Preparation of PVA nanocarriers. The rifampicin-loaded NSs were prepared according to a modified solvent displacement technique (Xxxxx et al., 1989). This method required 50 mg PVA-40 to be dissolved in 5 ml of methanol using stirring for 12 h for complete dissolution of the polymer. Different amounts of rifampicin (5, 10, 25 and 50 mg) were dissolved in this polymeric solution and injected into a dispersing phase (30 ml) containing citrate buffer (pH 4.8), 1 % w/v of PVA-80, at a speed of 0.5 ml/min using a 5 ml syringe housed in a syringe driver (Precidor, Infors, Basel, Switzerland). PVA nanocapsules were prepared using a similar method, with few modifications (Xxxxx et al., 1989). PVA (50 mg), phospholipids (75 mg) and rifampicin (5, 10 and 25 mg previously dissolved in 0.33 ml of benzyl benzoate) was added to 15 ml of acetone to form the organic phase. To form the nanocapsules the acetone mixture was injected into 30 mL of an aqueous solution composed of poloxamer 188 (0.5 % w/v in citrate buffer, pH 4.8) under moderate homogenization (5,000 rpm). Both mixtures used to form NSs and NCs were homogenized at 5000 rpm for 10 min using a Silverson L4RT laboratory mixer (Silverson Machines Ltd, Waterside, UK), stirred overnight in the fume hood for complete evaporation of the organic solvent and filtered through 1 µm cellulose filter to remove any large aggregates. Drug-free nanoparticles were prepared according to the same procedure omitting the drug. The drug-loaded nanoparticles were separated from the free drug by an ultrafiltration/centrifugation technique using 15 ml centrifugal filter devices (Amicon ultra 15®) with MWCF (molecular weight cut-off) of 100 kDA (Xxxxxx Scientific, UK) at 3000×g for 1 h (MSC centaur 2 centrifuge, DJB Labcare Ltd., UK). In this system, the non-encapsulated rifampicin is passed through the filter and the nanoparticles retained on the filter are re-suspended in 1 ml of water.
