Bioactivities of Chitosan and Its Derivatives - Chitosan Based Hydrogels: Functions and Applications - page 164

(a) (b) (c)

CH 2 OCH 2 CH 2 OH CH 2 OCH 2 CH 2 OH CH 2 OCH 2 CH 2 OH

O O O

O O O

HO HO HO

NH 2 NH

C=O
CH 3

NH
C=O

CH 3

H 3 C

Hydrophobically modified glycol chitosan conjugate (HGC)

Figure 3.28
Chemical structure of HGC nanoparticles prepared by chemical coupling of hydrophilic glycol chitosan with
hydrophobic 5β-cholanic acids. (Adapted from Maeda, H. et al. 2000. J control Release 65: 271-284.)

nanoparticles might protect the active lactone ring of CPT against hydrolysis under physi-
ological conditions, due to the encapsulation of CPT into the hydrophobic cores in HGC
nanoparticles. The freshly prepared HGC and CPT-10 wt%-HGC nanoparticles were well
dispersed in aqueous conditions and the particle sizes were about 254 and 288 nm, respec-
tively, which are confirmed by DLS measurements. Transmission electron microscopy
(TEM) images also revealed that HGC and CPT-10 wt%-HGC nanoparticles were almost
spherical in shape. Also, CPT-HGC nanoparticles were well dispersed and their particle
sizes were maintained up to 2 weeks at 37°C in PBS, indicating thermodynamical stability
in aqueous media. The loading efficiency of CPT was above 80% when the drug was pres-
ent at less than 10 wt% of nanoparticles, whereas a marked decrease in the loading effi-
ciency (to less than 45%) was seen when the drug was present at 20 wt%.
Protection of the CPT lactone ring from hydrolysis: The proportions of CPT in the lactone ring
and carboxylate forms are critical in predicting tumor response to CPT because the lactone
form has much higher antitumor efficacy compared to the carboxylate form. The protection
effect of CPT-HGC nanoparticles on the CPT lactone ring form against hydrolysis under
physiological conditions (PBS, pH 7.4, 37°C) using reversed-phase high-performance liquid
chromatography (HPLC) was evaluated. As shown in Figure 3.29a, free CPT dissolved in
PBS exhibited significant lactone ring opening due to rapid hydrolysis. The carboxylate
form and the lactone ring form of CPT were detected at 8.7 and 11.5 min, respectively, in
analytical HPLC spectra. Only 39% of CPT remained in the lactone ring form after incuba-
tion in PBS for 6 h, indicating that the unprotected CPT lactone ring was rapidly converted
into the inactive carboxylate by hydrolysis. On the other hand, about 89% of the lactone ring
was preserved after 6 h incubation in PBS when CPT was incorporated into HGC nanopar-
ticles (Figures 3.29b and c). This implies that the many inner cores of HGC nanoparticles
efficiently protected the lactone form of CPT from hydrolysis.
In vivo tumor-targeting characteristics of CPT-HGC nanoparticles : To estimate the in vivo
characteristics of CPT-HGC nanoparticles, HGC nanoparticles were labeled with the near-
infra-red (NIR) fluorophore, Cy 5.5 (exciting wavelength = 675 nm, emission wave-
length = 695 nm), which yields a strong fluorescence signal in vivo . To estimate in vivo
tumor targeting of CPT-HGC nanoparticles, 10 wt% of CPT was encapsulated into Cy
5.5-labeled HGC nanoparticles. The freshly prepared Cy 5.5-labeled CPT-HGC nano-
particles had the same physicochemical characteristics (particle size, in vitro stability,
morphological shape, and drug-loading efficiency) as unlabeled CPT-HGC nanoparticles,
whereas they presented a strong NIR fluorescence signal. After the i.v. injection of Cy