3.1 Ethanol Injection (Good for Cationic Liposomes)
The method is probably one of the easiest ones available. It was originally reported by Batzri and Korn in 1973 [14]. It involves the injection of a small volume of ethanolic solu- tion of lipids into a large volume of water. The force of the injection ensures homogeneous mixing of the lipids, as does the immediate dilution of the ethanol in the large excess of
water. This procedure can generate mainly small unilamellar vesicles (SUVs) with diameters around 25–50 nm. This process is probably the best for making anionic and cationic liposomes since at least some of the lipids dissolve readily in water. The charged lipid is dissolved in water while the hydrophobic one is dissolved in ethanol. The exact steps are as listed here using the example of a formulation prepared in-house:
1. Weigh out 12 mg of DDAB (dimethyl dioctadecyl ammonium bromide) into a clean glass test tube.
2. Dry down 4 mg of dioleoylphosphatidylethanolamine (DOPE) solution (in chloroform) using argon.
3. Dissolve the DDAB in 1 mL of absolute ethanol.
4. Dissolve the DOPE in 1 mL absolute ethanol. Use a 37°C bath to aid dissolution.
5. Prepare eight small glass test tubes with 0.75 mL nanopure water in each.
6. Add 0.125 mL of the ethanolic solution of DDAB to four glass test tubes.
7. Vortex one of the DDAB-containing tubes on high for 10 s.
8. Carefully, inject 0.125 mL of ethanolic DOPE into the vortexing tube in one swift motion with the tip of the pipettor below the surface of the vortexing liquid.
9. Vortex for further 10 s.
10. Extrude liposomes through a 220-nm polycarbonate filter 12 times through a poly(ether sulfone) (PES) membrane.
11. Transfer contents to storage vial and label appropriately.
12. Store liposome at 4°C.
3.2 Sonication (Good for Neutral Liposomes Used for Membrane Studies)
Lipids are mixed together and either sonicated using a probe or in a water bath. This procedure may be used for both charged and uncharged liposome manufacture, as sonication is a rather harsh method for rearranging the different lipids in a 3-D spherical conformation. The most important factor is to prevent heating of the sam- ple during sonication, a phenomenon that could result in lipid degradation, leading to poor-quality liposomes. The exact steps are as listed as follows:
1. Dissolve lipids in a 20-mL glass vial and place on ice.
2. To create an ice bath for the sonication of lipids (to prevent heat-induced chemical degradation of lipids), fill a 50-mL Falcon tube three-fourths full of ice and pour in water to fill in the gaps between the pieces of ice.
3. Set probe sonicator to constant speed.
4. Immerse clean probe at least 1 cm into nanopure water and sonicate for 2 min to clean probe.
5. Now place probe into lipid solution and perform no more than 3 × 2-min bursts of sonication to prepare unilamellar vesicles (ULVs). Place vial on ice between sonication bursts. The opaque solution should change to an opalescent hue.
6. Centrifuge phospholipid vesicles (PLVs) to remove titanium bits that may have come off during sonication.
7. Store at 4°C.
3.3 Dried-Reconstituted Vesicles (Good for Sterically Stabilised Vesicles)
Lipids are mixed together and solvent is removed using freeze-drying. Then an aqueous solution is introduced and the lipid cake around the vessel wall is reconsti- tuted. This method works best for manufacture of neutral liposomes, as the hydro- phobic lipids readily dissolve in solvents such as chloroform and are deposited dry on the wall of the rotavapor vessel. Then the material to be encapsulated is dissolved in an aqueous solution and the dry film on the vessel wall is hydrated with this solution. The exact steps involved in the preparation of hydrogenated soy phosphatidylcholine (PHSPC)- and cholesterol-containing vesicles at a molar ratio of 60 to 40% are as listed here:
1. Prepare the following buffer: 10 mM Tris-HCl, 1 mM EDTA, 150 mM NaCl, pH = 7.4, osmolarity = 300 mOsm/kg.
2. Prepare the lipid film as follows: in a 500-mL flask with 0.401 g PHSPC and 0.13328 g cholesterol, add 7 mL chloroform. Drying is carried out at 40°C, on setting “4.5” of the rotavaporator, for 5 h, with the pump minimum pressure set at 66 mbar.
3. Drug agent to be encapsulated is added to 6 mL of the earlier-mentioned buffer.
4. The dried lipids are hydrated with the 6 mL of buffer with dissolved drug agent, and beads are added to aid homogeneous mixing. The mixing is done under nitrogen, at 40°C, on setting “4.5” of the Buchi Rotary Evaporator with water- cooled condensor coil, for 2 h.
5. Freeze and thaw (50°C) the preparation five times at 4 min for each cycle.
6. Sonicate preparation for 30 s.
7. Extrude preparation at 45°C, using 0.4-µm polycarbonate filter membranes at 200 psi. Change membrane if flow stops.
8. Sonicate formulation for 3 s.
9. Extrude through 200-nm filters at least six times.
10. Sonicate preparation for 1 min.
11. Extrude through 100-nm filters at least six times.
12. Separate lipid-encapsulated ones from free drug molecules using an appropriate column. In-house, for separation of free oligonucleotides from encapsulated ones, a Sepharose CL-2B, fractionation range 70,000–40,000,000, is used.
13. Concentrate liposome preparation if formulation is deemed to be too dilute using Vivaspin 20 mL Concentrator tubes with 100,000 MWCO PES.
14. Store the final preparation at 4°C