The concentration has always been around 2.5mM and I haven't ever had this problem..

I look at some paper and they mentioned that gave a protocol from lipid manufacture.

Why do not you contact with your lipids manufacture?

Have you tried to perform another type of homogenization first? such as sonication or vortex (longer times). Maybe you have difficult aggregates?

Yes, I have. The suspensions were sonicated prior to extrude them.

if you have working with saturated lipids, you may increase the temperature (e.g. above the transition temperature of the lipid with the highest Tm) of the dispersion to extrude it. It would be easier if you downsize following a sequence of membranes (e.g. 800 nm followed by 400 nm followed by 200 nm or 100 nm)

She said she used a temperature above the Tm, but it's good if you check out that data or your equipment, is it properly heating the liposomal suspension? Also what Dr Cocera said is important too, maybe try to employ a bigger pore size first (1 um, for example). Have you tested the size and PDI BEFORE any extrusion? Other thing: the PS changes the zeta potential to anionic mV values so if you are encapsulating a polycation maybe it's making a complex? Have you tested your zeta potential?

Thank you all for your answers!

Yes, I extrude liposomes at about 10°C above the highest Tm. I don't believe it's a matter of temperature, because with DPPC:DPPE 1:1 I haven't had any problem even if DPPE has a higher Tm than DPPS..

I'm not even encapsulating anything at the moment, there is only 10mM phosphate buffer..

I have already tried to use 400nm before 100nm.. it worked with 400nm pores, but it didn't with 100nm.

Have you tried to reduce lipid quantity? Maybe it's too concentrated? Or reducing the concentration of the buffer.

The buffer has a low concentration. I will try to reduce the concentration by half. Thank you

Hello, I agree with all the previous comments, particularly with Dr. Cocera, the gradual reduction in size is critical. What type of extruder are you using?, according to my experience with a 10 ml extruder (now from the Lipex Extruder Company) several lipid mixtures required a really high nitrogen pressure and temperatures of almost 75 ° C (far from only 10 degrees above of higher transition temperature) to pass through. Both parameters are not easy to reach in a manual extruder. Allow your vesicle solution reach the desired temperature for at least 15 minutes before extrude. If you have the possibility to verify concentration and composition after preparing the vesicles, try changing the PC filter with a new one after the first pass, sometimes the polycarbonate filters are clogged in the first extrusions.

Hi Francesca,

It is true that you are extruding a quite concentrated lipid dispersion (~13mM). However it should be doable to extrude them.

Here what I recommend:

1) When hydrating the lipid film for the first time: Incubate the dispersion at T>Tm for at least 30 min. You will see everything homogenizes well and rather easily. This will diminish the chances of yielding aggregates that will not be extruded. Later you can conduct 10 freeze-thaw cycles (always remember with the thawing at T>Tm)

2) Dont sonicate your MLVs to "ease" the extrusion. Freeze-thaw cycles are intended to swell the vesicles and more importantly to have a constant salt gradient throughout the different layers of your MLVs. This gradient will be destroyed upon sonication.

3)Extrude at T>Tm and be patient, phosphatidylserine is a tough lipid.

4) I never worked with PBS buffer because it interferes in my phosphate quantification assays. But PS can combine with some salts and precipitate, check your ionic strength in solution.

Wish you the best of luck

Great recommendations. Good luck.

Thank you all for your precious advices! I will surely follow them!

Hi,

I had similar problems when extruding charged liposomes (PS has negative charge at pH 7.4) in a low ionic strength buffer. An increase of the extrusion pressure was necessary.

With DPPS I would not expect any difficulties, nor with its mixtures with PCs unless you have divalent cations or multivalent in the solution (a little EDTA might help with contaminating calcium, so I would suggest that, unless EDTA ruins your experiments). With LiposoFast (from Avestin) I have had no difficulties, although with higher concentrations manual help for this pneumatic extruder was occasionally necessary. The doable concentration depends on the exact extruder. With our extruder it was possible to reach about 20 mM though the pneumatic pressure was often then not enough especially for the first two-three passes through the filter. Some extruders allow 50 mM concentration to be reached. 2 mM concentration is doable for all, so you can try to extrude at lower concentration and larger volumes.

Hi all,

At this stage I can come up with two suggestions:

1. using alternative membrane for the extrusion of lipids / phospholipds (e.g. nitrocellulose, polyester or polyurethane instead of polycarbonate filters) - in case there is any chemical interaction hindering smooth extrusion. Still you need to observe the requirements of working above phase transition temperature (Tc), as explained in the attached paper (see section: 3.2. Extrusion Method).

[Pls note: Tm is melting temperature; for "Phase Transition Temperature" need to use Tc]

2. use alternative methods to prepare nanoliposomes (or to downsize your liposomes) - as explained in the attached manuscripts.

You may also find the following articles useful, published in 2018, reviewing some of the available nanoliposome preparation methods:

Nanoliposome technology for the food and nutraceutical industries

S Khorasani, M Danaei, MR Mozafari - Trends in Food Science & …, 2018 - Elsevier

***********************

Enhanced efficacy and bioavailability of thymoquinone using nanoliposomal dosage form

MR Mohammadabadi, MR Mozafari - Journal of Drug Delivery Science and …, 2018 - Elsevier

I am going to suggest an entirely different rationale for why you may be having difficulties extruding your DPPC:DPPS 1:1 and DPPE:DPPS 1:1 vesicle preparations. In both cases, the DPPS one in particular, you are working with HII phase formers which, while they have saturated acyl chains, above the Tm , will likely attempt to form an HII phase particularly in the presence of calcium (PS particularly a problem). Part of the problem is that these lipids when hydrated from a thin layer of dried lipid do not all hydrate equally and you may not be getting the 1:1 mixture in your lipid dispersion as you are thinking. As such, you may be getting a mixed dispersion of DPPC lipid MLV and DPPS lipid in HII phase...on warming.

The phase behaviour of these lipid species is quite complex based on the presence of saturated acyl chains. Another question, have you tried melting your dispersion in a Calorimeter to see what the melting transition looks like. This could be very informative as to what your vesicle species look like: two unique transitions from two unique vesicle populations versus one broader transition (DPPS has a transition at 54C and DPPE has a Tm of 64C for the lamellar phase and 118C for the HII phase). This is typically significant when the pH of your buffer system is below pH of 8...the transition for HII phase formation for PE lipid species.A question for you, have you ever looked at your preparation in Negative Stain TEM? If an HII phase problem, you will see them.

I would suggest you try two things: a) chelex treat your buffer to remove any calcium contaminants to see if these kinds of HII phase tubes are a problem and b) use a buffer pH higher than pH 8. A question for you, have you ever looked at your preparation in Negative Stain TEM? If an HII phase problem, you will see them.

best of fortune to you