Liposome retention in size exclusion chromatography
© Ruysschaert et al; licensee BioMed Central Ltd. 2005
Received: 24 November 2004
Accepted: 10 May 2005
Published: 10 May 2005
Size exclusion chromatography is the method of choice for separating free from liposome-encapsulated molecules. However, if the column is not presaturated with lipids this type of chromatography causes a significant loss of lipid material. To date, the mechanism of lipid retention is poorly understood. It has been speculated that lipid binds to the column material or the entire liposome is entrapped inside the void.
Here we show that intact liposomes and their contents are retained in the exclusion gel. Retention depends on the pore size, the smaller the pores, the higher the retention. Retained liposomes are not tightly fixed to the beads and are slowly released from the gels upon direct or inverted eluent flow, long washing steps or column repacking. Further addition of free liposomes leads to the elution of part of the gel-trapped liposomes, showing that the retention is transitory. Trapping reversibility should be related to a mechanism of partitioning of the liposomes between the stationary phase, water-swelled polymeric gel, and the mobile aqueous phase.
Retention of liposomes by size exclusion gels is a dynamic and reversible process, which should be accounted for to control lipid loss and sample contamination during chromatography.
Liposomes are self-assembled phospholipids enclosing a droplet of the aqueous medium in which they are formed . Liposomes have numerous applications namely as in vivo drug delivery vehicles . Drugs interact with liposomes in several different ways depending on their solubility and polarity characteristics. They can be inserted in the lipid chain bilayer region, intercalated in the polar head group region, adsorbed on the membrane surface, anchored by a hydrophobic tail or entrapped in the inner aqueous compartment. A prerequisite for the use of drug-loaded liposomes is to be able to separate encapsulated from free drugs. Recently, we encapsulated enzymes in liposomes to enhance the enzyme stability with respect to dilution and proteases [3, 4]. Size exclusion chromatography (SEC) is an old and widely used tool to separate small solutes from liposomes or to narrow the size distribution [5, 6]. For example, Sepharose 2B, 4B Sephacryl S-1000 and high performance exclusion gels of the TSK-PW series are suitable for separating small unilamellar vesicles from larger ones [6–10]. In 1977, Sorensen et al.  showed loss of lipid material during SEC of liposomes. In 1980, Van Renswoude et al.  observed by microscopy the retention of fluorescent liposomes between and on the polymer beads. The loss of lipids could reach 20 to 40% of the deposited material [8, 13]. These findings suggested a step of gel pre-saturation with lipids to avoid loss during SEC . To achieve high quality separation, the column pre-treatment is preferentially carried out with sonicated liposomes as their small sizes ensure efficient penetration of the lipids within the gel pores .
Here we show that when liposomes containing a hydrophilic protein pass through a SEC column, lipids and protein are simultaneously retained suggesting that the liposomes retained remain whole. Retention is, however, transitory and reversible: there is an equilibrium between retained liposomes and eluting ones. A pre-saturation step should thus be performed with liposomes of the same composition as that of the liposomes to be chromatographied in accordance with previous recommendations .
SEC elution pattern of liposomes
Relationship between retention of lipids and encapsulated enzyme
Retention depends on gel exclusion limits
Elution by extensive washing step, flow inversion or column repacking
Direct observation by scanning electron microscopy (SEM)
Elution of retained lipids by liposomes in the mobile phase
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.
What is retained, lipids or liposomes?
Lipid retention has been observed since the beginning of the 80's  and usually pre-saturation steps are used to avoid this phenomenon. However, a question remained: are liposomes or lipids retained? To address this, we compared the retention of lipids and the retention of encapsulated materials, enzyme or calcein. It appeared that intravesicular material is also retained suggesting that lipids are retained as intact liposomes without leakage (Fig. 1, 3 and 9). Furthermore SEM observations of the beads clearly showed the presence of lipid aggregates resembling liposomes, attached to the beads (Fig. 7). The next question should be: why are liposomes retained in SEC gels, whereas their size and shape would predict their exclusion? A first hypothesis is a direct interaction of lipids with the beads at the liquid/solid interface of water and the poly-dextran beads, this interaction may promote lipid reorganization as in liquid/air monolayer experiments. As full liposomes are retained in columns, this hypothesis seems less probable because such an interaction would destroy the liposome integrity and its contents would be released. A second hypothesis arises from liposomes' flexibility: they may be deformed and thereby pass through the polymer net and remain stuck . Several experiments favor this hypothesis. i) Retention depends on the bead pore size (Fig. 4) ii) Partial elution can be performed by inversion of the buffer flow (Fig. 6), depacking and repacking the gel in the column or extensive washing (Fig 5).
Is it possible to saturate a column to avoid liposome retention?
Figure 2 shows a saturation plateau at 60 nmoles lipids per mL of G25 suggesting that once the column has been saturated, SEC can be performed without liposome retention. However, if all the pores are filled with liposomes, like in the picture in fig 7C, the efficiency of chromatography may be affected. Furthermore, saturation depends on the washing step, the longer it is, the less the column remains saturated. Saturation is not static but seems to be the result of a dynamic process resulting from exchanges between free and entrapped liposomes. In conclusion, saturation of a column should be used with caution bearing in mind the reversibility of the saturation process, which can cause contamination of the samples to be analyzed.
Interaction of liposome with beads and leakage
Two observations suggest that retention of liposomes inside the beads is associated with liposome reorganization. First, retention of enzyme appeared to be higher than lipid retention suggesting that entrapped liposomes lose their lipids (Fig. 3) and second, free liposomes passing through the column are able to elute fixed liposomes (Fig. 8 and 9), most probably by fusion and fission. Is there some leakage of encapsulated molecules during SEC? Andersson and Lundahl  hypothesized leakage of glucose from the liposome due to interaction between the liposomes and the gel bead surfaces, and disruption of liposomes by shear forces from the liquid flow. However, simple permeation of glucose across the lipid bilayer is significant and does not allow permeation to be distinguished from leakage during the several minutes needed to perform SEC. However, our enzymatic test did not provide evidence of any liposome leakage or internal content release with time. This strongly supports that the transitory retention of lipids does not induce dramatic vesicle leakage or bilayer permeability enhancement and rather agrees with intact liposome trapping.
Relation with immobilized-liposome chromatography
Immobilized-liposome chromatography is used for studying the partitioning of compounds into phospholipid bilayers. Several methods are used to bind liposomes to the matrix. Specific ligands such as hydrophobic ligands  can be coupled to the gel beads. Liposomes can be formed in the presence of beads using the detergent dialysis method. In this method, lipids, detergent and beads are mixed and dialyzed. Elimination of detergent molecules during the dialysis results in the formation of liposomes entrapped in the beads . A third method comes close to liposome chromatography: beads are mixed with liposomes and binding is achieved by several cycles of freezing and thawing . Entrapped liposomes are thought to be suspended in micro cavities of gel beads . Our results suggest that part of the liposomes spontaneously rearrange in gel beads without outer stress. Buffer flow would be sufficient to push the liposomes inside the bead net and to immobilize them.
How should SEC be performed with liposomes? As retention is inversely related to the bead exclusion limit, the larger-pore gels should be preferred for liposome separation from small molecules as suggested by Grabielle-Madelmont et al . As retention is proportional to the gel volume, the bed volume of the column has to be calculated at minima for optimal separation, which also avoids sample dilution. The pre-saturation step diminishes liposome retention but as there are liposome exchanges, the purity of the sample may be affected if the retained liposomes are different from the free liposomes. Two ways are possible: (1) for analytic purposes, it is thus better to inject the liposomes to be sized several times until gel saturation and then perform size analysis (2) for preparative purposes like separation of loaded liposomes from non-encapsulated material, the problem is trickier and the experimental conditions should be adapted to minimize loss of lipid and contamination.
The lipids, egg-phosphatidylethanolamine (PE), brain Lα-phophatidylserine (PS), Lα-phosphatidylethanolamine-N-(lissamine-rhodamine-B-sulfonyl) (Rhod-PE), Lα-phosphatidylethanolamine N-(4-nitrobenzo-2-oxa-1,3-diazole) (NBD-PE), were purchased from Avanti Polar Lipids, Birmingham, AL, USA. Free rhodamine or free NBD which may contaminate labelled lipids were eliminated from liposome solution by chromatogaphy or dialysis. Egg-phosphatidylcholine (PC), was from Lipoid, Ludwigshafen, Germany. Calcein (high purity) was from molecular probes, Leiden, the Netherlands. Sephadex G-25 Medium, G-75 Medium and G-100 Medium, Sephacryl S-100 HR and Sepharose 4B were from Amersham Biosciences, Biogel P2 (45–90 μm) was from Bio-Rad.
Unless otherwise stated, all experiments were performed using 145 mM NaCl, 2.5 mM HEPES, pH 7.4 buffer as aqueous phase. Chromatography gels were washed before use by 0.5% Triton X-100 (TX-100) (Merck), water and equilibrated with buffer.
1 μmole egg-PC dissolved in CHCl3 was placed in a 10 mL glass tube. Then this was dried under a stream of N2 and under vacuum for three hours to form a dry lipid film. 200 μL of buffer were added to the lipid film and vortexed to peel off the lipid. The liposome suspension obtained was frozen in liquid N2 and thawed in a 25°C water-bath 10 times. Buffer was then added to obtain a 1 mM lipid dispersion, which was passed 10 times through a 0.2 μm polycarbonate filter (Schleicher & Schuell, Dassel, Germany). Labeled liposomes were prepared according to the same procedure except that the initial lipid film was obtained from a mixed chloroform solution of egg-PC and 1 mol% Rhod-PE or NBD-PE. Liposome size was estimated to be 230 nm (diameter) by dynamic light scattering.
To follow lipid elution in SEC, we measured the lipid content in each fraction by two methods: a phosphate assay to measure all lipids and a fluorescent assay to measure labelled lipids. As we found a good correlation between the two methods, we used only the second one allowing studies with lower amount of lipids to analyze retention of liposomes. Rhod-PE (excitation wavelength 550/emission wavelength 590 nm) and NBD-PE (excitation wavelength 470/ emission wavelength 530 nm) were used as membrane fluorescent markers. Calcein was used at 500 μM at an excitation wavelength of 492 nm and emission wavelength of 517 nm. Fluorescence was measured using a lamp LPS220 and a photomultiplier detection system PDS 810 from photon Technology International.
To obtain liposomes loaded with 5–10 enzyme molecules, 1 μmole of dried eggPC was dissolved with 200 μL of enzyme solution containing 4 nanomoles of Drosophila melanogaster acetylcholinesterase (AChE) in buffer. The liposome dispersion obtained was frozen in liquid N2 and thawed in a 25°C water-bath 25 times to allow the entrance of the protein [3, 4]. Non-encapsulated AChE was removed by inverse affinity chromatography, passing the liposome dispersion through a column filled with procainamide gel that retains free AChE. Elution contained at least 90% encapsulated enzyme and less than 10% free enzyme. Liposome containing fractions were pooled and lipid concentration was adjusted to 1 mM. Extrusion was performed 10 times through a 0.2 μm filter. AChE activity was measured with the sensitive method of Ellman  with 1 mM acetylthiocholine after dissolution of liposomes with 0.1% (w/v) TX-100.
To obtain liposomes loaded with calcein, 1 μmole of dried eggPC was dissolved with 1 mL of buffer containing 500 μM of calcein. The liposome dispersion obtained was frozen in liquid N2 and thawed in a 25°C water-bath 25 times to allow the entrance of the calcein and extrusion was performed 10 times through a 0.2 μm filter. Non-encapsulated probe was removed by passing the liposomes through a Sephadex G-100 column. The fluorescence of the fractions was measured in the presence of TX-100 to avoid calcein self-quenching.
1 cm diameter columns were used with two different methods. In the first one, the gel was freshly packed and the liposomes were deposited when the eluent level reached the top of the gel. Buffer flow was driven by simple gravity. The sample was allowed to penetrate the gel entirely before adding repetitive small buffer volume to proceed to the elution. In the second one, swollen gel was incubated in a buffer containing liposomes with gentle agitation for two hours. The gel was then packed into the column and non retained liposomes were eluted by successive additions of small amounts of buffer.
Sample preparation for scanning electron microscopy (SEM)
For SEM analysis, liposomes containing 60% PC and 40% PE were prepared as previously described. PE was chosen to allow cross-linking by glutaraldehyde. After passing and washing the liposomes on G-25 fine beads were fixed with 2% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) (Sorensen buffer) containing 6 mM CaCl2, for 4 h, at 4°C. After an extensive wash in the same buffer, samples were removed, post-fixed for 1 h at room temperature with 1% osmium tetroxide in 0.1 M cacodylate buffer (pH 7.4), dehydrated in a graded ethanol series, dried by critical point drier with an EMSCOPE CPD 750 and coated with gold-palladium for 3 minutes at 100 Ångströms / minute, and observed with a S-450 scanning electron microscope (Hitachi) at an accelerating voltage of 10 kV.
List of abbreviations
Size Exclusion Chromatography
Scanning Electron Microscopy
We acknowledge financial support through RTN Network Nanocapsules with functionalised surfaces and walls (HPRN CT-2000 00159) and Lipoids for the gift of egg-PC lipids.
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