I'm gonna make use of Amicon ultra-15 MWCO 100 kDa for the final collection of my nanoparticles after their preparation. Now I wonder that up to how many times it is possible to use them for this purpose, as they are quite expensive for me ;-) ..
Usually once. In my case I could have never been sure to remove all nanoparticles from them ever after 1 hour of vigorous shaking. If you can resuspend your particles after centrifugation, just centrifuge them, if not, use dialysis.
There are 3 reasons why it is difficult to reuse these units: damage to the membrane, clogging and microbial growth. You can prevent microbial growth by storing the membrane with a preservative solution, such as 0.02% sodium azide. Preventing damage to the membrane means never touching the membrane with any solid object. Clogging will become apparent when the filtration rate drops substantially. I don't know of any way to reverse this.
Thanks so much dear fellows
Sad news! so I just can be hopeful that my supervisor would spare more bucks!
My nanoparticles (NPs) are resuspendable after centrifugation but simple centrifugation is not efficient in collection of the smaller NPs, and this affects the yield of my entire preparation process. So I wanted to make sure of collecting all NPs in a fast and efficient way. Unfortunately dialysis couldn't be a choice as it's laborious and time consuming and that the NPs would start releasing their drug content.
It seams that the challenging part of ultrafiltration is reconstitution of the encapsulated particles. Any further detailed technical suggestions regarding this issue please??
Also I need to wash out the remaining emulsification surfactant and input drug. So I thought that using Amicon filters might be helpful so as to wash the collected NPs right after with water or PBS for 2-3 times. Do you have any idea or suggestion regarding this issue??
Thank you
I think in this case you just need to optimize the centrifugation condition in a way to reach a completely clear supernatant. The particles that you may have in a clear supernatants are the ones with a very small size that you should not concider them as your particles. I mean, although you still may have particles in supernatant but they are not of the size of majority of your particles, they are just the ones that cause standard errors in every experiments.
Thank you Ms. Sabaeifard
You know? the fact is that I don't have access to a high speed Falcon centrifuge, thus I have to divide the whole volume of sample into 2 ml microtubes and centrifuge them at 15000 rpm for 33 min. this is my optimized speed and time as the supernatant becomes quite clear, even when the drug is doxorubicin (red-orange colored) I see no color or opaqueness. But the real problem is collection of the tiny sediments from so many microtubes (sometimes 20 ones!), which even with neat and careful handling adversely affects collection efficiency. Also the washing issue remains a problem if I want to do it this way too. Therefore, as our animal study stage is close, I reckoned that using Amicon filter might be helpful.
If there is a preparative ultracentrifuge available at your university, you could pellet the nanoparticles very quickly in one tube.
I have the same problem regarding adverse nanoparticle loss following ultracentrifugation. Did you find an optimal time and speed in the end? I am trying to maintain a main particle population of ~150-300nm using 15000rpm for 20mins but quite simply it doesn't work well. Also, If I spin for too long then the pellet becomes difficult to resuspend again. Any suggestions for spin speed and time or membrane filtration? Thanks in advance, S
There is maximum centrifugal force of centrifugal filter. For example Amicon ultra-15 has Maximum relative centrifugal force of 4000 x g from swinging-buket rotor and 5000 x g from fixed-angle rotor.(http://www.merckmillipore.com/KR/en/product/Amicon-Ultra-15-Centrifugal-Filter-Units,MM_NF-C7715?bd=1).
You should check your centrifuge and rotor to find relation of RCF and RPM.
You can increase speed of filtrate volume to increase RPM over maximum force of filter. But, this can damage membrane and you sample can overflow into centrifuge.
Some people reuse filter unit for same material If there is no damage in membrane.
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