Bioactivities of Chitosan and Its Derivatives - Chitosan Based Hydrogels: Functions and Applications - page 168

When FGF-2 was added to hydrogels, most of the FGF-2 molecules retained in the hydro-
gels remained biologically active, and were gradually released on biodegradation of the
hydrogels in vivo [241,242]. The study showed that the controlled release of biologically
active FGF-2 molecules from FGF-2-incorporated chitosan hydrogels caused induction of
angiogenesis, and collateral circulation possibly occurred in healing-impaired diabetic
(db/db) mice and in the ischemic limbs of rats. The controlled release of paclitaxel from
photocross-linkable chitosan hydrogel in vitro has been described and its antiangiogenesis
and antitumor effects in vivo were shown to support the possible use of chitosan hydrogel
incorporating paclitaxel as a regional delivery for tumor treatment [243]. Thus, photocross-
linkable chitosan hydrogel and injectable chitosan/IO 4 − heparin hydrogel are excellent car-
riers for controlled release of drug reagents such as FGF-2 and paclitaxel. To prepare a
chitosan hydrogel incorporating paclitaxel, 1 mL of Taxol containing paclitaxel (6 mg/mL)
in a vehicle composed of Cremophor EL and ethanol at a 50:50 (v/v) ratio was mixed into
1 mL of 40 mg/mL Az-CH-LA aqueous solution (at a final concentration of 20 mg/mL
Az-CH-LA) using a vortex. While about 35-40% of both paclitaxel and vehicle were
released from the photocross-linked hydrogel within 1 day, the releases were observed
over a period of 4 days with a half-release time of 45 h [243].
Paclitaxel is a potent inhibitor of angiogenesis, cell migration, and collagenase produc-
tion in addition to its antiproliferative effect on tumor cells. Chitosan hydrogel incorporat-
ing paclitaxel was found to inhibit the growth of Lewis lung cancer cells (3LL), human
umbilical vein endothelial cells (HUVECs), and human dermal microvascular endothelial
cells (HMVECs) with half-cell growth-inhibition concentrations of 15, 15, and 1 ng/mL,
respectively. Washing chitosan hydrogels incorporating paclitaxel with the culture
medium for longer than 15 days resulted in loss of the ability to inhibit cell growth except
in the case of HMVECs. HMVEC growth was inhibited in the presence of the washed chi-
tosan hydrogel incorporating paclitaxel for longer than 21 days. On the other hand, the
chitosan hydrogel incorporating paclitaxel had a significantly lower inhibitory effect on
fibroblast (human dermal fibroblast) growth than 3LL, HUVECs, and HMVECs.
A measurable tumor volume (about 100 mm) was formed at 12 days after subcutaneous
implantation of 3LL cells. UV-laser irradiation for 30 s was able to convert the injected vis-
cous Az-CH-LA aqueous solution (0.2 mL) into the same insoluble hydrogel through
insertion of an optical crystal fiber connected to a He-Cd laser. As shown in Figure 3.32
[244], administration of paclitaxel alone and chitosan hydrogel alone reduced subcutane-
ous induced tumor growth of 3LL cells to various extents over 7 days. The chitosan hydro-
gel incorporating paclitaxel more strongly inhibited tumor growth to less than 5% of the
control group value ( p < 0.0001 versus control) than paclitaxel alone or chitosan hydrogel
alone. The inhibitory effect on tumor growth of the chitosan hydrogel incorporating pacli-
taxel lasted for 14 days and subsequently the tumor in most mice grew again. However, a
second application of the chitosan hydrogel incorporating paclitaxel 10 days after the first
application had an additional antitumor effect.
To evaluate the antiangiogenesis effect of chitosan hydrogel incorporating paclitaxel,
immunohistochemical staining of murine CD34 was carried out in 3LL cells treated with
chitosan hydrogel incorporating paclitaxel, chitosan hydrogel alone, paclitaxel alone, and
untreated control tumors. On day 8, in paclitaxel-treated and control mice, many CD34-
positive stained vessels were diffusely located and clearly formed tube-like structures
were found in the tumor. On the other hand, CD34-positive vessels were significantly
fewer in the tumors treated with chitosan hydrogel incorporating paclitaxel. As shown in
Figure 3.33, chitosan hydrogel incorporating paclitaxel significantly reduced the number
of CD34-positive vessels compared with other treatments, suggesting that chitosan