Stirring for 30 min at 25, pre-determined Renin protein: drug molar high. A The concentration of ethanol in the final L solution was maintained b5% v / v dispersions obtained contained casein protein and celecoxib: drug molar ltnissen in the range of 1:0.5 to 1:16. These dispersions are all transparent. As a contr On an L Solution of celecoxib was in 100% ethanol was added dropwise, under the same conditions in Hepes buffer. All dispersions contr The drugs were turbid, suggesting the poor dispersibility and L Solubility of celecoxib in f Ssriger L Solution, casein-free. 2.2. Freeze-drying of proteins and protein complexes were drug micellar dispersions followed by freezing in liquid nitrogen by drying in a Christ Alpha 1 April lyophilizer for 24 h, lyophilized. The samples were stored at 4 for more than 6 months then stored in HEPES buffer or a w Ssrigen L Solution, back to the original level, or an hour Higher concentration of 50 mg / ml. Resuspend is carried out by weighting the powder, adding a measured amount of buffer or DDW, and stirred for 30 minutes at room temperature.
The resulting suspensions were transparent and stable for at least 3 weeks. 2.3. Turbidity measurements of turbidity was measured using a spectrophotometer Ultrospec 2100 Amersham Biosciences each at a wavelength Length of 600 nm with an optical path length Length of 1 cm carried out. The Notch Pathway samples were observed for 10 mg / ml casein 1:08 moleratio with celecoxib and 10 mg / ml casein Mizellenl Solution as a control. Further measurements on samples were lyophilized and resuspended performed in these samples. 2.4. DLS and zeta potential, size-distribution rights E and zeta potential were at using a combined DLS and zeta potential analyzer, 25 For DLS measurements, the intensity t of the scattered light with an avalanche photodiode detector, detects used at a fixed angle of 90 °. Wavelength Length 658 nm laser at 90 mW in operation. Single, dual or tri-modal distributions were calculated from the intensity Tsschwankungen of light by Nicomp cumulants analysis of autocorrelation function calculated scattered. The data analysis was performed using the method CONTIN. Zeta potential measurements were performed in triplicate on formulations in HEPES buffer, pH 7, 10 mg / ml protein, carried out protein: molar ratio of 1:8 drug before and after resuspension in the same buffer. 2.5. A microscope optical microscopy Olympus BX51 light was operated in differential optical Nomarski interference contrast. A decrease of 5 l was applied to a Objekttr Makers and set with a sliding lid.
The images were digitally in a mag AREA 10-60 times taken with an Olympus DP71 digital camera with an optical microscope. The image processing was with cell A. 2.6. Direct imaging cryogenic transmission electron microscopy samples were either in an automated vitrification device controlledenvironment t or in a system prepared glass house, under the conditions of controlled temperature and humidity POSE to avoid the loss of volatiles. 5 l of the suspension was placed on a copper grid coated with a film TEM carbon atoms perforated placed and then transferred with filter paper to form a thin film of liquid sample. The grid was immediately in liquid ethane at its freezing temperature fell form a vitrified sample, and then stored until assayed in liquid nitrogen. Some specimens were examined in a Philips CM120 transmission electron microscope has.