextran with respect to elastin) to the aqueous phase in the inverse
emulsion in which the microgels were synthesized, under the same conditions as the
blank samples. In order to determine the amount of Texas Red-dextran taken up by
the micro-conetworks, the extracted Texas Red-dextran in the separated water was
quantified by measuring the absorbance at 596 nm and using a standard calibration
curve. The difference from the feed was taken as the amount loaded to the microgels.
To study the release of loaded dextran at 20 and 40
C, the microgels were taken up in
10 mL PBS buffer at pH 5 by shaking the cuvettes at 100 rpm. After leaving for 0, 1,
5, 10, 15, 25, 36, 48 or 72 h, the microgels were centrifuged. The release was
monitored by measuring the extinction of the supernatants. The sedimented
microgels were resuspended in fresh buffer solution. All studies were performed
in triplicate and the cumulative release was normalized to the original uptake:
Encapsulation efficiency% ¼ D t =D 0
ð
ÞÂ100,
ð4Þ
where D t is the actual amount of the dextran in the microgel at time t and D 0 is the
initial amount of dextran in the microgel.
3 Results and Discussion
Solubilized native elastin with a molecular weight range of 10–60 kDa was
dispersed in an inverse miniemulsion and crosslinked by reaction of PEG-DGE or
BS3 with the ε-amino groups of lysines in the sequence of the elastin peptide
fragments (Scheme 1).
PEG-DGE crosslinked microgels can be regarded as a conetwork of the hydrophilic PEGs and the thermoresponsive elastin. In contrast, the linking groups of the
BS3 crosslinked microgels are hydrophobic and are expected to promote precipitation above the coacervation temperature. The crosslinking density was varied by
adding different amounts of the telechelic linkers. In Table 1, the amount of linker
is given as a molar ratio of elastin and linker based on the molecular weight of
M n ¼ 60,000. Ratios of 4–0.5 crosslinker per elastin molecule correspond to
relatively low crosslinking degrees because of the lower molecular weight fraction
in the elastin sample. It must be noted, however, that the microgels were fractionated by separation of very loosely crosslinked and very small particles during the
isolation/sedimentation procedure. Thus, the crosslinker ratios can only be taken as
an indication of the relative variation in crosslinking density. Figure 1 depicts the
most densely crosslinked microgels (elastin to crosslinker ratio of 0.5) by scanning
electron microscopy (SEM) cryo-electron micrographs as quenched from ambient
422
S. Singh et al.