Epidermal growth factor (
EGF)-conjugated copolymer
micelles were prepared from a mixture of diblock copolymers of methoxy poly(
ethylene glycol)-block-poly(
delta-valerolactone) (MePEG-b-PVL) and
EGF-PEG-b-PVL for targeted delivery to
EGF receptor (EGFR)-overexpressing
cancers. The block copolymers and functionalized block copolymers were synthesized using PEG as the macroinitiator and HCl-
diethyl ether as the catalyst. The MePEG-b-PVL and the carboxyl-terminated PEG-b-PVL (HOOC-PEG-b-PVL) copolymers were found to have molecular weights of 5940 and 5900, respectively, as determined by gel permeation chromatography (GPC) analyses. The HOOC-PEG-b-PVL copolymers were then activated by
N-hydroxysuccinimide and subsequently reacted with
EGF to form the
EGF-PEG-b-PVL copolymers. The efficiency for the conjugation of
EGF to the copolymer was found to be 60.9%. A hydrophobic
fluorescent probe,
CM-DiI, was loaded into both the nontargeted MePEG-b-PVL
micelles and the targeted
EGF-conjugated PEG-b-PVL
micelles. The effective mean diameters of the CMDiI-loaded nontargeted and the CMDiI-loaded targeted
micelles were found to be 32 +/- 1 nm and 45 +/- 2 nm, respectively, as determined by dynamic light scattering (DLS). The zeta potentials for the nontargeted
micelles (no
CM-DiI-loaded),
CM-DiI-loaded nontargeted
micelles, and
CM-DiI-loaded targeted
micelles were found to be -6.5, -8.7, and - 13.5 mV, respectively. Evaluation of the in vitro release of
CM-DiI from the MePEG-b-PVL
micelles in
phosphate buffer saline (0.01 M, pH = 7.4) containing 10% (v/v)
fetal bovine serum at 37 degrees C revealed that approximately 20% of the probe was released within the first 2 h. Confocal
laser scanning microscopy (CLSM) analysis revealed that the targeted
micelles containing
CM-DiI accumulated intracellularly in EGFR-overexpressing MDA-MB-468
breast cancer cells following a 2 h incubation period, while no detectable cell uptake was observed for the nontargeted
micelles. Results obtained from the confocal images were confirmed in an independent study by measuring the intracellular
CM-DiI fluorescence in cell lysate. In addition, the presence of free
EGF was found to decrease the extent of uptake of the targeted
micelles. Nuclear staining of the cells with
Hoechst 33258 indicated that the targeted
micelles mainly localized in the perinuclear region and some of the
micelles were localized in the nucleus. These results demonstrate that the
EGF-conjugated copolymer
micelles developed in this study have potential as vehicles for targeting hydrophobic drugs to EGFR-overexpressing
cancers.