M ANEJO DE EMISIONES ATMOSFÉRICAS

A number of methodologies are in use for lab- and large-scale preparation of unilamellar and multilamellar liposomes ranging from 20 nm to several µm in diameter (reviewed in Mozafari, 2005). Some of the more common methods are detergent dialysis (Millsman et al., 1978), sonication (Papahadjopoulos and Watkins, 1967), reverse-phase evaporation (Szoka and Papahadjopoulos, 1978), high pressure homogenisation (Mayhew et al., 1984) and extrusion (Olsen et al., 1979; Hope et al., 1985). Clearly the method chosen depends very much on the intended application of the liposomes, particularly since residual organic solvents or detergents could prove severely problematic in certain downstream applications. Regardless of the method used though, the underlying principle of how liposomes form remains the same: hydrophilic/hydrophobic interactions between lipid-lipid and lipid-water molecules in a system perturbed by some physical means such as shaking, sonication, extrusion, etc., cause the spontaneous arrangement of the lipid and water molecules into the most energetically favoured conformation: the bilayered vesicle form (Mozafari, 2005).

Consideration of the expected applications of a model membrane system that would be used to characterise the structural and functional interactions of CLIC1 with membranes (in future work in this laboratory) generated five basic requirements for the model membrane system under study here:

i) The system had to be as biomimetic as possible in terms of lipid composition and size, which affects the extent of membrane curvature. ii) The encapsulation capacity of the liposomes had to be large enough to

enclose experimentally significant quantities of protein or other molecules, such as fluorescent dye or chemical modifiers.

iii) The size distribution had to be as narrow as possible to prevent the generation of extraneous noise in certain experiments (such as isothermal titration calorimetry, for example).

iv) The liposomes had to be relatively stable over time, especially in light of the prohibitive cost of purified phospholipids. Having to discard any portion of a single preparation is not ideal.

v) The system had to be amenable to different buffer conditions which would be used to explore the ideal environment for a large fraction of CLIC1 to insert into the membrane.

With these conditions in mind, the extrusion method of liposome preparation first described by Olsen et al. (1979) and improved by Hope et al. (1985) was chosen, using a freeze-thaw protocol and 400 nm polycarbonate membranes. The development of the small volume extrusion apparatus by MacDonald et al. (1991) appreciably extended the applicability of this method. Large unilamellar vesicles prepared by the extrusion technique (LUVETs) have no residual organic solvent or detergents, have a sharp size distribution, can be prepared in sizes ranging from 50-1000 nm, resemble biological membranes in that they are unilamellar and have a relatively good encapsulation efficiency, are relatively stable in that their lipid arrangement is at equilibrium, and are reasonably easy to prepare (MacDonald et

al., 1991).

Three types of lipid composition were used for LUVETs in this study:

i) Asolectin (Sigma-Aldrich product number 11145), a crude and relatively inexpensive extract from soybean comprising approximately 20-24 % phosphatidylcholine (PC), 18-22 % phosphatidyl- ethanolamine (PE), 12-15 % phosphatidylinositol (PI) and 4-7% phosphatidic acid (PA). This product was used initially to optimise the extrusion process and encapsulation of fluorescent dye. Asolectin (Sigma P5638) was successfully used by Tulk et al. (2000) to demonstrate functional reconstitution of CLIC1 into liposomes and planar lipid bilayers.

ii) A combination of ~60 % PC, ~10 % PI, ~10 % PE, ~10 % phosphatidylserine (PS), ~10 % cholesterol and the remainder sphingomyelin, based on approximate values gained from several

studies of typical nuclear membrane lipid compositions (Table 1). This lipid mixture is attractive for its resemblance to typical nuclear membrane composition, but in practice is almost impossible to reproduce batch to batch due to the very small masses being weighed out and the extreme stickiness of the lipids themselves. Thus this mixture was only used once or twice.

iii) A 4:1:1 mol/mol ratio of PE:PS:cholesterol (hereafter referred to simply as PE:PS:chol) determined by Singh and Ashley (2006) to produce the most reproducible ion channel activity in the presence of CLIC1 of all lipid mixtures tested. While Singh and Ashley (2006) used palmitoyloleoyl phosphatidylethanolamine (POPE) and palmitoyloleoyl phosphatidylserine (POPS), the phospholipids used in this study were purified L-α-PE Type IV and L-α-PS from soybean (Glycine max) (Sigma-Aldrich P8193 and P0474, respectively). Oleoyl is a monounsaturated C18:1 fatty acid, and palmitoyl a 16- carbon (C16:0) saturated fatty acid chain. The exact fatty acid composition of L-α-PE Type IV is unknown, but it contains 65 % C18 unsaturated fatty acids, primarily linoleic acid (C18:2), and 12.5-28.5 % saturated fatty acids. The fatty acids in the L-α-PS used are primarily linoleic acid. L-α-PE Type IV and L-α-PS rather than POPE and POPS were used in this study simply because of the logistical reason of availability, but they were chosen because their fatty acid chain lengths were at least similar.

Glassware used in the preparation of liposomes was cleaned thoroughly in phosphate- and chlorine-free detergent (Contrad™), rinsed in distilled water (dH2O), twice in deionised water (DI water) and twice in methanol to remove

excess water. Tubes and beakers were then sealed with parafilm and stored for no longer than 30 days prior to use. LUVET preparation followed the basic protocol described by MacDonald et al. (1991). Lipids were stored at -20 °C and brought to room temperature prior to opening the containers. Lipid mixtures were made up in concentrations from 20-100 mg/mℓ. The appropriate mass was weighed out

under nitrogen as far as possible and the storage container then flushed gently with nitrogen and sealed with parafilm before being placed back in the -20 °C freezer. Asolectin was solubilised in 1 mℓ chloroform and PE:PS:chol in 2:1 chloroform:methanol. For stocks, no more than four batches of lipids were solubilised and then stored layered under nitrogen in a Schott bottle with the lid sealed with parafilm at -70 °C. The lipids were dried under a gentle stream of nitrogen while rotating the tube by hand or using a vortex mixer. Residual nitrogen was removed overnight under vacuum of at least 300-500 mtorr. The lipid film was hydrated while shaking at a temperature above the phase transition temperature (Tm) (~18 °C for asolectin and less than that for PE:PS:chol) in 30-

500 mM potassium acetate or potassium nitrate in CLIC1 storage buffer (50 mM sodium phosphate, 0.02 % NaN3, 1 mM DTT), pH 5.5 or 7.0, used for dialysis

that week; or in 200 mM KCl in CLIC1 storage buffer. In cases where N- (ethoxycarbonylmethyl)-6-methoxyquinolinium bromide (MQAE) was encapsulated in liposomes, the hydration buffer contained 10 mM MQAE. The hydration phase with shaking or vortexing causes the formation of multilamellar vesicles (MLVs) as the lipid film lifts off the inner surface of the glass tube (Figure 2.3).

Once it could be seen that the lipid film had been fully removed from the inner surface of the tube by the hydration buffer, the solution was subjected to 10 freeze-thaw cycles in an ethanol/ice bath and lukewarm water bath (45 °C for asolectin, 30 °C for PE:PS:chol). Cycles were 1-3 minutes each for asolectin, or 1-2 minutes freezing and very brief thawing for PE:PS:chol. The freeze-thaw cycles ensure equilibration between trapped and bulk solutions (Mayer et al., 1985) and have also been shown to increase encapsulation efficiency of liposomes (Manojlovic et al., 2008). Extrusion was performed using an Avanti Mini- Extruder (Avanti Polar Lipids, Alabaster, USA) equipped with two 1 mℓ Hamilton syringes with removable needles (The Hamilton Company, Nevada, USA). The mini-extruder apparatus fits into a heating block which was maintained at 42 °C for asolectin and 22 °C for PE:PS:chol during extrusion.

Figure 2.3. Multilamellar vesicles formed during hydration of vacuum-dried lipid film.

(1) A lipid mixture solubilised in organic solvent is dried to a film around the bottom of a glass tube under a stream of nitrogen. The tube is rotated or vortexed while drying, resulting in layers of lipid drying over each other. (2 and 3) When hydrated in aqueous buffer these layers peel off to form multilamellar vesicles. Image from Lasch et al. (2003).

Multilamellar dispersions were extruded through a single 400 nm Nuclepore polycarbonate track-etched membrane (Whatman) 11-75 times. Dead space was reduced by pre-wetting the extruder parts by passing 0.22 µm-filtered buffer through the apparatus several times prior to extruding lipids. An odd number of passes was always used so that any large particles or other contaminants remaining after extrusion would be left in the first syringe. After extrusion the asolectin liposome suspensions were centrifuged at 13 400 rpm in a benchtop Eppendorf Minispin for 30 minutes to pellet larger particles. The PE:PS:chol mixture was found not to form any pellet at this stage so these mixtures were not centrifuged in subsequent preparations. The supernatant was aspirated into a clean tube, layered with nitrogen and the lid sealed with parafilm. Liposomes were

stored at 4 °C sealed under nitrogen at all times. They were used within 3-4 days of preparation.