Study On Targeting Liver Using Reconstituted High-density Lipoprotein-drug Complex Mediated By Apolipoprotein A-â… 

High-density lipoprotein (HDL) particles can deliver cholesterol from peripheral tissues to the liver through apolipoprotein A-Ⅰ (apo A-Ⅰ), which specifically binds to the scavenger receptor class B type1(SR-B1) receptor on the surface of hepatocytes.Therefore, HDL is considerable potential for development and value for application as a targeting liver delivery system.In this study, we first compared thin-film method, pH gradient method with ammonium sulfate-gradient method to formdoxorubicin-HDL complex (rHDL-DOX) using the apoA-Ⅰ as a carrier and a hydrophilic anti-cancer drug-doxorubicin hydrochloride as a model. Results showed that the mean diameter and encapsulation of Dox·HCl of HDL particles prepared by optimized thin-film method was59.70nm±4.50nm and20.20%±4.17%while by optimized ammonium sulfate-gradient method was113.80±10.29nm and83.33%±8.45%. pH gradient method was not suitable to prepare rHDL-DOX because of poor controllability. The product had a sustained release pattern and in vitro and the freeze-dried nanoparticles with a good placing stability.Next, we investigated the cytotoxicity and absorption of rHDL-DOX against several cells lines including BEL7402, HepG2, SGC-7901, CQN, HEK-293. The MTT assays showed that rHDL-DOX had higer cytotoxicity on BEL-7402which could absorbed much more DOX than other cells lines. An experiment about apo A-Ⅰ’s function on medicating drugs transported into cells showed rHDL-Rh6G with apoA-Ⅰ could present much more fluorescence intensity than OL-Rh6G. These results suggested that the combination of rHDL and hepatoma cells, which is stonger than other cells, was medicated by apoA-Ⅰ. In addition, an in vitro hemolysis assay showed that rHDL-Dox caused no limited hemolysis and is a safe drug delivery system.The pharmacokinetics experiment was performed to access the liver targeting from the other side. The t1/2β of rHDL-DOX was1.25times as OL-DOX. The AUC(0-∞) value of rHDL-DOX was2.58times as OL-DOX. The value of relative uptake ratio (Re) was1.824compared with free-DOX. These datas suggested rHDL-DOX nanopariticles could carry much DOX into liver to metabolize incadicating a targeting liver in vivo characteristics.In order to verify the effect of rHDL-DOX on liver cancer’s treatment, we compared its cytoxicity to OL-DOX and free dox by MTT assays. Meanwhile, for in vivo efficacy study, a orthtopic liver tumor model was established on nude mice by measuring the change of body weight and tumor. The IC50value of rHDL-Dox in BEL-7402and HepG2cells was6.143ug/ml and3.108ug/ml, respectively, indicating that the latter cell type was more sensitive to the nanoparticle preparation. The IC50value of rHDL-Dox in BEL-7402and HepG2cells was6.143ug/ml and3.108ug/ml, respectively. Moreover, the IC50values of OL-Dox and free Dox in BEL-7402and HepG2cells were9.879,12.60,6.728, and8.321μg/ml, respectively. Taken together, these data indicated that the hepotama cell lines were more sensitive to the rHDL-Dox than the OL-Dox and free Dox nanoparticles. For treatment group of rHDL-DOX with a high dose, the weight of mice’s body and tumor with a diameter of1.55cm was20.70±1.39g,1.37±0.49g, respectively. There is a weight reductions of44.1%,7.9%and3.7%for rHDL-DOX(10mg/kg), OL-DOX (10mg/kg) and rHDL-Dox (5mg/kg), respectively, compared to control.Taken together, our findings indicate that rHDL is a safe and effective drug delivery system for targeting liver. Our study work about the development of rHDL, which is cinducive to decrease anti-cancer drugs’side-effects while expanding the scope of application, will lay the foundation for thenext research about the liver targeing mechanism and provide a new thought for a research on anti-cancer drug.