I have been working on EE studies for my liposomal drug. Size of the particle is around 70-100nm and I measure my drug concentration using UV-Vis method. I have tried centrifuging at ~17,000g for 6 hours but found that my particles are still in the supernatant. With this said, I am not able to determine the actual free drug content in my supernatant. Anyone with ideas how to separate encapsulated drug from free drug in my formulation?
Thanks
Another possibility could be ultrafiltration. I have used it to separate liposomes in the same size range from their supernatant using a 30 kDa molecular weight cut off filter (http://www.merckmillipore.com/DE/de/product/Amicon-Ultra-0.5-Centrifugal-Filter-Unit,MM_NF-UFC503024).
I would recommend that using dialysis method and subsequently centrifuging the sample would help you in a better way.
Christina Nadler Hi Chirstina, thanks for the suggestion. I'll try to use this method and may I get an idea on the speed and time length for this centrifugation?
Thanks
Hi Muhammad Saad Khan , I actually tried to dialyze my formulation, in a way where my free drugs will also be dialyzed out. What I did next was to try to lyze my pellet in order to determine the EE but I had problem lysing them. I guess what you meant here is to dialyze my formulation but keeping free drugs in, and then centrifuge and measure supernatant like how I did?
I have used 20800xg, 4°C for 2.5 h for the filtration and 425xg, 4°C, 4 min to collect the solution from the filter. My liposomes pellet with the settings of the filtration though, which is probably why the filtration is slow and it takes 2.5 h for me to reach the retention volume (around 20 ul for the filter mentioned). If yours don't pellet, the filtration should be a lot of faster.
Christina Nadler Thank you so much for the answers. Mine doesn't really pellet and I'll try using this method.
Hello. I have the same question. I am trying to measure the encapsulation/association efficiency into LysoPS nanoparticles of a monoclonal antibody (IgG1) MW 148000 Da. Size of the particle is around 150nm.
I tried discontinous dextran gradient ultracentrifugation but I think that IgG1 precipitates in dextran (recently found out), so i was thinking what other method could I use. In the lab gel chromatography has been used but the previous person that tried with this formulation was having issues with having too diluted fractions. Does anybody have a suggestion for what could be improved in a gel filtration chromatography or what other method could I use to separate?
Thanks
Hello Dr. Corvo. Thanks for you answer.
In the lab we have 30cm height column with (I think) 2.6mm diameter. I read the procedure my labmate used and she loaded half of the column only. I haven't done any chromatography myself yet , I am trying to plan the "most appropriate conditions" to see if I can get some results.
Ultracentrifugation is not something we "trust" in the lab. The reasons that I heard from other are that the nanoparticles seemed to be clotting the pores of the filter (I am not sure if this was proved) so people were not having consistent results.
For the my particulate case of an IgG and nanoparticles of ~200nm size, what type of filter will you recommend? What cutoff size?
Thanks I appreciate all the comments. I am new in this and I am trying to plan my experiments with enough support background information.
Hi Jocelyn Ooi ,
As others noted, you may need much higher forces to sediment these particles.
One additional thing to keep in mind is the density of the particles - liposomes are obviously lipid-based, so the buoyant density can be quite low, which may hinder sedimentation. In this case, one potential solution is to do a flotation, rather than sedimentation experiment where you would increase the density of the sample solution, load the sample in the bottom of a tube, layer less dense solutions on top, and centrifuge - if the particles are less dense than the media they're in, they'll float upward toward the top of the gradient over time. This is a very common practice for lipoprotein isolation and is also used for purifying exosomes.
Hello Dr. Luisa Corvo
I am planning to do centrifugation for next week as you suggested. I found what you were saying about using centrifugation without filters as a common technique to measure entrapment efficiency.
You mentioned you used 2mL of sample and added 20mL of buffer? Why do you have to add more buffer? And why 20mL? Is there any specific calculation to decide the amount of extra buffer? I can imagine that additional buffer gives more room to the protein to separate... Usually, we prepare 1mL of liposomes. I was thinking to centrifuge the whole 1mL or 500uL because I need some volume for another thing. Do you think is appropriate to do it this way?
The particles are around 150-200nm and the protein I am quantifying is a monoclonal antibody.
I read in a paper that they used 14000rpm for 20 minutes. The volume used is not mentioned. They loaded bevacizumab in liposomes.
I appreciate your comments,
Thanks
Dear Milagros
I think you took it wrong because you went "I read in a paper..."
Dr. Corvo said ultra centrifuge and you said about filter and 14000rpm so there's a paradox here.
Your statement refers to Amicon tubes so just be careful.
In best of my knowledge there's no specific way to set a specific volume of liposome and buffer.
Regards
Hi Alireza Mordadi Mordadi. My first questions were about the filters but later I understood what Dr. Luisa was saying about centrifugation without filters, just centrifugation. In the last question I question I asked, the paper I mentioned didn't use any filter, just centrifugation. I was asking about the conditions they used and about why she mentioned adding 20mL more of buffer based on her experience.
Thanks for the comment.
Regards
Dear Dr. Luisa Corvo
. Thanks for your comments, they have been very useful. I will try that and see how it goes.- Badges
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