SEC elution pattern of liposomes
In order to quantify the amount of lipid retained on a freshly prepared column (not previously saturated with lipids) we injected 400 μL liposomes incorporating 1 mol % Rhod-PE as fluorescent label and loaded with 5 to 10 acetylcholinesterase molecules as internal material. The sample was passed through an 8 mL G25 column. The majority of lipid and enzyme was excluded from the pores of the gel beads and eluted together at the void volume of the column (from 6 to10 mL, Fig. 1). To determine the amount of lipid and enzyme retained in the gel, TX-100 was added to the elution buffer such that the final eluent concentration was 0.5% (w/v). This caused co-elution of a significant amount of lipid and enzyme. Control measurements performed either with free enzyme, or with a mixture of free enzyme and empty liposomes, did not show any retention of enzyme under similar conditions. A further experiment was performed with calcein-loaded liposomes. Identical results were obtained: TX-100 treatment led to the concomitant elution of lipids and calcein under a broader peak than in the case of enzyme-loaded liposomes, due to the smaller molecular mass of calcein (see below). Retention of lipids and of hydrophilic encapsulated materials were identical, suggesting that intact liposomes were retained during SEC.
Column saturation
We observed lipid retention when the chromatography gel was incubated with liposomes prior to being poured into the column. For convenience, we used this method to characterize the relationship between the amount of lipid added to the gel and the amount retained. 2 mL of swollen G25 were incubated in 5 mL buffer containing various concentrations of Rhod-PE labeled liposomes to evaluate the parameters underlying liposome retention. Apparently lipid retention onto the gel beads shows a saturation limit (Fig. 2). It appears that saturation is obtained by passing at least 5 μmole of lipids per ml gel.
Relationship between retention of lipids and encapsulated enzyme
Liposomes loaded with AChE were incubated with 5 mL of fresh G25 in 9 mL buffer. Different concentrations of liposome were used to obtain different retention efficiencies. After 2 h, the gel was poured into a column, washed and eluted with TX-100. The lipid and enzyme content of the fractions were quantified. We observed a linear correlation between lipid and protein retention (Fig. 3). In a control experiment, when liposomes and free enzyme were loaded together on the column, no enzyme retention was observed. This suggests that retention of membrane and intravesicular content are linked, consistent with the hypothesis of non-damaged liposome retention. Furthermore, we observed greater retention of AChE than of lipids suggesting liposome reorganization during the washing step.
Retention depends on gel exclusion limits
Labeled liposomes were incubated with different gels and retention was estimated by elution with TX-100. Retention appeared to depend on the pore size of the beads responsible for the size exclusion (Fig. 4). The smaller the pore sizes, the greater the amount of liposomes retained. The exclusion limit of the gels in our study was significantly smaller than the liposomes size. Exception was noted for Sepharose 4B, this gel reached the range of 60 nm diameters exclusion limit, which is enough to allow small liposomes to penetrate the pores of the SEC gel and to obey an effective permeation process. This chromatography may explain the apparent higher retention than expected. On the other hand, retention was independent of the size of the gel beads since G25 beads of different sizes, fine (20–80 μm diameter), medium (50–150 μm) and coarse (100–300 μm) exhibited the same retention efficiency (data not shown).
Elution by extensive washing step, flow inversion or column repacking
In order to distinguish binding from kinetic trapping, we modified the washing volume. 400 μL of liposome solution were loaded onto 8 mL G25 gels and washed with different amounts of buffer. The remaining retained liposomes were quantified with TX-100 elution. Fig. 5 shows that retention decreased as the washing step was increased.
To test if liposomes were trapped between the beads due to column packing, elution was applied backwards. First 400 μL of Rhod-PE labeled liposomes were loaded on 8 mL G25 and washed. After exclusion of non-retained liposomes, the buffer flow was inverted. The elution profile (Fig. 6) showed that some lipids were eluted by the flow inversion. However, only a small proportion of the retained liposomes were eluted as evidence by the amount of lipids eluted by adding TX-100. Identically, depacking a gel and pouring it into a column once more resulted to partial elution of the retained liposomes.
Direct observation by scanning electron microscopy (SEM)
SEM photographs of sephadex beads were performed to observe how lipids are retained in SEC columns and to investigate how lipids bind to the beads, as individual molecules, as membranes or as liposomes at the surface or inside the beads. Liposomes containing 40% of PE and extruded at 200 nm were passed onto a Sephadex G-25 fine column. After intensive washing, samples diluted in Sorensen buffer were fixed with 2% glutaraldehyde. Without liposomes, the beads appeared homogeneous (Fig 7 A). In contrast, beads originating from the column were covered by lipid aggregates resembling liposomes. Thus, it seems that entire liposomes are retained on the beads. Some liposome aggregation appeared on the beads which may result from the glutaraldehyde cross-linking. Liposomes appeared to be larger than expected with a 500 nm diameter compared to the 250 nm estimated by dynamic light scattering before loading. This difference suggests that liposomes fused either during the chromatography process or during fixation. It also appeared that some beads retained few liposomes (Fig 7 B) while others were completely covered (Fig 7 C). This heterogeneity might result from heterogeneity of column saturation from the top to the bottom.
Elution of retained lipids by liposomes in the mobile phase
We tested if liposomes could be dissociated from the column by other liposomes. Liposomes labeled with fluorescent lipids and containing AChE, were loaded on a G25 column. Following exclusion of non-retained liposomes, unlabeled liposomes were loaded on the column. It appeared that some retained lipids and enzymes were co-eluted by the unlabeled liposomes (Fig. 8). Enzymes eluted by liposome were encapsulated because their activity was detectable only by using TX-100 in the solution. TX-100 disrupted liposome bilayers and allows the enzyme substrate to reach AChE which is unable to cross the lipid membrane [16]. The same experiment was performed with an intravesicular tracer of smaller size: calcein. This fluorescent probe was loaded in rhod-PE labeled liposomes. The elution profile (Fig. 9) shows one peak for excluded liposomes. Passing unlabeled liposomes resulted in an elution of both retained labeled lipids and calcein.
In order to test lipid exchanges between liposomes in the column, the previous experiment was repeated replacing the unlabeled passing liposomes by NBD-PE labeled liposome on Rhod-PE ones retained in the gel. As previously, passage of NBD-PE labeled liposomes, led to the elution of Rhod-PE liposomes. In addition, we observed elution of NBD-PE liposomes by adding TX-100. Thus, it appears that binding and release of liposomes on Sephadex columns is a dynamic process.