Conventional nanocarriers are associated with systemic toxicity and poor bioavailability of the antitumor drugs due to undesired specificity. The objective of this study is to introduce a folic targeting ligand on the surface of a polymeric nanocarrier to enhance the delivery of the doxorubicin (DOX) via ligand-mediated endocytosis. Hence, the folate decorated-micelle based on the star-shape FOL-PEG-PCL copolymer was synthesized. The chemical structure of the copolymer was characterized by proton nuclear magnetic resonance spectroscopy, gel permeation chromatography and differential scanning calorimetry, respectively. A generalized biocompatibility test of the micelle was evaluated using MTT assay, in vitro hemolytic test, nitric oxide production and reactive oxygen species generation, respectively. When DOX was encapsulated in the micelle, the drug loading efficiency and drug loading content were found to be 90% and 13%, respectively. The average particle size of the DOX-loaded micelle, determined by dynamic light scattering was 148.2 nm. The intracellular uptake experiments showed that human breast cancer cells (MCF-7) could uptake a similar amount of DOX from two dosage forms: free DOX and DOX-loaded FOL-PEG-PCL micelle. The uptake of DOX-loaded FOL-PEG-PCL micelle was higher than that of free DOX in MCF-7/adr cells, adriamycin-resistant cell line. The uptake of the micelle in MCF-7 was found to be time-dependent; e.g. caveolae/lipid-raft mediated endocytosis and then folate receptor-mediated endocytosis was observed. This study demonstrates that the FOL-PEG-PCL micelle was non-toxic and the DOX-loaded FOL-PEG-PCL micelle could be a potential carrier for cancer treatments.

The triblock copolymer is composed of two identical hydrophilic segments Monomethoxy poly(ethylene glycol) (mPEG) and one hydrophobic segment poly(ε-caprolactone) (PCL); which is synthesized by coupling of mPEG-PCL-OH and mPEG-COOH in a mild condition using dicyclohexylcarbodiimide and 4-dimethylamino pyridine. The amphiphilic block copolymer can self-assemble into nanoscopic micelles to accommodate doxorubixin (DOX) in the hydrophobic core. The physicochemical properties and in vitro tests, including cytotoxicity of the micelles, have been characterized in our previous study. In this study, DOX was encapsulated into micelles with a drug loading content of 8.5%. Confocal microscopy indicated that DOX was internalized into the cytoplasm via endocystosis. A dose-finding scheme of the polymeric micelle (placebo) showed a safe dose of PEG-PCL-PEG micelles was 71.4 mg/kg in mice. Importantly, the circulation time of DOX-loaded micelles in the plasma significantly increased compared to that of free DOX in rats. A biodistribution study displayed that plasma extravasation of DOX in liver and spleen occurred in the first four hours. Lastly, the tumor growth of human breast cancer cells in nude mice was suppressed by multiple injections (5 mg/kg, three times daily on day 0, 7 and 14) of DOX-loaded micelles as compared to multiple administrations of free DOX.

This study reports on the cellular uptake of folate tethered micelles using a branched skeleton of poly(ethylene glycol) and poly(ε-caprolactone). The chemical structures of the copolymers were characterized by proton nuclear magnetic resonance spectroscopy, and Fourier transform infrared spectroscopy. Doxorubicin (DOX) was utilized as an anticancer drug. The highest drug loading efficiencies of DOX in the folate decorated micelle (DMCF) and folate-free micelle (DMC) were found to be 88.5% and 88.2%, respectively, depending on the segment length of the poly(ε-caprolactone) in the copolymers. A comparison of fluorescent microscopic images of the endocytosis pathway in two cell lines, human breast cancer cells (MCF-7) and human oral cavity carcinoma cells (KB), revealed that the micelles were engulfed by KB and MCF-7 cells following in vitro incubation for one hour. Flow cytometric analysis revealed that free folic acid can inhibit the uptake of DOX by 48%-57% and 26%-39% in KB cells and MCF-7 cells, respectively. These results prove that KB cells are relatively sensitive to folate-tethered micelles. Upon administering methyl-β-cyclodextrin, an inhibitor of the caveolae-mediated endocytosis pathway, the uptake of DOX by KB cells was reduced by 69% and that by MCF-7 cells was reduced by 56%. This finding suggests that DMCF enters cells via multiple pathways, OPEN ACCESSPolymers 2014, 6 635 thus implying that the folate receptor is not the only target of tumor therapeutics.