Lissamine rhodamine B

NanoART of IDV, RTV and EFV were prepared by high-pressure homogenization using an Avestin C-5 homogenizer (Avestin, Inc., Ottawa, ON, Canada). A range of surfactants were used to coat the drug crystals including Lipoid E80® (an egg phosphatide mixture of phosphatidylcholine, phosphatidylethanoloamine and the hydrolyzed lyso [single aliphatic chain]; Lipoid GmbH, Ludwigshafen, Germany), a block copolymer of ethylene oxide and propylene oxide (poloxamer 188 [P-188]; Spectrum Chemicals, Gardena, CA, USA), 1,2-distearoyl-phosphatidyl-ethanolamine-methyl-poly(ethylene-glycol) (DSPE-mPEG₂₀₀₀; Genzyme, Cambridge, MA, USA), poly(lactic-co-glycolic acid) (PLGA; ratio 50:50 of lactide to glycolide; Sigma-Aldrich, St Louis, MO, USA), (1-oleoyl-2-[6-[(7-nitro-2–1,3-benzoxadiazol-4-yl)amino]hexanoyl]-3-trimethylammonium propane) (DOTAP; Genzyme) and cetyltrimethyl ammonium bromide (CTAB; Sigma-Aldrich). To coat the nanosized drug crystals, either alone or in combination, each surfactant (or copolymer) (weight/weight percent) was made up of Lipoid E80 (1.4%), P-188 (0.5%), DSPE -mPEG₂₀₀₀ (0.2%), PLGA (12%), DOTAP (0.1%) and CTAB (0.5%). The nanosuspensions were formulated at a slightly alkaline pH of 7.8 using either 10 mM sodium phosphate or 10 mM HEPES as a buffer. Tonicity was adjusted with glycerin (2.25%) or sucrose (9.25%). Drug was added to the surfactant solution to make a concentration of approximately 2% (weight-to-volume ratio [%]). Lissamine rhodamine B 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium salt (rDHPE; Invitrogen, Carlsbad, CA, USA) was used to label nanoART, which appeared as red fluorescence. In order to synthesize nanoART, a suspension was prepared by adding crystalline drug to a surfactant solution and mixing for 4–7 min using an Ultra-Turrax T-18 (IKA® Works Inc., Wilmington, NC, USA) rotor-stator mixer to reduce initial particle size. The suspension was homogenized at 20,000 pounds per square inch for approximately 30 passes or until desired particle size was reached. For DOTAP-containing suspensions, the homogenized suspension was centrifuged (12,100 × g for 30 min at 5°C) to pellet the drug particles. The supernatant was decanted and surfactant-containing DOTAP was added to the drug pellet. The drug was resuspended by mixing with an Ultra-Turrax T-18.
In the case of EFV–PLGA NPs, EFV (1.25 g) and PLGA (6 g) were dissolved in dichloromethane (50 ml) and added to a 1% poly(vinyl) alcohol solution (500 ml). Particle size was achieved by sonicating at 50% amplitude for 10 min using a 400/600 W sonicator with a ¾ inch high gain probe. The solution was stirred overnight to evaporate the dichloromethane-hardened particles. The suspension was then centrifuged, washed with 18-Ω water and decanted twice. The particles were suspended in 10% mannitol before being frozen or lyophilized for storage. For all nanosuspensions, particle size was measured using a HORIBA LA 920 light scattering instrument (HORIBA Instruments Inc., Irvine, CA, USA; RRI = 1.08 for IDV and 1.20 for RTV and EFV). ζ-potential was measured by diluting 0.1 ml of the suspension into 9.9 ml of 10 mM HEPES, pH 7.4 on a Malvern Zetasizer Nano series instrument (Malvern Instruments Inc., Westborough, MA, USA). Final drug content of the formulations was determined by RP-HPLC (data not shown).