Yes you have to. you can also sonicate your vesicles if you want smaller liposomes (50nm).

Depends on several factors.

Are you working with a blend of lipids (eg long chain saturated phospholipid and cholesterol)? Then it is advisable to generate a homogeneous blend by dissolving the lipids in organic solvent and making a film or freeze-drying first. This homogeneous lipid film (cake) should be dispersed in aqueous medium at temperatures above the phase transition temperature of the phospholipid. Subsequent high-pressure homgenization may be carried out at ambient temperatures if the product after finished homogenization is equilibrated above phase transition temperature overnight again.

Dear Dhruv Butani

You can use Avanti Mini Exruder: http://www.avantilipids.com/index.php?option=com_content&view=article&id=185&Itemid=193

and plycarbonate membranes for this extruder with pore sizes 0,03; 0,05 or 0,08 um:

http://www.avantilipids.com/index.php?option=com_content&view=article&id=506&Itemid=293

According to my experience with 0,1 um membranes the size of liposomes is rather uniform.

A practical suggestion:

They suggest syringes of different sizes. Syringe of 250 ul is the best, while syringe of 1 ml is rather difficult to use.

Best regards

Yury Tarahovsky

Yes, you have to maintain temperature above the Tm of the phospholipids. You can see an example in:

"Liposomes Prepared by High-Pressure Homogenizers." Methods in Enzymology, vol 367, Part A, Methods of liposome preparation. pp 28-46. See Figure 4, p.40

Total lipid content or concentration, pressure and total passage can affect on the size. Therefore, you can optimise your procedure. Diluted samples, higher pressure and at least 10 times passage can be useful.

In any case, you will need to 'stress' the vesicles in order to break down their bilayers and thus diminish the final vesicle size.

In principle, any source of stress compatible with maintaining the used lipid(s) stability will serve your purpose. The most commonly used methods are high-shear homogenisation (for large dispersion volume) and ultrasonication or extrusion (both for small dispersion volume). Each of the three methods requires some special kit (e.g. rotor-stator homogeniser for the former, a tip/horn sonifier (NB: a bath will not do!) or an extrusion device (such as an Extruder(R) (Avestin) for the latter. The benefit of extrusion is that it is especially gentle and therefore material / compounds preserving. Its down side is the sensitivity to poorly / not extrudable material or contaminations, which tend to clog the nano-porous filter through which you nee to push the crude (large vesicles) dispersion.

Inclusion of a cosolvent (such as ethanol (Vierl et al Biophysical Journal 67: 1067-1079 (1994)) or some other alcohol (Loebbecke & Cevc, Biochimica et Biophysica Acta 1237: 59-69 (1995) ), if acceptable in the final preparation () or of charged lipids facilitates small vesicle formation: the former by reducing bilayer lysis tension and the latter by increasing repulsion between adjacent bilayers - which is both helpful. (For a thorough analysis of discussion of the role of lysis tension see e.g. Hunter & Frisken Biophysical Journal 74:2996–3002 (1998); Frisken et al Langmuir 16: 928 - 933 (2000)); for some experimental data on lysis tension see Rawicz et al Biophysical Journal 79: 328–339 (2000).) It is also better to keep the vesicles durding extrusion / sonication / high-pressure homogenisationat, rather than somewhat above, the chain melting phase transition, to exploit the transition induced fluctuations (Cevc et al. Studia biophysica 138:57-70 (1990); Cevc & Richardsen Advanced Drug Delivery Reviews 38: 207–232 (1999)) as bilayer-breaking 'catalysts'. If feasible and tolerable, processing at a much higher temperature affords the best results (Cevc, patent appl. WO2012126966).

More specifically - and focussing on extrusion as the gentlest vesicle manufacturing method (Hope et al. Biochim. Biophys. Acta. 812:55–65 (1985) - you will have to use a sufficiently high driving pressure (depending on the starting vesicle / utilised pore-size mismatch -> see the cited papers by Hunter and/or Frisken for more details) to obtain vesicles with the average diameter having approx. 2x the pore size (see e.g. fig. 2 in Cevc et al. Langmuir 19: 10753-10763 (2003)). If the starting dispersion contains (many) vesicles larger than 100 nm, you should extrude the dispersion several times in a sequence, using smaller nominal pore size for the purpose.

To obtain vesicles with 2r < 100 nm you will consequently need to extrude the coarser dispersion through (a) filter(s) with the average pore diameter < 50 nm at T~Tm or T>> Tm. Bear in mind, however, that many vesicles (including the most popular phosphatidylcholine vesicles) do not tolerate bilayer curvatures corresponding to vesicle diameter < 70-80 nm and hence - rather sooner then later - fuse (typically to form vesicles with the average diameter > 100 nm). Furthermore, at T< Tm many kind of uncharged vesicles also tend to aggregate spontaneously. If kept below pretransition or even subtransition temperature, they will ultimately form non-spherical, multi-facetted structures or a multilamellar, non-vesicular mesophase. Charging-up vesicle bilayer (either by inclusion of ionised phospholipids (see Cevc Chemistry and Physics of Lipids 57: 293--307 (1991) for 'compatibility list') and/or of charged water soluble molecules with an affinity for lipid bilayers (Cevc WO2012126966) increases the final dispersion colloidal stability and may resolve the problem.

Sonication can be the fastest, but stability wise the most 'aggressive' , method useful for making small vesicles. Even such extremely 'stressfull' preparation method typically cannot create very small (< 45 nm) vesicles, the smallest tolerable size of a vesicle being determined by the maximum tolerable vesicle-forming bilayer curvature. The latter increases with the used lipid headgroup polarity, and charge (Cevc Cliemica Scripra 25: 96-107 (1985)), meaning that you should choose your starting lipid(s mixture) judiciously aiming to obtain small vesicles at the end.

Before filter, keep running multiple times with homogenizer. 

It may depend on temperature, concentration w.r.t organic solvent aqua mixture, method of preparation for drying w.r.t time.

This is not possible with a homogenizer but possible with an extrusion device or ultra sonication.

Thanks all of you for your valuable answer.