After preparing the oil (10mL DCM + 720 mg PLGA 75:25 acid terminated + 72 mg CsA drug) and water (90mL of 0.5%PVA) phase solution, I used the ultrasonic emulsifier while the beaker was placed in ice (100 Watt for 3.5 minutes). I always obtained a stable, milk-like solution with no visible particles at the bottom. Then, I stirred the solution for DCM evaporation for 18 hours.
- However, after centrifuging the four 50ml tubes of the same sample at 10xg for 15 minutes (without cooling option), all the pellets were super-solid and did not re-disperse in water, even with long sonication period on ice.
- After three unsuccessful washing attempts (since no pellet dispersion), I discarded the supernatant and froze the pellet itself at -80 degrees Celsius for 0.5-1 hour before starting lyophilization. Unfortunately, I ended up with a strongly crystallized pellet instead of a fine powder.
Regarding the second issue, I believe it can be resolved if I can successfully re-disperse the pellet and add particular amount of water before lyophilization.
Please, let me know how to re-disperse the pellet in water during washing &&& amount of water to be added to each pellet in the four tubes before starting lyophilization.
Thank you.
Well, where do I start?
10xg for 15 min is not enough to sediment PLGA nanoparticles with a size around 200 nm. If the pellet material will not redisperse, then the particles have aggregated due to the centrifugal force, which means they were not stable enough. Even if you produce nanoparticles, if you do not add a cryoprotectant, you will end up again with aggregated material after lyophilization. Do not lyophilize nanoparticles, you will destroy them as single nanoentities.
Thank you, Omar Gonzalez-Ortega, for your answer.
So, I should use higher centrifugation speed (16k xg, for example) with shorter duration (10 min) to get the nanoparticles and avoid aggregation. You also suggest add cryoprotectant (Sucrose, for example) to the water phase to avoid aggregation during lyophilization.
Please, let me know if you have further comments.
Thank you.
If you are sedimenting particles at 10xg in 15 min, they are not close to being nanoparticles. You are most likely producing microparticles. When you produce particles with a size close to 100 nm you will realize that you need a higher relative centrifugal force and longer time for the particles to sediment.
The steric stability provided by PVA (you do not have enough PVA on the surface of the particles) is not enough as you cannot redisperse the particles. Your particle preparation protocol is therefore wrong.
A cryoprotectant does not prevent aggregation, it only allows to produce not to big aggregates. Do not dry nanoparticles with or without cryoprotectants.
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