Background: Ritonavir (RTV), lopinavir (LPV), and efavirenz (EFV) are considered appropriate HAART in the treatment of HIV-infected patients. We fabricated ART nanoparticles containing these 3 drugs and tested the release of the drugs from the nanoparticles over 14 days.

Methods:  RTV, LPV, and EFV (10 mg of each) were added to 200-mg poly-caprolactone polymer in methylene chloride. Pluronic-127 (200 mg) was dissolved in 1% polyvinyl alcohol. The 2 phases were combined in a water-in-oil-in-water emulsion using a Teflon emulsion tip at 15 W (10 minutes). The organic phase was evaporated overnight, washed, frozen at –80°C and lyophilized. Nanoparticles were tested for particle size and charge using photon correlation spectroscopy. Drug loading, entrapment efficiencies, and in vitro release were determined. ART nanoparticles (10 mg) were placed in phosphate buffered saline (PBS) (pH 7.4 and 4.0) and 100 µL PBS was removed, centrifuged, and analyzed by HPLC for RTV, LPV and EFV concentrations. ART nanoparticles (10 mg) were placed in PBS (pH 7.4 and 4.0) and 9.9 mL were removed and replaced with 9.9 mL of fresh PBS. Samples were taken every 2 hours for 8 hours, and then 1, 2, 3, 4, 5, 7, 9, and 14 days. Drug levels were analyzed by high performance liquid chromatography. Intra-day and inter-day variability were both <10%.

Results:  Results are presented as mean ± SEM. Nanoparticles size was 340.4±12.4 nm. Mean zeta potential was –23.7±1.36. ART loading averaged 38.3%. Loading efficiency was 18.2%, 30.5%, 46.4% for RTV, LPV, and EFV, respectively. Entrapment efficacy averaged 97.4%. Release of RTV, LPV, and EFV occurred in vitro over 14 days. Peak ART levels for pH 4.0 (simulating endozome pH) occurring at day 7 with levels of 8.2±0.39 µg/mL for RTV, 8.5±0.4 µg/mL for LPV, and 12.0±1.5 µg/mL for EFV. Peak RTV, LPV, and EFV levels at pH 7.4 were 5.8±0.9, 6.4±0.9, and 8.4±1.5 µg/mL, respectively, occurring at 4 hours. The ART levels at 14 days were all >3.5 µg/mL. Where 9.9 mL PBS was removed and replaced, in vitro measurements at the peak level (pH 4.0 and 7.4) were all >4.0 µg/mL, at 0 hours. Day 14 levels for RTV, LPV, and EFV were all >1.0 µg/mL. Electron microscopy locates nanoparticles in microglia cells.

Conclusions:  Results of these experiments demonstrate that RTV, LPV, and EFV release from our nanoparticle formulation occurs over at least 14 days. ART nanoparticles could be a delivery system for multiple anti-retroviral agents in the future.