Characterization of liver cancer-targeted doxorubicin coupling segmented copolymer nanomicelles and its inhibitory effect on hepatocellular carcinoma HepG2 and Huh7 cells

Objective To investigate the characterization of doxorubicin (DOX) coupling segmented copolymer nanomicelles with dual effects of passive and active targeting to liver cancer and its antineoplastic function in vitro. Methods DOX was covalently conjugated to a terminal hydroxyl group of poly lactic-co-glycolic acid-poly ethylene glycol (PLGA-PEG) diblock copolymer to form DOX-PLGA-PEG. The formation of amido bond was determined by using fourier transform infrared spectroscopy (FTIR) and magnetic resonance method. Amphiphatic diblock copolymer DOX-PLGA-PEG could self-assemble to form nanomicelles in an aqueous phase by dialysis method. The DOX-PLGA-PEG targeted micelles decorated with liver cancer HAb18 F (ab')2 specific antibody were prepared by using physical bonding method. The size and the scattering scope of nanomicelles was determined by using granulometer and dynamic light scattering (DLS). Micelle morphology was examined by using scanning electron microscopy (SEM). The drug loading rate and entrapment rate of DOX-PLGA-PEG micelles or targeted micelles were measured by using ultraviolet spectrophotometry method, and stimulated release in vitro experiment was done. After administration of 2 mg/ml DOX-PLGA-PEG or targeted micelles, cell morphology change of liver cancer HepG2 and Huh7 was observed by using the phase-contrast photomicrography. After administration of 1 mg/ml DOX-PLGA-PEG or targeted micelles, cell survival was analyzed by using plate clone formation assay. Results The spectrum peak was around 1 575 cm-1 under the observation of FTIR, which was accord with the location of the peak of amido bond. Activating with p-nitrophenyl chloroformate, DOX was covalently conjugated to PLGA-PEG to produce DOX-PLGA-PEG via a carbamate linkage between the primary amine group in DOX and the terminal hydroxyl group in PLGA-PEG. DLS measurements showed that the diameter of DOX-PLGA-PEG micelles and targeted micelles was (55.0±6.3) nm and (87.6±9.3) nm, respectively, and polydispersity index was 0.098 and 0.142, respectively. SEM micrographs revealed that these nanomicelles had a spherical morphology and relatively smooth surface. Drug loading rate of DOX-PLGA-PEG micelles and targeted micelles was (2.4±0.2)% and (2.2±1.9)%, and the entrapment rate was (91.7±5.3)% and (87.5±4.8)%, respectively. The drug release curve in vitro of DOX-PLGA-PEG micelles and targeted micelles exhibited a biphasic pattern characterized by a fast initial release, followed by a slower and continuous release. The amount of the drug release rate was about 30% within 5 d, and 25% within 6 h. After 2 mg/ml DOX-PLGA-PEG micelles and targeted micelles, the cell morphology of liver cancer HepG2 and Huh7 had the impaired change, and the part of the cells were dead, the clonality decreased. The effect of targeted micelles was more significant compared with DOX-PLGA-PEG micelles. After DOX-PLGA-PEG micelles and targeted micelles, the survival rates of HepG2 and Huh7 cells were decreased with time, and the effect of targeted micelles was more effective compared with DOX-PLGA-PEG micelles (all P < 0.05). The 50% effective inhibition of the targeted micelles and DOX-PLGA-PEG micelles was obtained in 2.4 d and 5.5 d, respectively for Huh7 cells. At these time points, DOX concentration was 1.15 μg/ml and 1.24 μg/ml, respectively. The 50% effective inhibition of targeted micelles and DOX-PLGA-PEG micelles was obtained in 3.3 d and 7.4 d, respectively for HepG2 cells. At these time points, DOX concentration was 1.20 μg/ml and 1.31 μg/ml. Conclusion DOX nanomicelles with dual effects of passive and active targeting can release a large number of active drugs in vitro, which plays an obvious inhibitory role in the cell proliferation of liver cancer HepG2 and Huh7 cells. Key words: Liver neoplasms; Doxorubicin; Drug delivery systems; Molecular targeted therapy; Targeted micelles