Dear Pharmacis,

The answer is NO. You can not proceed without centrifuge and washing. Please follow the following steps:

Nanoparticle Collection

1-Split the hardened nanoparticles into two Oak Ridge centrifuge tubes (30 ml nominal volume) and balance to within 0.1 g.

2-Centrifuge nanoparticles in a fixed-angle rotor for 15 min at 17,000 x g. Longer centrifugation times will result in the collection of a higher fraction of smaller nanoparticles.

3-Discard the supernatant, being careful not to disturb the nanoparticle pellet (water may be poured orpipetted off). Add 15 ml diH2O and use a water bath sonicator and/or vortexer to completely resuspend the nanoparticles.

4-Combine the contents of the two centrifuge tubes into one and repeat steps 2.1-2.3 2x more, for a total of three washes in 30 ml of diH2O each time. The fluid volume of the last pellet resuspension should be 4-5 ml.

A weight ratio of 1:2 trehalose:polymer may be added at this point as a cryoprotectant (freeze a small aliquot of nanoparticles without trehalose for SEM imaging). Ice crystals that form during the freezing process may damage the particle surface and induce aggregation, and inclusion of trehalose has been shown to improve the uniform and complete resuspension of PLGA nanoparticles13.

5-Transfer the nanoparticles to a preweighed 5 ml centrifuge tube and freeze at -80 °C for a minimum of 30 min.

6-Moving quickly so as not to let the frozen contents melt, uncap the tube and cover the top by securing lab tissue across the top with a rubber band. **If any melting occurs, refreeze before placing in the lyophilizer.

7-Lyophilize 72 hr for a 5 ml volume. Store lyophilized particles in Parafilm-wrapped tube at -80 °C.

http://www.jove.com/video/51015/plga-nanoparticles-formed-single-or-double-emulsion-with-vitamin-e

Hoping this will be helpful,

Rafik

Dear Pharmacis,

The answer is NO. You can not proceed without centrifuge and washing. Please follow the following steps:

Nanoparticle Collection

1-Split the hardened nanoparticles into two Oak Ridge centrifuge tubes (30 ml nominal volume) and balance to within 0.1 g.

2-Centrifuge nanoparticles in a fixed-angle rotor for 15 min at 17,000 x g. Longer centrifugation times will result in the collection of a higher fraction of smaller nanoparticles.

3-Discard the supernatant, being careful not to disturb the nanoparticle pellet (water may be poured orpipetted off). Add 15 ml diH2O and use a water bath sonicator and/or vortexer to completely resuspend the nanoparticles.

4-Combine the contents of the two centrifuge tubes into one and repeat steps 2.1-2.3 2x more, for a total of three washes in 30 ml of diH2O each time. The fluid volume of the last pellet resuspension should be 4-5 ml.

A weight ratio of 1:2 trehalose:polymer may be added at this point as a cryoprotectant (freeze a small aliquot of nanoparticles without trehalose for SEM imaging). Ice crystals that form during the freezing process may damage the particle surface and induce aggregation, and inclusion of trehalose has been shown to improve the uniform and complete resuspension of PLGA nanoparticles13.

5-Transfer the nanoparticles to a preweighed 5 ml centrifuge tube and freeze at -80 °C for a minimum of 30 min.

6-Moving quickly so as not to let the frozen contents melt, uncap the tube and cover the top by securing lab tissue across the top with a rubber band. **If any melting occurs, refreeze before placing in the lyophilizer.

7-Lyophilize 72 hr for a 5 ml volume. Store lyophilized particles in Parafilm-wrapped tube at -80 °C.

http://www.jove.com/video/51015/plga-nanoparticles-formed-single-or-double-emulsion-with-vitamin-e

Hoping this will be helpful,

Rafik

Thank you sir. Now I understood it.

Please careful consider that the settings for the centrifugation step era not valid for all type of nanoparticles.

Each type of nanoparticles requires different conditions, the influence of the type of suspending medium should be also considered.

Thank you sir. Can you provide any article or material related to it?

No. You can not determine entrapment efficiency of nanoparticles without washing and centrifugation. Because these lyophilized suspension contain powder of free drug also it may nanoparticles containing surface adsorbed drug.  Washing is also required to remove surface adsorbed drug. 

Thank you Giulia  and all researchers. Is it possible that free drug remain even after three times washing?

of course it should be this way.

If drug is very strongly bound to nanoparticles, you will always dilute free drug by the ratio of washing liquid to liquid bound to particles.

In addition there maybe an adsorption equilibrium, i.e. drug will dissolve into washing liquid.

Titus, I am not getting this. Can you please elaborate more....... 

If drug is very strongly bound to nanoparticles, you will always dilute free drug by the ratio of washing liquid to liquid bound to particles.

In addition there maybe an adsorption equilibrium, i.e. drug will dissolve into washing liquid.

Regards

lets put in more easier terms

if you are washing a sediment and remove washing liquid in a first step the remaining washing liquid will have the same concentration as the washing liquid removed. Washing in a second step with pure liquid, this dilutes the washing liquid remaining from the first step and so forth. That is washing liquid will still contain substance to be removed according to the liquid dilution achieved in several steps. If in addition drug adsorbed will desorb you will have a higher concentration in washing liquid than expected by consecutive dilution steps only.

Titus I got your point. Thank you so much.  Have you worked on probe soincator for nanoparticles  preparation?

Did not worked on nanoparticle preparation only on dispersion and characterization.

My synthesis background is weak.

Its ok sir.