| dc.description.abstract | The purpose of this study was to determine the mechanism of uptake and release of doxorubicin from HA nanoparticle. We used human head and neck squamous cell carcinoma MDA-1986 as a model cell line. MDA-1986 was imaged after incubation with HA-DOX (NPs), doxorubicin, HA-CY7 or HA-DOX-CY7 at 15 minutes, 1, 6, 24, and 48 hours' time points using an inverted fluorescence microscope. Also, the cell nucleus was stained with DAPI stain, and the cells were imaged after incubation with HA-DOX conjugates, doxorubicin, HA-CY7 or HA-DOX-CY7 for 6 hours. Furthermore, the lysosomes were stained to determine if the lysosomal pathway is the major degradation pathway of the nanoparticles and the carrier. To determine the internalization mechanism, four inhibition conditions were used: excess HA to block HA receptors, anti CD44 antibody (Hermes-1) to block the CD44 receptor, chlorpromazine to inhibit clathrin mediated endocytosis, and reduced temperature of 4°C. The fluorescence intensities of the cells co-incubated with the nanoparticle or the carrier with different inhibitors were compared using the inverted fluorescence microscope. The quantitative analysis of cells treated with HA-DOX and co-incubated with different inhibition conditions (10 mg/mL HA, chlorpromazine, Hermes-1 and 4°C) showed significant decrease in the fluorescence intensity except in cells pretreated with 5 mg/mL HA. Also, the quantitative analysis of cells treated with HA-CY7 and co-incubated with different inhibition conditions (excess HA, chlorpromazine and Hermes-1) showed significant decrease in the fluorescence intensity. However, the quantitative analysis of cells treated with free doxorubicin or HA-DOX-CY7 showed significant decrease in the fluorescence intensity in only cells pretreated with 25- µM chlorpromazine or cells incubated at 4 °C. The suggested internalization mechanism of HA-DOX conjugates is an active transport mechanism mediated mainly by the CD44, one of the HA receptors, through a clathrin-dependent endocytic pathway. On the other hand, a sulfated-HA derivative has similar uptake profile as the parent HA-CY7, and it is mainly localized in the cytosol. | |
