A Folic Acid Conjugated Micelles Actively Targeting To The Active Hepatic Stellate Cells For Liver Fibrosis Therapy

The active hepatic stellate cells (HSCs) have been the important targets for antifibrotic therapy since these highly proliferative myofibroblastic cells secrete and accumulate too much extracellular matrix (ECM) in the liver, which results in the liver fibrosis. To develop an antifibrotic drug system which can either carry lots of hydrophobic and unstable therapeutic agents but also specifically target the active HSCs to improve the efficancy and unwanted side effects of drugs, we have designed a PEG-PCL formed nanomicelle coupled with folic acid as the ligands to actively target the active HSCs. In vitro and in vivo experiments have been performed to confirm that this drug system presents good targeting to the active HSCs and the fibrotic liver. Camptothecin has been then carried with this delivery system and examined its inhibitory effects on the proliferation and myofibroblastic phenotype of the active HSCs to assess its potential application for liver fibrosis therapy.Firstly, the amphiphilic poly (ε-caprolactone)-poly (ethylene glycol) copolymer with FA ligands (FA-PEG-PCL) were synthesized and self-assembled into the stable micelles by a solvent evaporation method.The physiochemical properties of materials and micelles were evaluated through various analyses including 1HNMR, FT-IR, DLS and TEM. Cytotoxicity analysis and measurement of hemolysis rate were then performed and the results showed that the prepared FA-PEG-PCL micelles possess excellent biocompatibility.Next, we studied the cellular uptake of the nile red loaded micelles with or without FA targeting ligands by the activative HSCs in vitro. Compared to the micelles without FA coupling, the FA-conjugated micelles exhibit significantly higher cellular uptake both by the immortalized active HSC-T6 cells and by the culture-activated primary HSC cells, for the first time suggesting that the folic acid may be a novel ligand to effectively target the myofibroblastic HSCs which express more folic acid receptor (FR) than the quiescent ones. More importantly, biodistribution studies with the CCl4 acute injured mice revealed that the FA-conjugated micelles were significantly higher accumulation in the injured liver, further indicating that this developed nano micelles can provide the drug with the specifically and actively targeting to the liver fibrosis.Lastly, we prepared the CPT-loaded micelles and evaluated their physicochemical properties. In vitro drug release profiles exhibited that the drug release profile of the micelles was partially controlled in the pH-dependent manner. As expected the FA-CPT-micelle showed stronger inhibitory effects on the active HSC than the FA unconjugated CPT micelles, as shown either by more HSC-T6 cells killed in vitro cytotoxicity assays or by more downregulation of several fibrotic genes such as a-Sma and Collagen lal. In sum, we have generated a novel FA-conjugated drug delivery system to carry the antifibrotic drugs including CPT, and then to specifically act on the myofibrotic HSCs and possibly on the fibrotic liver for liver fibrosis treatment.