1. Methods and Materials: Microscopic & Drug Distribution Studies
Presented By:
Rich Dominiak
Laura Kuczynski
John Roszko
February 14, 2006

2. Microscopic Study Procedure
1st – 10 µm thin sections were obtained using a cryogenic microtome set at -25 °C with a microtome knife
2nd – 1 mL of embedding medium was poured onto the chucks
30 seconds later it became an
opaque solid

3. Procedure cont’d
Next, about 3 mm x 1 mm x 1 mm of release matrix was taken from various location in the original 1 cm x 1 cm x
1 mm slab.
These pieces of the matrix were then placed on the planed embedding medium, which was in contact to the planed surface.

4. Procedure cont’d
More hardening medium was added to the planned surface.
Sections 10 µm in thickness were cut and stuck to the microtome knife
They were then taken off of the knife with a glass slide at room temperature

5. Final Matrix The final sections were 3 mm x 1 mm x 10 µm.
The 1 mm represents the original depth of the matrix.

6. Observation
They were observed under scanning electron microscopy (SEM).
Dried to enable high vacuum conditions for SEM.

7. Drying Drying Procedure Water to 100% ethanol
100% ethanol to 100% amyl acetate
Cooled to 4 °C and filled with liquid CO2
Exhausted the CO2 vapor while adding CO2 liquid
Temperature and Pressure increased over 20 minutes to 40 °C and 55 atm, respectively (when amyl acetate was gone)

8. Drying cont’d
After 20 min it is assumed all amyl acetate is gone because all the CO2 is vapor
Pressure and Temperature went back to ambient conditions
Sample is ready for analysis

9. Drug Distribution Studies
Separating matrix into four sections
Removed and frozen on dry ice to terminate drug release
Serially cut with cryomicrotome into four sections for analysis
The samples were set in 0.9% NaCl solution for 3 days
The medium was then filtered and protein concentration was determined by UV spectroscopy at 220 nm

10. Questions ?