Hi

look at:

Purification, Fragmentation and Isotopic Labelling of Monoclonal Antibodies

JAMES W. GODING, in Monoclonal Antibodies (Third Edition), 1996

and consider, these kind of beads that you wanna use separate agent basic on their molecular weight.

on the other hand 10ml is a big volume for a simple lab working.

Hi Aslude,

First of all, small molecules removal from liposomes has been performed all the time since the 1970s, so there is plenty of experience. There are several approaches and several concerns/issues/opportunities.

1. Regular size exclusion chromatography can be extremely efficient, and will remove all of external small molecule, such as a fluorescent dye. Typically, column volume is ten-twenty times the sample volume, or even more, if you want clear separation between your first (liposome) peak and the second peak of the free dye, with nothing in between. The downside is dilution. Concentration of your liposomes after the separation will drop by several times.

2. Alternative is spin column. This was published by Fry, White and Goldman, Rapid separation of low molecular weight solutes from liposomes without dilution. Anal Biochem. 1978 Oct 15;90(2):809-15. Disposable syringes with several ml of gel are used; purification level is ~100-fold (in terms of the amount of free small molecule that passes through the column without liposomes). Dilution is almost nonexistent. So, using several syringes at the same time will get you there; some centrifuges allow running a dozen of tubes at once, or more. So you can get your 10ml with ten syringes – but maybe you could just increase lipid concentration and reduce the volume? 50-100mg/ml lipid may be feasible...

3. When internet was relatively new, before www was widely used, there were so-called “usenet newsgroups”, and in one of those, bionet.molbio.methds-reagnts, I had written a small note on the subject of spin columns, it can be found here: http://www.bio.net/mm/methods/1993-June/005901.html - it has some more technical details.

4. In a lab class module here at UVA that I teach BME undergraduates for almost a decade, on making and targeting of liposomes, we use 100mg/ml calcein, which is obviously more than 0.1 M. And there is one more issue – some calcein sources seem to have some (polymer?) impurity, so G25 might have too narrow pores to retain this impurity, and would not give the students a clear separation between the liposome band and the free dye, as they travel through the columns. We use bio-gel A with lower numbers, which will be rigid enough even for a spin column. Incidentally, if your iron oxide nanoparticles are small enough, you might be able to get rid of excess of those, too, if you use a gel with large enough pore size.

5. Perhaps magnetically collecting and washing these liposomes may avoid chromatography altogether?

Hope this helps, please let me know if you have more questions.

Sincerely,

Sasha Klibanov

Hi Asude,

Look at: https://www-ncbi-nlm-nih-gov.insb.bib.cnrs.fr/pubmed/16533059

We use Sephadex G-75 and 40 mM calcein, but 100 mM should work too. For 10 ml sample you should use a column of 16 × 600 mm. 

Thank you for all the answers and especially Mr. Klibanov for taking your time to reply so extensively!

Asude,

thanks!

Glad I could help! Best regards to Twan and Fabian!

Sincerely,

Sasha

The GE Healthcare chromatography handbooks are also a very good resource for general theory on almost any chromatography separation method you can think of. They also describe in detail the different media that are available from GE and provide simple protocols to get you started in the right direction.

Their PDF on size exclusion methods is very thorough. I prepared fluorescien loaded LUVs in grad school (some time ago) and used their basic SEC guidelines to separate the vesicles from the free fluorescien.

https://www.gelifesciences.com/en/us/solutions/protein-research/knowledge-center/protein-purification-methods/chromatography-principles.

If I have only one clear peak in DLS (of given liposomes) after extrusion of lipid suspension, do I still need the gel filtration? It seem not? which sephadex G50 is best, e.g. medium, fine or coarse. Please suggest. Alexander Klibanov Alexander Klibanov

I do not think 100 encapsulation, so I think yes.

thanks

Pramod, the goal of gel filtration is to remove non-entrapped materials, such as calcein dye as above. If they are small molecules (as pure calcein dye) they are most likely too small to be picked up by DLS as a separate peak. You are right on 100% encapsulation: it depends on the lipid concentration and liposome size, but passive entrapment (if you first make MLVs in the dye solution aqueous medium and then Nuclepore them) is never 100%, most likely under 50%, often 10%...

Best of luck with your experiment! A.K.

thanks, Alexander Klibanov I am planning to use sephadex G 50 fine for SE for 100-200nm liposomes. You are absolutely correct that the small uncapsulated dye molecules are not captured using DLS. Do you recommend any specific literature stating, which sephadex for which size of particles. For my knowledge sephadex G50 and G25 both will work! but do not have any specific literature on that.

thanks

Pramod

Pramod, in an ideal world, you would be perfectly correct. Unfortunately sometimes calcein that we happen to use is far from pure, and looks like it contains a polymer (we ran into this issue here, in a BME lab class module that I teach). That polymer seemed to be large enough to go out of the column with the void volume of G25 or Bio-Gel P6, ie with liposomes. So the bigger the pores, the better. Then again, G75, that was mentioned above, might not be sturdy enough if you plan a spin column. Things are always more complicated than they look, sorry. But that is our real world, and you can make it work for sure! :-).

thanks Alexander Klibanov .

Hello everyone,

Its very nice and useful to see all the above discussion. I think I have a very similar confusion and thought I could probably add on my question here to the discussion. I am trying to load DPPC liposomes with fluorescein molecules. I would like to remove the encapsulated free fluorescein from the encapsulated liposomes.

1) I have the Sephadex G25 spin columns from GE healthcare (cytivia). Is this column good enough to separate these two molecules apart?

2) The diameter of my liposomes are 150-400 nm (from DLS) depending on my batch. I am sorry this could be a silly question but I am not sure how to convert the size to molecular weight. I see that most of the Sephadex/sepharose beads or spin columns are based on molecular cut-off. The molecular weight of DPPC lipids are 700 g/mol but I know it has to be converted to molecular weight in Daltons to say which cut-off of Sephadex would suit me.

Can someone please help me understand this?

Shwathy, liposomes are much bigger than each individual DPPC molecule. A 100 nm liposome will have over 1e5 of lipid molecules in it, and plenty of water inside the inner core. So there is many orders of magnitude size difference between liposome and fluorescein. Therefore it is guaranteed that liposomes will not be going into the core of the granules of any sephadex gel. And fluorescein dye individual molecules are going to enter the granules and be retained for longer in flow (unless for whatever reason fluorescein forms a precipitate - eg if buffer pH is wrong or buffer composition includes an undesirable counter-ion and it forms a water-insoluble salt). Other than that - should work. Run a control which does not have liposomes first.

Great thanks Alexander. That helps a lot. That exactly what I thought and went ahead and ordered the Sephadex G25 spin columns. But I am a bit worried now pondering if I should have ordered the G50 columns. The questions still comes back to how do I calculate the molecular weight of a liposome? I do understand they are much bigger but how much bigger? I have seen conversion of FITC dextran molecular weight to nanometers by a hydrodynamic equation. Is there something like that for liposomes?

Shwathy Ramesan , the molecular weight of developed liposomes is not a constant value as you may get size variation (as per batch), but as Alexander suggested, you should be able to resolve the dye and liposomes using G50 or G25. both will work. The main target is to remove small molecules ( fluorescein) which will run far slow than the liposomes in column.

https://www.researchgate.net/post/which_Sephadex_is_most_suitable_to_separate_out_75_kDa_protein_from_liposomes important figure

Hello Pramod, thanks a lot for that answer. I will probably give a go with sephadex G25 and will see.

Alexander Klibanov Pramod Kumar I am using Sepharose CL-4B for separation of unencapsulated drug from my PLGA nanoparticle formulation. The size of the nanoparticles obtained in eluate after the separation from freshly prepared columns differs from the subsequent runs in the same column. Does that mean, I should use the Sepharose columns for single time use only? What is the general practice of using Sephadex column? How many times the samples are run on single column?

Bivek, this may be an issue with the concentration of liposomal lipid vs column volume. Sepharose can adhere some lipid initially (not everything comes out, if you load low concentration on a large column). With further runs, when the gel adsorption sites are saturated, this problem goes away. You can reuse the column verymany times, just have in mind the possibility of such lipid exchange. Also, Sepharose is best to separate liposomes from larger things, like proteins. Small molecule drug removal can be done with gels that have smaller pores (Eg Sephadex - which can also be reused).

Hello! I am beggining to work with liposomes, and to practice I wanted to start by encapsulating calcein with pure DMPC, DPPC or DSPC liposomes. I do them by thin film method, and then extrusion (sometimes with, sometime without freeze-thaw).

When I tried different purification methods, I finally came up with the spin column protocol you spoke here about. It seemed to work (I had a yellow eluted fraction, while the calcein seemed to remain in the column), but when I tried to measure the fluorescence intensity increase when adding Triton X-100, it did not increase (and even decreased). Is it because calcein is not quenched inside the liposomes? Maybe low encapsulation efficiency? If so, how could I increase it?

Thank you very much.

Alexander Klibanov Pramod Kumar

Hello Eduardo! Do you have cholesterol in your liposomes? If not, extrusion of "solid" lipid (eg DSPC) liposomes at room temperature is almost impossible, you have to heat it above 56C.

Where I would start - first, make MLVs by thin film method, then do centrifugation (they are large, they will sediment) and check calcein uptake and if fluorescence goes up when liposomes are broken by TX100. Next question is the control spin column - just calcein sample, no liposomes. Sometimes, if you use a small-pore gel, like G25, there is a polymer in some calcein preps, which will co-elute with liposomes, and it will not be dequenched when you add calcein. Hope this helps. And - do not try to extrude MLVs after centrifugation - in that case, internal cancan will leak.

Thank you very much for your rapid answer Alexander Klibanov

I perform the extrusion with the Avanti heating block, so yes, the temperature is above the transition temperature (most of my trials have been done with DMPC and DPPC because of this).

I will definitively try both suggestions and hope they shed light on the matter!

I am trying to purify my calcein-loaded giant multilamellar vesicles with Sephadex G25, but I found only membrane fragments.

Does anyone have any advice for me?

grazie

Hi,

I tried to purify my calcein loaded LUVs (around 100nm) after extrusion. Firstly, I used centrifugation but I got no pellet. So I would like to try chromatography but I am not which sephadex is suitable for separating calcein from my vesicles (100nm). I have G-50, G-100, and G-200. Do you have any idea what is better to choose?