If the drug has sufficient aqueous solubility, it can be removed by dialysis, if it is not tightly stuck to or inside the liposomes. GIve the dialysis at least 2 days, with rapid mixing. Alternatives to dialysis that are faster are gel filtration chromatography, ultrafiltration, and ultracentrifugation. In all cases, only drug that is associated with the liposomes will remain.

If the compound is not soluble in water and is not stuck to or inside the liposomes, it will probably precipitate. If the liposomes are small enough to stay as a suspension, you can separate the liposomes from the precipitated drug by pipetting the supernatant.

If the compound is stuck tightly to the outside of the liposomes or buried in the bilayer, it may be difficult or impossible to separate it, and it would be counted as liposomal even though it was not inside.

Thanks Dr. Adam.

Here, my drug is not water soluble and in general, we expect the lipophilic drugs get captured in lipid bilayer of liposomes. If I carryout dialysis, how would I know that the drug estimated is the unentrapped but not the drug released from the liposomes as release studies are also done using dialysis bag! Kindly advice. Thanks a ton. 

Dialysis is an equilibrium procedure. If the entrapped compound is in equilibrium between the liposome membranes and the aqueous medium then, given sufficient time, the compound will all be dialyzed out of the liposome sample whether or not it started out entrapped. The time scale maybe very slow if the equilibrium strongly favors binding of the compound to the liposomes.

One way to avoid confusion would be to use a rapid method to remove or measure unincorporated compound, both before and during the release study, rather than dialysis.  If the compound is not water soluble at all, however, it may be difficult to distinguish unincorporated/released compound from liposomal compound.

Do you prefer ultracentrifugation or colum separation? 

My preference for liposomes is gel filtration chromatography. I haven't had much success with ultracentrifugation. Either the liposomes float, or they form a rather loose pellet. Either way, it's hard to get a good separation from the liquid, and the yield can be poor. Gel filtration columns are easy to make and fast to run. When working on a small scale, I make 0.8 x 20-cm columns, and I don't reuse the resin, saving the trouble of cleaning it. The only disadvantage is that the sample gets diluted. This problem is avoided by dialysis, but that takes much longer.

Dear Adam, do you have a protocol of gel filtration chromatography? I'm interested in this method since centrifugation is failing me.

The protocol is very simple. I equilibrate the swelled gel filtration resin (Sephadex G-75 or Sepharose 4B, for example) with the desired buffer at room temperature, then pour it into an open-top column with a stopcock at the bottom and let it settle. The size of the column depends on the sample volume. I am working with samples of 100-200 µl, so I use a 0.8 x 20 cm column.  If you want to elute the column at 4oC, put it into the cold room and let it cool down. Otherwise, you can elute it at room temperature. Apply the sample to the top of the column and let it drain in, then elute by gravity with the same buffer used to equilibrate it. Keep the flow rate low for better separation. The liposomes will elute in the void volume. You may be able to see them come off the column because of their turbidity. Small molecules will elute later.

The larger the sample, or the more low-molecular-weight material you need to remove, the larger the column you should use. Long, thin columns give better separation than short, wide ones. Don't use a column that is larger than necessary because the larger the column the more dilute the liposome fraction will be.

Your explanation is awesome, thanks a lot.

Tangential Flow Filtration 

You can avoid dilution (which is happening during GFC/GPC) and you can further concentrate the liposomal solution (Ultrafiltration) and you can remove untrapped drug from liposomal solution and buffer exchange with final formulation buffer.

Advantages : 

1. Avoid dilutions and you can further concentrate the liposomal solution

2. Buffer exchange or Dia-filtration : When you select the right MWCO of your TFF membrane ( Cassette / Hollow fiber) , you can retain your desired liposomal solution in retentate side and untrapped drug and other low molecular weight impurities and salt will be removed in diafiltration. You can also use and customize dia-filtration buffer wisely.

Please write me on [email protected] if you need further support.

http://www.pall.co.in/main/laboratory/tangential-flow-filtration-product-selec-53300.page

http://www.pall.co.in/main/laboratory/literature-library-details.page?id=7046

If I want to keep my buffer intact but remove the untrapped drug is it possible? 

Yes. You can do diafiltration with your desired buffer.

So you can use your desired buffer as diafiltration buffer. 

Untrapped drug completely replaced by diafiltration buffer with 6-10 diafiltration volumes (normally) without disturbing your desired liposomal product. You can confirm the diafiltration completion with permeate output  pH , conductivity and other desired analysis.

http://www.pall.com/pdfs/Laboratory/02.0629_Buffer_Exchange_STR.pdf

http://www.bioprocessintl.com/wp-content/uploads/bpi-content/0105ar06_77637a.pdf

If your drug is hydrophilic enough (for example, curcumin) you can assume it is bound to your liposomes (entrapped into the membrane). You can also perform centrifugations if you have different sizes of the liposomes.

Dear Adam can I use 0.8 µm syringe membrane filter to separate untrapped paclitaxel from liposomes

Yes, if either (1) your liposomes are larger than 0.8 µm so that they don't pass through and you can verify that free paclitaxel does pass through, or (2) that your liposomes are smaller than 0.8 µm so that they do pass through but free paclitaxel does not.

Dear Adam Thank you very much for the explanation. The size of liposome is 0.2 µm . Should i use the syringe filter of 0.4 or 0.8 µm ?

Liposome preparations have a distribution of sizes, and filters do not have exact cutoffs. Even though the liposomes are 0.2 µm some of them may still get held back by a 0.4 µm filter, or even a 0.8 µm filter.

Even if most of the liposomes pass through the filter, what happens to the drug? Will it also pass through the filter, or will it get held back because it sticks to the filter?

My preferred method for getting rid of unincorporated drug from a liposome preparation is dialysis. The molecular weight cutoff of dialysis tubing is much smaller than liposomes, so there will be very little loss. However, you have to make sure that the drug passes through the membrane, or sticks to it.

If the drug has very low aqueous solubility, it may be possible to separate the unincorporated drug from the liposome be gentle centrifugation because it will simply precipitate.

Dear Prof. Adam B Shapiro, Drug release studies of nanocarriers like liposomes are usually performed by dialysis method. When we subject the liposomes to dialysis for like two days to get rid of the excess free drug, how can we be sure only the unentrapped drug comes off and not the drug released from the liposomes. Please share your insights. Thanks.

Vishnu Rompicharla, is your molecule hydrophobic? The problem with dialysis is that your molecule needs to be somehow soluble in the receptor media, because otherwise the diffusion would be intrinsically limited by the nature of the molecule

My thinking is that the dialysis method is valuable for measuring the content of stably entrapped drug. This means drug that is not in equilibrium between the internal and external environments. It will remain with the liposomes as long as there is no perturbation. Examples are (1) a hydrophilic drug trapped in the internal aqueous space because it is incapable of passing through the lipid membrane, (2) a lipid-permeant drug entrapped in the internal aqueous space because of a pH gradient, and (3) a substance that is so hydrophobic that it is essentially insoluble in water and therefore remains trapped in the lipid membrane. The dialysis method is useful for examples (1) and (2), but not (3) because the insoluble compound can't be dialyzed. Any of this type of compound that is not incorporated will simply precipitate from the liposome suspension.

The remaining situation concerns a drug that is not stably incorporated but is associated with the liposomes and is in equilibrium with the external medium. Such a drug would have some degree of aqueous solubility. If you dialyze the liposome-drug suspension, all of the drug will eventually be dialyzed away. In such a case, a better method to measure the amount of liposome-incorporated drug might be ultracentrifugation or ultrafiltration.

Dear Adam B Shapiro , thanks for the wonderful explanation. I was also wondering if the dialysis would cause any impact on the physical/chemical stability of the lipid material of the liposomes. How do we ensure that the lipid is not undergoing hydrolysis or oxidation in the dialysis bag. Please share your experience. Thanks.

David E. Bautista-Erazo , yes my question is for poorly soluble drug molecules. Your understanding is correct. So we add surfactants or solvents to solubilize the drug in the dialysis medium.

Vishnu Rompicharla if you are performin an in vitro release test, adding surfactants such as Tween can help you, but if it's just for separating free molecule it can damage your actual particles or liposomes.

In my experience, liposomes made with saturated lipids and cholesterol are stable during dialysis in a simple solution such as 5% glucose. Adding organic solvent or detergent will probably cause a change in the liposomes. If unsaturated lipids are used, there is a risk of oxidation. Dialysis should be performed at cold temperatures (4oC, typically) for the sake of preserving liposome quality.

Thank you for your explanation @AdamBShapiro. But what would you do if the drug is a heavy protein and the liposome size is the same than the protein (similar size for the membrane cutoff)? Is it possible to separate the water soluble protein and liposomes by ultracentrifugation?

Thank you very much.

Ultracentrifugation might work if you use a sucrose or glycerol gradient to separate the free protein from the liposomes on the basis of the difference in their buoyant densities. Gel filtration chromatography might work if the liposomes are a lot bigger than the protein. You would use Bio-gel A0.5m or something similar. If the protein is supposed to be inside the liposomes rather than on the surface, you might be able to use ion exchange chromatography to remove unincorporated protein (I think this may be the best approach if the circumstances are favorable.) Another approach to removing external protein is to digest it with protease.

Dear Adam Thanks for the explanation, I used centrifugation but still have the doubt about the results I got. Now I got Sephadex G 100 to remove untrapped peclitaxel from liposomes.

I would suggest using detergent to dissolve the lipids without precipitating the protein. I think I would choose something like 50 mM n-octylglucoside. 1M Tween-80 sound excessively high.

Gel filtration chromatography could probably be done on numerous samples if you make your own mini-columns and work out a simple elution scheme to separate the liposomes from the protein. (Personally, when I was in graduate school I used to prepare foe each experiment by pouring 40 small Sephadex G-50 desalting columns.)

It may depend on the type of lipids you are using (neutral, anionic, cholesterol). SDS will definitely keep the protein in solution, but it will denature it. If you don't want it to be denatured, use a non-ionic or zwitterionic detergent (examples: Tween-20, Triton X-100, n-octylglucoside, dodecylmaltoside, CHAPS).

Hi Vishnu Rompicharla and Adam B Shapiro

I am currently working on an SUV liposome (

Medina Widy since your liposomes are so small, I think you will need ultracentrifugation and more time. Are they sterically or electrostatically stabilized? What is the zeta potential?

You could try using gel filtration chromatography. The liposomes will come out in the void volume and small molecules will come out much later. I would suggest using an inexpensive resin, such as Sephadex G-50, that you can discard after a single use so that you don't have to clean the column. You don't need an FPLC. You can elute by gravity and collect the liposome fraction by hand.

David E. Bautista-Erazo thank you for the response! The zeta potential was between -20 mV and -25 mV.

Adam B Shapiro I'll discuss this method with my supervisor, thank you.

Medina Widy I also think that Shapiro's suggestion is good. Unless you have access to an ultracentrifuge easily which could be a little bit more easy but maybe time-consuming (30 min-1 hour)

Dear Medina Widy, in which method you are getting good results now for your sample? As I am facing the same issue that you mentioned earlier. My sample is hydrophilic in nature.

Anjali Khuntia my sample successfully separated by centrifugation with addition of ethanol

Dear Medina Widy Thank you for your answer. But I guess the addition of ethanol may disrupt the particle, as some phospholipid and cholesterol are soluble in ethanol. So some entrapped drug may release/leak out during centrifugation.

If the drug is practically insoluble in water , could I separate unincorporated drug aggregates from liposomes by filteration through 0.45 micropore filter and consider filterate the entrapped drug in liposomes - Neglecting unincororated drug which is soluble because it is very small as the drug is practically insoluble ?

Try a test using a free drug suspension to see if it is retained by the filter. Also, you should consider how well the liposomes pass through the filter. There are different types of filter membrane to try as well. Liposomes and free drug may stick more to some than others.

Thanks to Adam B shapiro your suggestion helps me too

Hello Mr. Adam B Shapiro and David E. Bautista-Erazo

currently i develops a liposome from soya phosphatidylcholine (unsaturated) contains a hydrophobic drug using ethanol injection method.

I've tried to centrifuge it but the method cannot precipitate the free drug and the liposome would also precipitate. I also tried membrane dialysis, but i am afraid the lipid would be oxidized. Would you please suggest me a method?

My lipid composition solely only from L-alpha-phosphatidylcholine (https://www.sigmaaldrich.com/catalog/product/sigma/p3644?lang=en®ion=ID)

Regards,

Handika-Biotechnology ITB, Indonesia

I think if you perform centrifugation your liposomes would easily precipitate since they are not stabilized by electrostatic repulsion. But your hydrophobic molecule should be on the supernantant along with small liposomes. Maybe try to adjust your centrifugation strenght to have a value between pelleting all your liposomes without damaging the standardized size.

Adam B Shapiro Thank you very much professor Adam

Adam B Shapiro If niosomes have high zeta potential reaching - 60 mV, could this be the reason why they are not separated after centrifugation for one and half hours at 13,000 rpm? Is zeta potential related to separation by centrifugation, not just particle size related?

Omar, zeta potential directly affects centrifugation efficacy. If you have highly charged particles they are going to need a stronger centrifugation. But if your particles are too small, you will have to use ultracentrifugation. I prepare liposomas of 1000 nm and they precipitate rapidly even if they have +60 mV... But smaller ones (less than 400 nm) are more problematic

David E. Bautista-Erazo Thank you very much doctor David

Another way to separate liposomes from external solutes is by gel filtration. The liposomes pass through the resin, such as Sephadex G-50, in the void volume while the external small molecules are retained longer. Analyze the encapsulated compound concentration in the eluted liposomes by whatever technique is appropriate. The easiest method is to use a radioactive compound because then you can just use scintillation counting, but there are many other ways.

Adam B Shapiro David E. Bautista-Erazo Dears,

I found your suggestions quite interesting. I have almost the same system as were already discussed here, A small lipophilic encapsulated molecule in the liposomal system. They are quite small, about 120 nm. For the encapsulation efficiency study, I need to separate the free target molecules from encapsulated. As I tried, they do not precipitate by micro centrifuge within 1h with 14000 rpm. Would you please kindly advise me?

I suggest you first try gel filtration chromatography with spin columns, which is easy, fast and uses little sample. You can purchase small, prepacked, disposable desalting columns that can be spun in a microcentrifuge. The assumption is that the free small molecule acts as a small molecule (rather than a high molecular weight aggregate) and stays in the resin, whereas the large liposomes pass through the column. There will be some loss of liposomes in the columns, so you will have to quantify them at the end. You should check to make sure the small molecule behaves as expected in the absence of the liposomes, i.e. it does not come through the column.

Here is one supplier of spin columns:

https://www.bio-rad.com/en-us/category/bio-spin-micro-bio-spin-size-exclusion-spin-columns?ID=58ee7212-1c1f-4cdc-b348-eb91a641eced

Anther approach you could use is dialysis. This takes much longer than gel filtration, but there is likely to be no loss of liposomes. It relies on the small molecule being able to diffuse through the pores of the dialysis membrane. You should test that assumption, of course. You can use small dialysis cassettes, such as these:

https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-purification-isolation/protein-dialysis-desalting-concentration/dialysis-products/slide-a-lyzer-dialysis-cassettes.html

Adam B Shapiro Thank you for your suggestion. I was thinking about membrane dialysis and wondering if the free hydrophobic molecules are willing to pass through the membrane due to their hydrophobicity features. It is a suspension in PBS, and the media of release is PBS, too.

It depends on the solubility of the compound in the buffer. You will have to try it. It is a simple experiment.

Density gradient ultracentrifugation, differential centrifugation would be ideal for liposome purification. You can use gel filtration chromatography, ultrafiltration techniques knowing the size of your liposomes.

Cheers,

Amal.

Ultrafiltration, mentioned by Amal Senevirathne, is another option. It uses pressure to force a solution through a membrane with tiny pores, similar to dialysis membrane. There are 2 ways of doing it. (1) using a stirred, nitrogen-pressurized ultrafiltration cell, or (2) with a disposable centrifugal ultrafiltration device. A potential disadvantage is that the liposomes can become concentrated against the membrane to form a gel, which may damage them. You would have to check that the free drug was able to pass through the membrane, which would block the liposomes. Then you could measure the free drug concentration in the filtrate.

One brand of centrifugal ultrafiltration device:

https://www.emdmillipore.com/US/en/product/Amicon-Ultra-15-Centrifugal-Filter-Units,MM_NF-C7715