2.2. Preparation of nanoparticles

A modified w/o/w-emulsion solvent evaporation technique was employed to prepare polymeric NPs. Briefly, PLGA and drug were dissolved in 2 ml of an organic solvent (DCM-ethanol) system which was developed during the formulation. Initially, solubility characteristic of TLM was studied by LC-MS/MS. The developed solvent system was further used for determining the solubility of PLGA. The primary (w₁) and secondary water (w₂) phase of the emulsion were prepared by dissolving PVA (polyvinyl alcohol) (emulsifier) in deionized water in different ratios (table 3). The primary water phase (w₁) was emulsified with the oil phase using high-speed homogenization (25 000 r.p.m.) for 5 min. Then the primary emulsion (w₁/o) was introduced into secondary water phase (w₂) containing 0.5% PVA by a syringe, maintaining the homogenization speed at 7000 r.p.m.. Additionally the prepared w/o/w emulsion was sonicated at 20°C for 30 min. After that, the emulsion was allowed to stay overnight (10 h) on the magnetic stirrer (at 320 r.p.m.) to evaporate the organic solvents. The NPs were collected by centrifugation method at 16 000 r.p.m. for 30 min at 4°C. Washing was done three or four times with deionized water until complete removal of the surfactant (PVA) and finally the NPs were collected by centrifugation for 10 min at the same speed and temperature as given above. The collected NPs were lyophilized to dry completely and stored at −20°C for future work.

BBD of three-variable systems.