Index
Materials
films of PLLA
films of PLGA
PBS (pH 7.4)
TC-treated 12-well plates
forceps
aluminum foil tape
automatic pipetters
Safety
1. Wear disposable nitrile or latex gloves at all times
2. Wear safety glasses at all times.
Experimental Design
· The following table lists the times when PBS must be changed and when polymer film samples must be removed from PBS, dried, and weighed, as described below.
· Three films of each type (PLLA and PLGA) will be dried and weighed at each appropriate time point.
Overall Strategy
· Measure degradation of PLLA and PLGA films in PBS over 4 wk period.
· Change PBS every 8 hrs for the first 24 hrs.
· Change PBS every day for the first 1 week (except Sunday).
· Change PBS every 2-3 days for remainder of study.
· Recover and remove 3 PLLA and 3 PLGA films every week.
Session 1
1. Determine the number of samples of each polymer that you will need. Consult the charts and protocol below. Remember, each measurement with each type of polymer should be in triplicate.
2. Prepare films as described in PLLA/PLGA Polymer Film Fabrication Part I.
Session 2 (or Friday for Tu/Th Group 5)
1. Detach films from glass coverslips as described in PLLA/PLGA Polymer Film Fabrication Part II.
2. Samples should be stored in vacuum oven.
1. See charts below.
NOTE: For M/W Groups 3, 5, and 6 and
Tu/Th Groups 1, 2, 3, and 6, “Day 1” will be Session 3 of the Polymer Module
and will fall on a M or Tu. Follow the
chart below.
|
TIME |
SESSION |
CHANGE PBS |
DRY SAMPLES |
WEIGH SAMPLES |
|
Day 1, ~ |
3 |
|
|
X |
|
Day 1, ~ |
|
X |
|
|
|
Day 2, ~8 am (after you wake up) |
|
X |
|
|
|
Day 2, ~ |
|
X |
|
|
|
Day 3 |
4 |
X |
|
|
|
Days 4, 5, 6, 7 (except Sunday) |
|
X |
|
|
|
Day 8 = Week 2 |
5 |
X |
X |
|
|
Day 10 |
6 |
X |
|
X |
|
Day 15 = Week 3 |
7 |
X |
X |
|
|
Day 17 |
8 |
X |
|
X |
|
Day 22 = Week 4 |
9 |
X |
X |
|
|
Day 24 |
10 |
X |
|
X |
|
Day 29 = Week 5 |
11 |
X |
X |
|
|
Day 31 |
12 |
|
|
X |
NOTE: For M/W Groups 1 and 2 and Tu/Th Group 4, “Day 1” will be Session 3 of the Polymer Module and will fall on a W or Th. Follow the chart below.
|
TIME |
SESSION |
CHANGE PBS |
DRY SAMPLES |
WEIGH SAMPLES |
|
Day 1, ~ |
3 |
|
|
X |
|
Day 1, ~ |
|
X |
|
|
|
Day 2, ~8 am (after you wake up) |
|
X |
|
|
|
Day 2, ~ |
|
X |
|
|
|
Days 3, 4, 5 (except Sunday) |
|
X |
|
|
|
Day 6 |
4 |
X |
|
|
|
Day 7 |
|
X |
|
|
|
Day 8 = Week 2 |
5 |
X |
X |
|
|
Day 13 |
6 |
X |
|
X |
|
Day 15 = Week 3 |
7 |
X |
X |
|
|
Day 20 |
8 |
X |
|
X |
|
Day 22 = Week 4 |
9 |
X |
X |
|
|
Day 27 |
10 |
X |
|
X |
|
Day 29 = Week 5 |
11 |
X |
X |
|
|
Day 34 |
12 |
|
|
X |
NOTE: For Tu/Th Group 5, “Day 1” will be Session 2 of the Polymer Module and will fall on a Tu. Follow the chart below.
|
TIME |
SESSION |
CHANGE PBS |
DRY SAMPLES |
WEIGH SAMPLES |
|
Day 1, ~ |
2 |
|
|
X |
|
Day 1, ~ |
|
X |
|
|
|
Day 2, ~8 am (after you wake up) |
|
X |
|
|
|
Day 2, ~ |
|
X |
|
|
|
Day 3 |
3 |
X |
|
|
|
Days 4, 5, 6, 7 (except Sunday) |
|
X |
|
|
|
Day 8 = Week 2 |
4 |
X |
X |
|
|
Day 10 |
5 |
X |
|
X |
|
Day 15 = Week 3 |
6 |
X |
X |
|
|
Day 17 |
7 |
X |
|
X |
|
Day 22 = Week 4 |
8 |
X |
X |
|
|
Day 24 |
9 |
X |
|
X |
|
Day 29 = Week 5 |
10 |
X |
X |
|
|
Day 31 |
11 |
|
|
X |
Day 1
1. Films should have been stored in the vacuum oven after their fabrication and removal from the glass coverslips. Weigh each sample film and record.
2. Set aside films for initial time point measurement and store in plates with aluminum foil tape.
3. Record the transparency, color, shape, and any unique features of each film. Estimate the diameter of each film.
4. Place one film in each well of a TC-treated 12-well plate. (Wells should NOT be covered with aluminum foil tape.)
5. Add 3 mL of PBS to each well.
6. Push each film to the bottom of the well with your finger or a pipet tip to ensure that both sides of the film are wetted.
7. Place the 12-well plates on shaker set at 100 rpm in the 37 oC room in Keck 227.
8. Save the 12-well plates with aluminum foil for use later in the experiment.
Day 1 and following
1. Measure the pH of the PBS solution in wells containing PLLA and PLGA films. You may group samples, as you deem appropriate. When you feel reasonably confident that the pH is constant, you may stop routinely measuring pH.
NOTE: The byproducts from the in vitro degradation of PLLA and PLGA are acidic, and therefore may change the pH of the PBS solution. An increased pH may, in turn, accelerate the degradation rate of the polymers.
2. Change PBS solution as often as given in the charts above. To change solution, remove PBS using a pipet; replace with 3 mL of fresh PBS. Be careful not to “suck up” the film into the pipet when you are removing the PBS.
3. Record the transparency, color, shape, and any unique features of each film. Estimate the diameter of each film.
4. Push each film to the bottom of the well with your finger or a pipet tip to ensure that both sides of the film are wetted.
5. As films become more degraded, they will be harder to manage. Ideally, the films should be flat; however, too much manipulation to unfold or unwrinkle a film may lead to its disintegration.
Days 8, 15, 22, 29
1. Retrieve plates with aluminum foil circles. If more wells or plates with foil are needed, cut aluminum foil tape into circles that fit snuggly into the bottom of the wells of a 12-well plate. Attach aluminum foil circles to the bottom of the wells.
2. Carefully remove films for appropriate time point from the 12-well plates containing PBS using tweezers. As films become more degraded, they will be harder to manage. They may collapse when you lift them out of the well; do NOT try to unfold or unwrinkle, as they may disintegrate.
3. Place films in foil-covered wells.
4. Leave films in hood to air dry until the end of the afternoon in lab.
5. Dry films in vacuum oven for a minimum of 24 hrs. Films may stay in the vacuum oven for several days or weeks.
Days 10, 17, 24, 31 or Days 13, 20, 27, 34 (M/W Groups 1
and 2 and Tu/Th Group 4)
1. Weigh the recently dried samples.
2. Record the transparency, color, shape, and any unique features of each film. Estimate the diameter of each film.
3. After weighing the samples, return them to their foil-covered wells. Keep the films in the vacuum oven until the end of the experiment (Day 31) for GPC testing.
Day 31 or Day 34 (M/W Groups 1 and 2 and Tu/Th Group 4)
1. Select two or three films of each polymer type (PLLA, PLGA) from each time point. Dissolve the films together in 1 mL chloroform to 15-25 mg/mL in a 5 mL GPC vial.
2.
Ensure that the polymer is
completely dissolved; this may take up
to 1 hr. Tiny flecks of material may
remain in some vials (particularly the ones containing the PLGA films). It is
imperative that these solutions are filtered before they are run on the GPC.
3. Measure the retention time of each polymer sample using the GPC. See “GPC for PLLA and PLGA.