Methods and Materials: Microscopic & Drug Distribution Studies

Presented By:Rich DominiakLaura KuczynskiJohn Roszko

February 14, 2006

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

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.

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

Final Matrix

The final sections were 3 mm x 1 mm x 10 µm.

The 1 mm represents the original depth of the matrix.

Observation

They were observed under scanning electron microscopy (SEM).

Dried to enable high vacuum conditions for SEM.

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)

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

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

Questions

?