| [Object]To explore the combination of UA and DOX;To prepare the UA/PluronicF127/TPGS mixed nanomicelles,and its particle size,drug loading and encapsulation efficiency were characterized.TPGS-DOX was synthesized by chemical methods,preparing UA/pluronicF127/TPGS-DOX mixed nanomicelles.To investigate its release behavior in vitro and its freeze-drying process;To study the effect of UA/pluronicF127/TPGS mixed nanomicelles on HepG-2 cells.To study the pharmacokinetic behavior in rats after tail vein injection,and to calculate the PK parameters.[Method]MTT method was used to investigate the cell inhibition of monotherapy and combination therapy on different cells,using the Jin’s formula to select the best proportion;UA/pluronicF127/TPGS mixed nanomicelles was prepared by thin film hydration method,according to the results of the single factor and the L9(34)orthogonal test prescription,the preferred process prescription was determined;TPGS-DOX were bounded by amide bond,which was using to prepare UA/pluronicF127/TPGS-DOX mixed nanomicelles;The drug release behavior was investigated by membrane dialysis;to determine the freeze-drying process prescription by investigating the freeze-dried protective agent;The cell inhibition of nanomicelles against HepG-2 and HepG-2/ADR was investigated by MTT method.Using Annexin V-FITC cell apoptosis detection kit and PI single staining to detect the cell apoptosis and cell cycle arrest of nanomicelles by flow cytometry;The plasma concentrations were determined by HPLC after single intravenous administration;The pharmacokinetic parameters were calculated by DAS2.0 software and analyzed using SPSS 17.0 statistic software.[Results]The quality ratio of UA to DOX was determined to be 50:1;The optimized formulation of UA/pluronicF127/TPGS mixed nanomicelles was obtained by orthogonal design,particle size was115±9.42nm,entrapment efficiency was 89.00+0.38%,drug loading was 6.59 ± 0.03%;TPGS-DOX was synthesized successfully,according to the combination ratio the UA/pluronicF127/TPGS-DOX mixed nanomicelles were successfully prepared;The drug release in vitro results showed that it has certain sustained release effects;5%lactose and 5%mannitol was chosen to be the freeze-dried protective agent;HepG-2 and HepG-2/ADR cells were treated with different concentration groups(high,medium and low)for 48h,the cell inhibition rate of nanomicelles was significantly higher than that of free UA+DOX when acting on HepG-2/ADR,while there was no significant difference observed when acting on HepG-2;The reversal index of nanomicelles compared with free DOX was 4.644 times;After being treated with nanomicelles for 48h,HepG-2/ADR cells were stained with Hoechst 33342,the result was that apoptotic cells were bright,the nuclei were concentrated,which was significantly stronger than the free UA+DOX group and other control groups.Test results of Annexin V-FITC/PI double staining was nanomicelles can significantly induce early apoptosis and late apoptosis against HepG-2/ADR cells compared with the free drug group.The result of PI single staining showed that nanomicelles blocked G0/G1 phase and G2/M phase significantly against HepG-2 ADR cells;The pharmacokinetic parameters of the rats in vivo,AUC(0-∞),AUMC(0-∞),MRT(0-∞),VRT(0-∞),Vz,CLz of UA+DOX emulsion group and nanomicelles group at the same dosage were significant differences(P<0.01).Such as the AUC of UA in nanomicelles was 1.71 times than that of emulsion group,while DOX was 3.36 timesthan emulsion group.While the average residence time of the two drugs in the nanometer micelle group was also significantly prolonged.[Conclusion]UA/pluronicF127/TPGS-DOX mixed nanomicelles were successfully prepared by film hydration method;UA was encapsulated in hydrophobic core formed by micelles self-assembled,DOX was chemically conjugated to TPGS,therefor the proportion of the two drugs can be controlled;UA/pluronicF127/TPGS-DOX release in vitro and pharmacokinetics in vivo shown its good sustained release effect;UA/pluronicF127/TPGS-DOX induced early apoptosis and late apoptosis and blocked cell growth of G0/G1 phase and G2/M phase significantly on HepG-2/ADR. |
