This organic phase added drop by drop (2 ml/min) in external aqueous phase containing surfactant PVA in a fixed concentration (0.5% w/v) at 13,500 rpm (Omni GLH homogenizer). This suspension was then processed in high pressure homogenizer (Gea Niro Soavi, Italy) for eight cycles. A subsequently

organic solvent from external aqueous phase was removed under reduced pressure. The formed REPA-EC polymeric nanoparticles were recovered by centrifugation (R243A, Remi) at 18,000 rpm Tenofovir for 20 min followed by washing thrice with distilled water and washed nanoparticles were subjected to freeze drying (Scanvac, Denmark). The viscosity of internal phase was measured by Brookfield rotational digital viscometer DVLV II at 25 °C. The obtained REPA-EC NPs were dispersed in distilled water by sonication and vortex mixing for 30 s and the particle size (Z-average mean) and zeta potential were determined by using Nano series Malvern Instruments, UK. The percentage yields of dried nanoparticles were calculated by using Eq. (1) equation(1) Percentageyield=MassofnanoparticlesrecoveredMassofpolymers,drugandformulationexcipients×100

Accurately weighed freeze dried nanoparticles were dissolved in dichloromethane. Then REPA was extracted in 50 ml phosphate buffer (pH 7.4) solution. After the evaporation of DCM and removal of precipitated polymer by filtration, the amount of drug in phosphate buffer was measured using Ultraviolet selleck chemicals spectroscopy (U2900, Hitachi, Japan) at 275.5 nm. Encapsulation

efficiency (%) and drug content (%, w/w) were represented by Eqs. (2) and (3) respectively. equation(2) Encapsulationefficiency(EE%)=MassofdruginnanoparticlesMassofdrugusedinformulations×100 equation(3) Drugcontent(%,ww)=MassofdruginnanoparticlesMassofnanoparticlesrecovered×100 The shape and surface characteristics of nanoparticles were investigated and photographed using Field Emission-Scanning Electron Microscopy (FE-SEM) (S4800, Hitachi, Japan). Appropriate samples were mounted on stub, using double sided adhesive carbon tapes. Samples were gold coated and observed for morphology, at acceleration voltage of 1.0 kV. The samples (REPA, EC and nanoparticles) were homogeneously mixed with potassium bromide and infrared spectrums were recorded in region of 4000-400 cm−1 by using infrared spectrophotometer (IR-8400, below Shimadzu Co. Ltd., Singapore). X-ray diffraction of samples was carried out using Model-D8 Advance, Bruker AXS GmbH, Germany diffractometer. A Cu Kα source operation (40 kV, 40 mA) was employed. The diffraction pattern were recorded over a 2θ angular range of 3–50° with a step size of 0.02° in 2θ and a 1 s counting per step at room temperature. Accurately weighed samples were suspended in 100 ml phosphate buffer saline (pH 7.4). The solution was stirred at 50 rpm with temperature adjusted to 37 ± 1 °C. At programmed time intervals 5 ml samples were reserved and centrifuged at 20,000 rpm for 30 min.