3.2 Assembly
of the LEPS Construct
1. Dissolve (DOPC), (DOPG), (DGS-NTA-Ni), cholesterol,
(DSPE-PEG2000) at a molar ratio of 3:3:1:4:0.1 to a total
lipid mass of 500 μg in chloroform.
2. Add 1 mL of warm solution of Pneumococcal polysaccharide
19F serotype with the concentration of 0.6 mg/mL in PBS (see
Note 1) to the lipid solution and sonicate at an amplitude of
50% for 1 min.
3. Using rotatory evaporator, evaporate the solvents at 50
C
until a thin white film comprised of the lipids and Pneumococcal polysaccharide 19F serotype.
4. Rehydrate the film by rotating it with 1 mL of double-filtered
PBS at the same temperature until the thin film is fully dissolved (Fig. 1).
5. Pass the dissolved lipids 10–12 times through a polycarbonate
membrane (200 nm pore size) at 60
C using a handheld
extruder (see Note 2). This process should yield unilamellar
liposomes of a defined size (200 nm) (Fig. 2).
6. To purify postextrusion liposomes from free/unencapsulated
polysaccharides and small vesicles, Transfer 500 μL of postextrusion sample to the 300 kDa microcentrifuge tubes and
centrifuge for 5 min at 12,000 rcf at 4
C.
Fig. 1 Initial multilamellar liposome production
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