Impact of Reverse Micelle Loaded Lipid Nanocapsules on the Delivery of Gallic Acid into Activated Hepatic Stellate Cells: A Promising Therapeutic Approach for Hepatic Fibrosis

Gallic acid (GA) is a polyphenolic compound with proven efficacy against hepatic fibrosis in experimental animals. However, it suffers from poor bioavailability and rapid clearance that hinders its clinical investigation. Accordingly, we designed and optimized reverse micelle-loaded lipid nanocapsules (RMLNC) using Box-Behnken design that can deliver GA directly into activated-hepatic stellate cells (aHSCs) aiming to suppress hepatic fibrosis progression.

Methods
GA-RMLNC was prepared using soft energy, solvent free phase inversion temperature method. Effects of formulation variables on particle size, zeta potential, entrapment efficiency (EE%) and GA release were studied. In-vivo biodistribution of GA-RMLNC in rats and in-vitro activities on aHSCs were also explored.

Results
Nano-sized GA-RMLNCs (30.35 ± 2.34 nm) were formulated with high GA-EE% (63.95 ± 2.98% w/w) and physical stability (9 months). The formulated system showed burst GA release in the first 2 h followed by sustained release profile. In-vivo biodistribution imaging revealed that RMLNC-loaded with rhodamine-B accumulated mainly in rats’ livers. Relative to GA; GA-RMLNC displayed higher anti-proliferative activities, effective internalization into aHSCs, marked down-regulation in pro-fibrogenic biomarkers’ expressions and elevated HSCs’ apoptosis.

Conclusions
These findings emphasize the promising application of RMLNC as a delivery system in hepatic fibrosis treatment, where successful delivery of GA into aHSCs was ensured via increased cellular uptake and antifibrotic activities. Continue reading here

Materials
Gallic acid (GA) powder, Kolliphor® HS 15 (a mixture of free polyethylene glycol 660 and polyethylene glycol 660 hydroxystearate), Kolliphor®RH40 (Macrogol glycerol hydroxystearate), stearylamine (SA), Sulphorhodamine B (SRB) dye, Rhodamine B dye and dialysis membrane with 12,000–14,000 molecular weight cut-off were purchased from Sigma-Aldrich, MO, USA. Dulbecco’sModified Eagle’s Medium(DMEM) and fetal bovine serum (FBS) were purchased from GIBCO, NY, USA. Hanks Balanced Salt Solution (HBSS), phosphate buffer saline (PBS) and trichloroacetic acid were purchased from Lonza Bioproducts, Verviers, Belgium. Recombinant TGF-β1 and 4′,6- diamidino-2-phenylindole (DAPI) fluorescent dye were purchased from Cell Signaling Technology, MA and Invitrogen Corporation, CA, USA, respectively. Sodium chloride, potassium dihydrogen phosphate (KH2PO4) and disodium hydrogen phosphate (Na2HPO4) were purchased from El-Nasr Pharmaceutical Chemicals, Egypt. Ethanol 96% was obtained from Piochem, Egypt. Rat tail type I collagen tissue culture plates were purchased from Greiner Bio-One, Germany. Labrafil M 1944 CS (Oleoyl macrogolglycerides, HLB = 9), Labrafac lipophile WL 1349 (caprylic-capric acid triglycerides, HLB = 2) were kindly supplied by Gattefossé, France. Lipoid® S75 (soybean lecithin at 70% of phosphatidyl choline) was a gift from Lipoid Gmbh -Ludwigshafen, Germany.