Design of bile-based vesicles (BBVs) for hepatocytes specific delivery of Daclatasvir: Comparison of ex-vivo transenterocytic transport, in-vitro protein adsorption resistance and HepG2 cellular uptake of charged and β-sitosterol decorated vesicles

Abstract

Daclatasvir is a new direct acting antiviral used in treatment of Hepatitis C virus, in an attempt to increase its hepatocytes specificity and uptake. It was encapsulated within bile based vesicles (BBVs) containing egg phosphatidyl choline, cholesterol and sodium deoxycholate fabricated by thin-film hydration method. A D-optimal mixture design was applied to study the effect of formulation variables on vesicular characteristics. The dependent variables picked were the particle size, polydispersity index, zeta potential and entrapment efficiency. The optimized bile based vesicles were subjected for further modifications to prepare miniaturized anionic (ABBVs), cationic (CBBVs) and Sito-G decorated BBVs (Sito-GBBVs) to be capable to penetrate liver fenestrae (<200 nm). The aim of the current work is to compare the potential of the ABBVs, CBBVs and Sito-GBBVs loaded with Daclatasvir for stability in simulated biological fluids, ex-vivo intestinal transenterocytic transport, HepG2 cellular uptake and resistance to blood protein adsorption. The miniaturized ABBVs, CBBVs and Sito-GBBVs showed acceptable stability in simulated biological fluids. CBBVs had the highest transenterocytic transport through intestinal membrane. The internalization of CBBVs into HepG2 cells was about 2.1 folds that of ABBVs and 1.45 folds that of Sito-GBBVs. ABBVs and Sito-GBBVs showed superior resistance to opsonization compared to CBBVs which showed significant increase in particle size (p˃0.05) due to protein adsorption. The miniaturized Sito-GBBVs constitute a promising strategy to overcome key biological barriers facing hepatocytes specific delivery of Daclatasvir.

Fig 1

Contour plots of the impact of variables on the vesicle size (Y1) (a), PDI (Y2) (b), ZP (Y3) (c) and EE (Y4) of BBVs preparations and Overlay plot for the impact of different variables on the responses.

Fig 2. TEM micrograph of the optimized DAC-loaded BBVs.

Fig 3. In-vitro release profiles of DAC from drug suspension, the ABBVs, CBBVs and Sito-GBBVs in phosphate buffer pH 6.8 with 0.75% Brij 35.

Fig 4

(a) Cumulative amount permeated per unit area of DAC from ABBVs, CBBVs, Sito-GBBVs and drug solution through non-everted rat intestine in 0.9% saline at 37°C. Data presented as mean ±SD (n = 3). Dotted line: before dilution with methanol. Solid line: after dilution with methanol. (b) Percent of transenterocytic BBVs delivered through intestinal rat epithelial cells.

Fig 5. Confocal laser scanning microscopy (CLSM) images of HepG2 cells after incubation with Rh B labelled BBVs for 2 h at 37°C.

(a) ABBVs, (b) CBBVs and (c) Sito-GBBVs.

Fig 6. Representative bar chart of particle size of DAC loaded ABBVs, CBBVs, Sito-GBBVs and conventional liposomes before and after 2h incubation with rat serum.