lycol 1000 succinate (TPGS) in the micelle composition, the burst release of 17-AAG from the micelles was significantly reduced. These PEG-DSPE/TPGS mixed micelles have the potential to be further functionalized to achieve active targeting of 17-AAG to tumor cells. 2. Materials and methods 2.1. Chemicals 1,2-Distearoyl- sn -glycero- Celecoxib phosphoethanolamine- N – [methoxy(polyethylene glycol)-2000] (ammonium salt, PEG-DSPE) 281 nm, respectively. The standard curve of 17-AAG in phosphate buffer was linear between 0.1 M and 10 M. Micelle samples were diluted with the HPLC mobile phase, and injected into the HPLC system. 2.4. In vitro release of 17-AAG from micellar nanocarriers The release of 17-AAG from micelles was evaluated by a dialysis method ( Shin et al., 2009 ). The drug-loaded micelles were prepared with 5.3–12.5 mM PEG-DSPE, 10.6–25.0 mM TPGS and 0.6 mM 17- AAG. Post micelle preparation, each sample was diluted with HBS, a volume of 1 ml of which was loaded into a 3 ml Slide-A-Lyzer dial- ysis cassette (Pierce, Rockford, IL) with a MWCO of 20,000 g/mol.
Each cassette was placed in 1.0 l phosphate-buffered saline (20 mM, pH 7.4), which was changed every 4 h to ensure the sink condition for the drug. A sample of 20–40 l was drawn from each cassette at 1 h, 2 h, 4 h, 6 h, 9 h and 12 h, which was replaced by the same volume of fresh HBS. The concentration of 17-AAG in each sample was quantified by HPLC as described above. The fraction of the drug remaining inside the dialysis cassette as a function of the release time ( t ) was fitted to the first-order kinetics using the following equation: C t / C 0 = e − kt , where C t and C 0 are the drug concentration within the buy Celecoxib dialysis cassette at the sampling time ( t ) and at the initiation of the study, respectively; and k is the first-order release rate constant. The release rate con- stant ( k ) was derived from the best-fit nonlinear regression (Sigma Plot, San Jose, CA), which was used to calculate the release half-life ( t 1/2, release = 0.693/ k ). 2.5. Micelle size measurement The size of the micelles was determined by dynamic light scat- tering using a ZETASIZER Nano-ZS (Malvern Instruments Inc., UK) equipped with He–Ne laser (4 mW, 633 nm) light source and 90 ◦ angle scattered light collection configuration. The hydrodynamic 2 172 T. Chandran et al. / International Journal of Pharmaceutics 392 (2010) 170–177 diameter of micelles was calculated based on the Stokes–Einstein we first examined the in vitro release profiles of 17-AAG from PEG- equation.
All measurements were repeated three times, and data DSPE micelles. The release of 17-AAG into the sink was examined by were analyzed in terms of volume-weighted particle size distribu- monitoring the drug concentration inside the dialysis cassette. For tion. drug molecules to be released into the sink, they need to be first liberated from the micelles and subsequently diffuse across the 2.6. Storage stability of 17-AAG-incorporating micelles purchase Celecoxib dialysis membrane. To ascertain that the dialysis membrane was not a significant barrier during the release process, the release of Freshly prepared 17-AAG-loaded PEG-DSPE/TPGS micelles were free 17-AAG was also studied as a control. We found that, the incor- incubated at 37 ◦ C for two weeks or stored at 4 ◦ C for five weeks.
poration of 17-AAG into PEG-DSPE micelles substantially reduced At predetermined time points, the samples were centrifuged at the release rate of 17-AAG into the sink, compared to that of free 17- 12,000 × g for 10 min, and the supernatant samples were stress reduction analyzed AAG ( Fig. 1 A), indicating that the release of 17-AAG from micelles for the changes in the particle size and drug content. is indeed the rate-limiting step during the drug release from the dialysis cassette. Importantly, the final PEG-DSPE concentration in 2.7. Cell proliferation assay the micelle dispersion inversely affected the release rate of 17- AAG from the micelles. As PEG-DSPE concentration was increased Human ovari