Characterization of loaded liposomes by size exclusion chromatography

J. Biochem. Biophys. Methods 56 (2003) 189 – 217 www.elsevier.com/locate/jbbm

Review

Characterization of loaded liposomes by size exclusion chromatography Ce´cile Grabielle-Madelmont *, Sylviane Lesieur, Michel Ollivon Equipe Physico-chimie des Syste`mes Polyphase´s, UMR CNRS 8612, Universite´ Paris-Sud, Chaˆtenay-Malabry Cedex 92296, France

Abstract This review focuses on the use of conventional (SEC) and high performance (HPSEC) size exclusion chromatography for the analysis of liposomes. The suitability of both techniques is examined regarding the field of liposome applications. The potentiality of conventional SEC is strongly improved by using a HPLC system associated to gel columns with a size selectivity range allowing liposome characterization in addition to particle fractionation. Practical aspects of size exclusion chromatography are described and a methodology based on HPSEC coupled to multidetection modes for on-line analysis of liposomes via label or substance encapsulation is presented. Examples of conventional SEC and HPSEC applications are described which concern polydispersity, size and encapsulation stability, bilayer permeabilization, liposome formation and reconstitution, incorporation of amphiphilic molecules. Size exclusion chromatography is a simple and powerful technique for investigation of encapsulation, insertion/interaction of substances from

Abbreviations: Ca, calcium; C16G2, diglycerol hexadecyl ether; Chol, cholesterol; CholHS-PEG2000, cholesterylhemisuccinate monomethoxypolyethylene glycol; Con A, concanavalin A; DCP, dicetyl phosphate; Dil, 1,1V-dioctadecyl-3,3,3V-tetramethylindocarbocyanine amide perchlorate; DG, dodecyl-h-D-maltoside; DPPC, dipalmitoylphosphatidylcholine; DMPC, dimyristoylphosphatidylcholine; EPA, egg phosphatidic acid; EPC, egg phosphatidylcholine; SEC, size exclusion chromatography; FITC-dextran, fluorescein-isothiocyanate dextran; Hepes, N-[hydroxyethyl]piperazine-NV-[2-ethanesulfonic acid]; HPSEC, SEC performed with a high performance chromatography (HP) equipment; LUV, large unilamellar liposomes; M-PEG-cholesterol, monomethoxypoly (ethylene glycol) cholesteryl carbonate; MLV, multilamellar liposomes; NSV, sonicated non-ionic monoalkyl surfactant-cholesterol liposomes; OD, optical density; OG, octylglucoside (n-octyl-h-D-glucopyranoside); PA, phosphatidic acid; PEG-Chol, poly(ethylene glycol) cholesteryl ether; SUV, small unilamellar liposomes; SPC, soy phosphatidylcholine; SPG, soy phosphatidylglycerol; Sph, sphingomyelin; 99mTc, technetium. * Corresponding author. Tel.: +33-1-4683-5644; fax: +33-1-4683-5312. E-mail address: [email protected] (C. Grabielle-Madelmont). 0165-022X/03/$ - see front matter D 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S0165-022X(03)00059-9

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small solutes (ions, surfactants, drugs, etc.) up to large molecules (proteins, peptides and nucleic acids) in liposomes. D 2003 Elsevier Science B.V. All rights reserved. Keywords: High performance gel exclusion chromatography; Gel filtration; Liposome; Liposome reconstitution; Encapsulation; Permeability

1. Introduction Liposomes are hollow microspheres, formed by self-assembly in water of phospholipids. Their membrane consists of one or several curved lipid bilayers, which entrap part of the aqueous medium in which they are suspended [1– 4]. Because of the amphiphilic character of the phospholipids and their organization in closed structures, liposomes can encapsulate hydrophobic molecules in the bilayer membrane or hydrophilic compounds in the aqueous internal cavity as well as amphiphilic substances (Fig. 1, Parts 1 –6). These special properties have generated numerous applications of lipid liposomes as models for biological membranes and as drug delivery vehicles for in vivo applications. Drugs can 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 (Fig. 1, Parts 7– 8). A prerequisite for the use of drug loading liposomes in vivo is to develop methods allowing to control liposome preparation, including particle size, stability, encapsulation rates and in the opposite to determine the leakage kinetics of the entrapped substances. The nature of the lipids and the methods of preparing liposomes define the chemical and physical characteristics of liposomes. Determination of their structural parameters, i.e., the lipidic bilayer organization, the mean number of lamellae per liposome, the average size and size distribution, the internal volume, are essential for their use as model systems and drug carriers. Small-angle X-ray diffraction and scattering, static and dynamic light scattering, freeze fracture electron microscopy, analytical ultracentrifugation, sedimentation field flow fractionation, size (or gel) exclusion chromatography, and measurements of internal volume have been used for liposome characterization. Conventional size exclusion chromatography (SEC) [5– 10] and high performance size exclusion chromatography (HPSEC) [11 – 14] are methods particularly interesting as they permit to physically separate liposomes from small solutes, on the one hand, and to subdivide liposomes into a series of subpopulations, on the other hand. This allows precise determination of size and size distribution. In addition, as this technique is not destructive for the samples, fractions after collection can be essayed for various complementary analyses. However, HPSEC presents major performances over conventional SEC regarding both analysis reproducibility and separation efficiency [11– 15]. In particular, it has been shown that HPSEC is a very powerful technique for liposome size distribution analysis, size stability study, liposome fusion and aggregation analysis [14].

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Fig. 1. Schematic representation of unilamellar liposomes (1) and positions of loaded substances (2 – 8). Hydrophobic markers are essentially located within the acyl chains of the bilayer; amphipatic labels are inserted in the polar head groups and water-soluble ones in the internal and external aqueous medium (2). Surfactant molecules, which are amphiphilic, partition between the liposome bilayer and the aqueous medium (3). Hydrosoluble polymers of various average molecular masses can be entrapped (4). Stealth liposomes may be formed with anchored (5) and covalently attached (6) polymers. Interaction sites of various molecules such as drugs, proteins or biological macromolecules (7, 8): water-soluble molecules can be loaded into the liposome interior or bound to the bilayer surface; amphiphilic molecules orient into bilayers; transmenbrane proteins span the lipid bilayer with sites exposed to the aqueous phase.

Different aspects and applications of conventional SEC and HPSEC in the field of liposomes and proteoliposomes have been previously reviewed [13,14,16]. In this paper, we focus on the analysis of loaded liposomes by SEC and HPSEC. We address certain practical aspects on size exclusion chromatography and present the potentiality of a methodology based on HPSEC coupled to multidetection modes for on-line analysis of stability and characterization of liposomes via label encapsulation. Some examples of applications are described which concern polydispersity, size and encapsulation stability, bilayer permeabilization, liposome formation and reconstitution, incorporation of amphiphilic molecules. They underline the relevance of the information provided by this technique.

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2. Conventional SEC and HPSEC A variety of designations, gel filtration, gel permeation, gel chromatography, molecular sieve chromatography, steric exclusion chromatography and size exclusion chromatography, is currently used to define separation of solutes or aggregates by their molecular size. According to Hagel [17], SEC refers to the designation of the separation principle, as it is a formally correct descriptive term of the process and the name gel filtration is used for the applications of SEC in aqueous solvents. In the following, the terms conventional SEC and HPSEC will be used for clarity. 2.1. Principle Conventional SEC and HPSEC are based on the same principle, i.e., on a sizedependent permeation of solubilized molecules or dispersed molecular aggregates through the appropriate gel. Sample resolution includes both a separation and a dispersion process [18]. The former includes any secondary retention mechanism additional to size exclusion mechanism. The latter, which causes the broadening of the chromatograms, depends on the molecular diffusion and the column packing. A true size exclusion mechanism assumes that interactive mechanisms such as partition and adsorption do not contribute to retention behavior. The retention of liposomes is mainly determined by liposome size and shape. It may be however affected by bilayer flexibility or rigidity, which depends on the lipid composition of the liposomes. Liposome interactions with the gel matrix may come from the chemical nature of the gel, the lipid bilayer composition and the encapsulated material, the presence of charged species contributing to increase this phenomenon. These interactions may affect the sample recovery from the column gel and thus change the characteristics and the composition of the analyzed liposomes. 2.2. Available gels Conventional SEC of liposomes has been performed on different soft gels: large-pore Agarose gels (Bio-gel A150, Sepharose 2B or CL-2B, Sepharose 4B or CL-4B), crosslinked dextrans (Sephadex). These gels are commonly used in the removal of small solutes non-entrapped in liposomes. By using of the correct gel, it is possible to perform a separation in which the liposomes are excluded and the solute totally included. The upper exclusion limit of these gels (e.g., 60-nm liposomes for Sepharose 4B [19], 100-nm liposomes for Sepharose 2B [7,15,19 – 22], 150-nm liposomes for Agarose-150-m, [23]; see also Table 1 in Ref. [16]) does not allow their separation in a wide range of size. Sephacryl S-1000, an allyl-dextran methylene bisacrylamide copolymer, allows to efficiently separate and size liposomes up to 200 –300 nm [8 –10,24,25]. This gel is suitable for conventional SEC and HPSEC chromatography. It presents a good mechanical stability for separations at moderate pressures and flow rates (0.3 – 0.5 ml/min) [11]. Liposome sizing with this gel has been done through the calibration of the column by using polystyrene monodisperse latex although these standards as solid polymer spheres are far from the liposome structure based on an aqueous core delimited by a lipid shell [9,26]. With better relevance, selectivity curve for Sephacryl S-1000 has been performed with

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unilamellar liposomes of diameter ranging from 17 to 181 nm [11]. Provided presaturation of the column with lipid (small unilamellar liposomes, SUV), good recovery and integrity of the liposomes have been demonstrated. Sephacryl gels S-500 Hr, S-400 Hr and S-300 Hr, which offer low exclusion limits (from 100 to 60 nm) [26 –28], could be used in combination with a Sephacryl S-1000 column. Different prepacked HPSEC gel columns have been used in the field of liposomes. The efficiency of TSK Type gels based on semi-rigid hydroxylated polyether (GDNA PW, G6000 PWXL, G6000 PW and G5000 PW, Toyo Soda, Tokyo, Japan) for liposome sizing were examined with egg phosphatidylcholine (EPC)/egg phosphatidic acid (EPA) (9/1 mol/mol) liposomes of different sizes obtained by sequential extrusion through 0.4, 0.2, 0.1 and 0.05 Am Nuclepore membranes. The TSK-G6000 PW gel was found the best efficient regarding sizing range (20 to over 500 nm) [11], lipid recovery [11,13,14] and average size and size distribution determination [14]. Combination of the G6000 PW column in series with G5000 PW and/or G4000 PW enables the separation of smaller diameter liposomes from each other and from low molecular mass substances [13,29]. The semi-rigid cross-linked polysaccharide Superose 6 presents a narrow sizing range from 1 to 1.5 nm micellar aggregates to 25-nm liposomes with a good lipid recovery [30]. 2.3. Technical considerations The methodology of chromatographic analysis is described in details in Refs. [13,14,16]. Here, we briefly presented certain points of importance to use this technique in a convenient and efficient way, i.e., the choice of gel and column, the material recovery, the type of detection and the column calibration. The choice of the column and gel bed is determined by the size range of the liposomal suspension and the type of separation or analysis to be performed. To prevent loss of material by adsorption of lipids on the gel, it is necessary to saturate the gel with lipids before analysis. Column pre-treatment is preferentially carried out with sonicated liposomes as their small sizes ensure an efficient penetration of the lipids within the gel pores, the elution buffer being generally that in which the liposomes are suspended. Presaturation is achieved when liposomes injected at constant sample loadings elute at the same elution volumes and yield peaks of constant areas. For a general viewpoint, the eluent should be isoosmolar with the internal and external aqueous media of the liposomes to avoid osmotic shocks, which may cause damage or shrinkage of the liposomes. In the presence of water-soluble molecules, the eluent should correspond to that in equilibrium with the loaded liposomes. With amphiphilic compounds such as surfactants, which can partition between the liposome bilayer and the aqueous continuum, the eluent should correspond to the effective continuous medium in equilibrium with the mixed aggregates, in order to maintain the actual characteristics of the system during the analysis (see Section 3.3.4). Size separation may be affected by deformability and/or packing down of the gel beads with soft gels. In particular, the flow rate should be adapted to avoid compression of the gel beads. The pression should not exceed 1 bar for soft gels and 10 – 15 bars for semi-rigid ones. Sample loading and gel volume are adapted to the preparative or analytical scale, respectively. The void and total

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volumes of columns can be determined with nonextruded multilamellar liposomes (MLV) and very small solutes (NaCl, sodium azide, glucose), respectively. When possible, on-line detection should be preferred as it ensures a precise description of the elution profiles. Otherwise, fraction collections must be collected for subsequent analyses. Liposomes can be directly detected by light scattering, refractive index, total lipid concentration or indirectly via fluorescent or radiolabel markers either within the bilayer(s) or in the internal aqueous space of the liposomes. Light scattering is sensitive to the morphology and size of the liposomes but does not enable to determine the amount of lipid since the relation between the intensity of the scattered light and the liposome size is far from being linear. Refractive index detection [13,14], phosphorus analysis, radiolabeled lipids (14C-DPPC, 3H-DPPC) or fluorescent lipids (diphenylhexatriene) [31] permit to quantify the lipid amount of the liposomes. Water-soluble radioactive or fluorescent or any chromophore labels allow to determine encapsulation efficiency, release content and kinetics of leakage. For applications involving liposomes loaded with water-soluble fluorescent labels (e.g., liposome leakage analysis), calibration curves of the loaded substances can be done by elution of these substances as a function of their concentration. The release content can be then directly determined from the chromatograms recorded, provided the release amount is in the concentration range for which there is a linear dependence between the fluorescence

Fig. 2. Application fields of HPSEC and conventional SEC in liposome technology from preparative uses to analytical ones. According to the purpose of analysis, the full arrows indicate the best potentialities of HPSEC and SEC, while dashed arrows show possible but less efficient routes.

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Fig. 3. Schematic representation of the different sequences of chromatograms obtained according to the liposome analysis performed. (A) Formation of loaded liposomes and removal of unencapsulated material; (B) determination of the liposome size and size polydispersity, of the size stability and impermeability; monitoring of liposome solubilization by surfactant molecules, studies of release kinetics; (C) liposome reconstitution. Conventional SEC is rather convenient on a preparative scale, whereas successive analyses require a HPSEC system.

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intensity and the label concentration. As liposomes and fluorescent molecules are separated by elution on the columns, it is not necessary to encapsulate a high amount of labels due to the high sensitivity of the fluorescence detection. This avoids the drawback of quenching or inner filter effects, which result in a deviation from linearity of the fluorescence intensity versus label concentration [32]. After removal of the nonentrapped molecules, the total amount encapsulated is determined by lysis of the liposomes with a very small volume of a concentrated surfactant solution for negligible dilution. The surfactant is chosen for its solubilizing and diffusing properties into the lipid bilayer. 2.4. Conventional SEC and HPSEC performances The advantages of HPSEC over conventional SEC result from the specificities of the available columns associated with the use of a HPLC system. This last allows faster run times, reduces sample size, significantly increases peak resolution, analysis reproducibility and separation efficiency [11– 13,15]. The application fields of both types of chromatography based on their separation and analysis efficiencies are reported in Fig. 2. HPSEC is recommended for liposome characterization (size distribution, size stability, integrity, liposome reconstitution), time resolved analyses (release kinetic, permeability, encapsulation stability) and removal of high molecular weight substances. Conventional SEC is quite adapted for liposome preparation and drug encapsulation (for instance by surfactant removal) and for removal of low molecular weight substances. Liposome separation into subpopulations can be performed by both techniques, except that the conventional SEC gel columns cover a narrow liposome size range. On a preparative scale, conventional SEC offers the possibility to adapt the column dimensions to the separation to be done and thus to minimize the dilution factor which may be a drawback with the prepacked HPLC columns. Recovery of liposomes at the void volume of the gel also enables to collect concentrated fractions. Another non-negligible aspect is the low cost of conventional gels, which do not necessitate regeneration in case of contamination. HPSEC is by far the more efficient on an analytical scale. It provides accurate information in a variety of studies. An overview of the different types of analysis, which can be performed, is summarized in Fig. 3 with the sequences of chromatograms related to the process under study.

3. Applications 3.1. Liposome loading There are a large variety of techniques for generating multilamellar and unilamellar liposomal systems [33]. The same methods are used to encapsulate materials within liposomes [34]. Some of the procedures are summarized here. Membrane markers or lipophilic substances are introduced in the lipid(s) by codissolution in an organic solvent followed by solvent evaporation. Water-soluble substances are dissolved in the aqueous buffer used for the liposomal preparation. Their localization with

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respect to the liposome bilayer is related to their affinities to the different regions of the liposome [35] (Fig. 1). Depending on the application, multilamellar liposomes, small unilamellar liposomes (sonicated liposomes, SUV) or the extruded liposomes obtained by successive extrusion down through polycarbonate membranes can be prepared. Another encapsulation procedure consists in forming loaded liposomes by depletion of surfactant molecules from a solution of mixed lipid – surfactant micelles. The drug can be either introduced in this mixed micellar solution or in the surfactant-free buffer used to generate the liposomes. Detergent removal can be done by SEC, dialysis or dilution with pure buffer. Encapsulation can also be achieved with preformed liposomes, the substance to be encapsulated being in this case added in the external medium of the liposome suspension. Several cycles of freezing and thawing are often applied to improve the entrapment. Whatever the liposome preparation procedure used, a fraction of the solute molecules is entrapped but most remains in free solution. Removal of unentrapped material is often performed by conventional SEC, dialysis or centrifugation. Generally speaking, the method chosen for the removal should not damage the liposomes, which may cause a partial loss of the encapsulated material. This may be the case when centrifugation is used. Due to the wide range of the centrifugation conditions applied in various studies (700  g to 150,000  g) [36], first a variety of liposome size may be obtained; second, at high g, small liposomes can be lost in the supernatant meanwhile the loaded solute can be extracted from the liposomes. Schubert et al. [37] have shown that the release of entrapped inulin from liposomes by addition of cholate was significantly lower when liposomes were separated from released inulin on a Sepharose CL-4B than that resulting from ultracentrifugation (140,000  g). Removal of substances by dialysis is limited by the cut off of the dialysis membrane. This is the case for macromolecules such as dextrans. For small molecules, the separation by conventional SEC is generally efficient since, with available gels (Sephadex, Sepharose), the liposomes are excluded and the solute totally included [36]. For solutes of high molecular masses, their effective separation from the liposomes is the challenge. Indeed, the gap between the respective elution volumes of the liposomes and the unentrapped molecules may become too narrow, leading to the overlapping of both elution peaks. Using Sephacryl S1000 columns and even more HPSEC with TSK-G6000, G5000 or G4000 PW HPLC columns in series [13,29] combined with a low elution rate significantly improves the separation. Whatever the technique used for the removal of the unencapsulated substance, control of its efficiency is important as the residual concentration of the free material in the external medium of the liposomes would interfere in further analysis of stability, release kinetic or permeability. 3.2. HPSEC methodology for analyzing loaded liposomes Here, we present a procedure developed in our laboratory [29]. The HPSEC experimental set-up (Fig. 4) consists of two TSK-PW columns in series (30  0.75 cm, Toyo Soda, Japan), one efficient in liposome sizing (G6000 PW), the other in that of smaller particles (G4000 PW). The HPLC apparatus is equipped with a Hitachi pump (Model L-6000) and a precision injection valve (Rheodyne). Elution is monitored on line by using a three-

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Fig. 4. HPSEC experimental set-up coupled with fluorescence (a) and 90j light scattering (b) on-line detections: (a) liposome characterization, (b) separation of unencapsulated probe from the loaded liposomes.

window circulating quartz cell (ref 176.353-QS, Hellma, France) thermostated at 25 jC and placed in a X-format spectrofluorimeter Fluorolog SPEX (FL1T11) monitored by a computer. Fraction collection is used if necessary. The HPSEC set-up was tested for separation efficiency with extruded EPC/EPA liposomes encapsulating water-soluble fluorescein-isothiocyanate dextrans (FITC-dextrans) of molecular masses ranging from 4400 to 147,800 [29,38]. Complete separation of the unentrapped FITC-dextrans from the FITC-loaded liposomes was achieved for all the polymers studied, the entrapping efficiency strongly decreasing with increasing polymer chain length [39]. The methodology is illustrated in Fig. 5 with the FITC-dextran 4400, as an example. The double on-line detection by fluorescence (excitation wavelength = 493, emission wavelength = 514 nm) and 90j light scattering at 493 nm enables simultaneous measurement of the marker concentration and the turbidity of the liposomes. Light scattering (90j) allows to detect the liposomes containing or not dextran (Fig. 5A). Fig. 5B displays the chromatogram of the liposomes prepared in buffer containing FITC-dextran 4400, which shows the efficiency of the separation of the free unencapsulated dextran from the loaded liposomes.

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Fig. 5. HPSEC methodology for loaded liposome analysis. Elution profiles on TSK-G6000 and -G4000 PW columns connected in series of extruded EPC/EPA (9:1, w/w) liposomes. (A) Without dextran encapsulated, (B) prepared in aqueous buffer containing 1 mg/ml of FITC-dextran 4400, (C) FITC-4400 liposomes after separation of the unencapsulated marker. Eluent: aqueous buffer (145 mM NaCl, 10 mM Hepes, pH 7.4). Sample loading: 200 Al, flow-rate: 1.0 ml/min. On-line detections: 90j light scattering at 493 nm (A); fluorescence (the excitation and emission wavelengths were 493 and 514 nm, respectively) (B and C). Redrawn from Ref. [29].

The fluorescence intensity measured along the elution profile centered at Ve = 13.45 ml depicts the fluorescent dextran encapsulated inside the liposomes, while that of the peak detected at Ve around 20 ml is related to the dextran in the external medium of the liposomes. On-line detection allows to select the fraction of the eluted liposomes, which is not contaminated by the marker as clearly shown by the elution profile of the liposomes after separation (Fig. 5C). This methodology also provides information on the characteristics of the loaded liposomes after the preparation. The fact that the elution parameters (Ve and halfheight Ve) of the dextran-loaded liposomes remain the same as that of the unloaded liposomes prepared by the same procedure demonstrates that encapsulation of dextran does

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not alter the size and polydispersity of the liposomes. By injecting the liposome suspensions containing the probe at different times, the stability of the liposome size versus time can be therefore controlled together with the stability of the encapsulation. 3.3. Application fields of conventional SEC and HPSEC 3.3.1. Liposome polydispersity A major physical characteristic of liposomes concerns the polydispersity of the suspensions. Several methods for size and size distribution analysis of liposome populations by using HPSEC on a TSK-G6000 PW column have been examined in detail in Ref. [14]. They can be applied to other types of column gels, provided the liposomes are not eluted at the void volume of the column and preferentially recorded by on-line detection for precise drawing of the elution profile. Liposome size can be determined from the selectivity curve of the column, but reliable size will be obtained only if the elution volume of the liposomes lie in the plateau region of the selectivity curve. Moreover, this last must be established with liposomes of various mean diameters corresponding to the separation range of the column. To improve the accuracy of the selectivity curve, it is recommended first to fractionate the liposomes through the column by collecting narrow fractions at the maximum of the elution peak, and then to rechromatography this fraction, its mean diameter being independently measured by quasi-elastic light scattering. Size polydispersity of a liposome preparation can be easily obtained by rechromatography of narrow fractions collected all along the elution profile (for details, see Ref. [14]). Procedures involving labeling of the lipid bilayers in conjunction to that of the internal aqueous volume also permit to determine the size distribution. Small unilamellar dipalmitoylphosphatidylcholine (DPPC) liposomes (Table 1, line 1) were labeled with 3 H-DPPC and 14C-mannitol. The external marker was removed by conventional SEC. The liposomes were analyzed by HPSEC by using a two-column system with a TSK-G6000 and a TSK-G5000 columns in series [14]. The turbidity was detected on line and liposome fractions were collected. The size distribution was obtained from the lipid concentration of each fraction determined from 3H counts and the corresponding effective liposome radius deduced from the ratio 14C/3H. The analysis indicated a narrow size distribution. The same approach was applied to characterize a heterogeneous liposomal DPPC suspension (Table 1, line 2) labeled with 14C-DPPC and encapsulating self-quenched calcein. The liposome size distribution was determined from the lipid concentration and the effective liposome radius of each collected fraction deduced from the dequenching calcein fluorescence/14C ratio. It is to note that the quantitative measurement of calcein after lysis of the liposomes should be done in a dilution regime for which self quenching or inner filter effect are completely suppressed to assess a linear dependence between the calcein concentration and the fluorescence intensity [32]. 3.3.2. Liposome stability In the field of drug delivery via liposomes, the following paradox should be resolved: drug should remain entrapped during storage as long and complete as possible, whereas the same drug should be released after administration at a controlled rate. To mimic stability of drug encapsulation, glucose, the sodium salt of quinoline yellow and the

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Table 1 Synopsis of conventional SEC and HPSEC studies: size parameters and stability of liposomes No. Lipids

Membrane marker

Aqueous marker

1

3

14

2

3

4

5

6

DPPC SUV

DPPC polydisperse vesicles Soja lecithin, hydrogenated soja lecithin, chol, MLV LUV SUV EPC/DCP/Chol (70/20/10), MLV LUV SUV EPC/Chol/PA (7/2/1), EPC/Chol/PA (7/7/1), 200-nm SUV, 70-nm SUV C16G2/Chol/DCP (47.5/47.5/5 wt.%), sonicated NSV

Applications Aim of analysis

References

C-mannitol Sephadex G25M saturated with DPPC SUV TSK-G6000 + G5000 PW 80 mM TSK-G6000 calcein PW

free marker removal

[14]

vesicle fractionation vesicle fractionation

size distribution size [14] distribution

oil scarlet

Na quinoline Sephadex G50 yellow glucose

vesicle/free probe separation

liposome stability

[40]

3

14 C-inulin horseradish peroxidase

Sephadex G200 vesicle/free Sepharose 2B probe separation

liposome stability kinetic of release liposome stability

[41]

vesicle stability

[42,43]

H-DPPC

14

C-DPPC

H-EPC

14

Column gel

C-DPPC, Tc

vesicle/free probe separation

99m

Sepharose 4B, TSK-G5000 PW TSK-G6000 PW

on-line size analysis

[15]

lipophilic dye oil scarlet were together encapsulated into SUV, large unilamellar liposome (LUV) and MLV liposomes of different lipid compositions (Table 1, line 3) [40]. Analysis of the kinetics of the substance release as a function of the average size of the liposomes, lipid composition, storage temperature and addition of adjuvants was done by conventional SEC on Sephadex G-50. The liposome suspensions were chromatographed at different time intervals and the released marker spectrophotometrically analyzed after lysis of the liposomes. The average particle size and a polydispersity index were independently measured by QELS. Stability of liposomes in biological fluids should also be controlled for their applications in vivo, as serum or plasma constituents have destructive effects on the bilayer structure. Modulating their phospholipid composition is one of the ways to increase the carrier potential of liposomes. Multilamellar, large and small unilamellar liposomes (Table 1, line 4) were labeled with 3H-phosphatidylcholine, 14C-inulin and horseradish peroxidase [41]. Conventional SEC was used for unentrapped marker removal and analysis of the aqueous label release from the loaded liposomes after incubation in serum or plasma. The leakage has been attributed to disruption of phospholipid bilayers. Conventional SEC has shown the variation of leakage according to the incubation conditions and the

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stabilizing effect of high cholesterol (Chol) content. Incorporation of sphingomyelin (Sph) into lipid bilayers enhances the integrity of the liposomes. The role of cholesterol as protective agent against serum destructive action was also examined with cholesterol-poor and cholesterol-rich negatively charged liposomes loaded with 14C-DPPC and technetium (99mTc) which is a liposome surface label (Table 1, line 5) [15]. Their stability in vitro (in saline and in serum) and in vivo was investigated both by conventional SEC on Sepharose 4B and HPSEC on a TSK-G5000 PW column. The 200and 70-nm liposomes were prepared by sonication so that they are able to elute at the void volume of the SEC and HPSEC columns, respectively. Their stabilities were assessed by co-elution of the radiolabels at the void volume of both columns. When instability occurs, both labels were, at least, partly shifted from the void volume on both columns and entered the gels over a wide molecular mass range. In this case, there were differences in the elution profiles of the labels on Sepharose 4B and G5000 PW columns which were attributed to the different void volumes of both columns and to the difference in the size of the two liposome preparations. The results have shown that insertion of a high ratio of cholesterol into the liposome bilayers prevented phospholipid loss when exposed to serum or in vivo, while cholesterol-poor liposomes were damaged in serum and strongly altered in vivo. Advantages of using a HPSEC system has been underlined, the TSK-G5000 PW being suitable for monitoring movement of phospholipids between liposomes and serum proteins and for detecting changes in liposome sizes. The size stability of non-ionic surfactant liposomes (NSV) containing cholesterol was analyzed versus time by HPSEC (Table 1, line 6) [42,43]. Two factors were analyzed: the evolution of the liposome mean diameter determined from the selectivity curve of the column [12] and their size distribution deduced from the shape of the elution profile. However, with the TSKG6000 PW column, the analysis is limited to NSV of small mean diameter ( < 120 nm) as unilamellar larger NSV ( z 170 nm) are completely retained by the gel column. This behavior illustrates the effect of the bilayer rigidity of the liposomes on the elution mechanism as mentioned above. 3.3.3. PEG-coated liposomes Another strategy to preserve liposomes against solubilizing agents and protein adsorption of the immune system is to modify the liposome surface by anchoring hydrophilic polymer chains. Two factors are important: the amount of the polymer bound to the liposomes and the coating efficiency. These data have been obtained by conventional SEC [44 – 46]. Incorporation of poly(ethylene glycol)cholesteryl ether (PEG-Chol) in liposomes and stability of PEG-Chol liposomes (EPC/PEG-Chol and/or Chol) were analyzed (Table 2, line 1) [44]. The liposome membrane was labeled with 1,1V-dioctadecyl-3,3,3V-tetramethylindocarbocyanine amide perchlorate (Dil) for monitoring PEGChol by fluorescence and the phospholipid concentration was obtained by a classical method. PEG-coated liposomes were chromatographically separated from free PEG-Chol molecules. Levels of PEG-Chol incorporation in liposomes were calculated from profiles obtained after gel filtration. The data have shown that PEG-Chol cannot be totally incorporated at a high molar ratio and the concentration values depend on the PEG chain length. Incorporation of cholesterylhemisuccinate monomethoxypolyethylene glycol (CholHS-PEG2000) into the membrane of contrast-carrying liposomes was investigated

No.

Lipids

Polymers

Membrane markers

Aqueous markers

Column gel

Applications

Aim of analysis

References

1

EPC/PEG-Chol and/or Chol extruded vesicles SPC/Chol/SPG (6/3/1), SPC/Chol (7/3)

PEG2200-Chol, PEG4400-Chol, PEG8800-Chol CholHS-PEG2000

3

Dil

Sepharose CL-4B

PEG incorporation liposome stability

[44]

iopromide Gd-DTPA

Sephacryl S400

CholHS-PEG2000 incorporation

[45]

DL-a-DPPC/Chol

hydrophobized polysaccharides

FITC-polysaccharide

Sepharose-4B

efficiency of anchoring

[46]

extruded vesicles 4

DMPC, DMPC/Chol (7/3) extruded vesicles

PNIPAM-Py, b-PNIPAM-Py

Sephacryl S1000 XK column

efficiency of anchoring

[48]

5

C16G2/Chol(50/50)/ M-PEG-Chol (50/50 – x/x) mol% SUV

M-PEG1000-Chol, M-PEG2000-Chol

TSK-G6000 PW

coated vesicle/ free polymer separation coated vesicle/ free polymer separation coated vesicle/ free polymer separation coated vesicle/ free polymer separation on-line analysis

PEG-Chol vesicle characterization

[49]

stability

[50]

2

3

(3/1 mol)

H-DPPC

calcein

TSK-G6000 + G4000 PW

free calcein removal calcein release

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Table 2 Synopsis of conventional SEC and HPSEC studies: polymer loading

203

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by conventional SEC on Sephacryl 400 [45]. These liposomes (Table 2, line 2) were generated by encapsulation of the water-soluble radiographic contrast agent iopromide (iopromide-liposome: soy phosphatidylcholine (SPC)/cholesterol/soy phosphatidylglycerol (SPG), 6:3:1 molar ratio), by a continuous extrusion process through polycarbonate membranes of decreasing pore sizes associated with freeze – thaw cycles [47]. The impact of surface modification of the liposomes on their properties was studied by conventional SEC and other methods. SEC has demonstrated successful incorporation of CholHSPEG2000 into liposomal bilayers as shown in Fig. 6. Various hydrophobized polysaccharides were used to form polysaccharide-coated liposomes (Table 2, line 3) [46]. SEC on Sepharose 4B was applied both to separate the polysaccharide-coated liposomes from free polysaccharide and to quantify the amount of polymer bound to the liposome surface, using FITC-labeled polymers. This amount was defined by the percent of the polymer recovered from liposomes to that of the initially added. The coating efficiency was investigated as a function of the hydrophobicity of the anchors (cholesteryl, hexadecyl and a,aV-dodecyldiglyceryl diether groups) and various polysaccharides, such as pullulan, dextran and mannan. Conventional SEC has demonstrated that the coating efficiency of liposomal surface was strongly related to the hydrophobicity of the anchors, the cholesteryl moiety being the best. In addition, the chromatographic analysis was able to evidence weak association between pullulan and liposomes. Thus, although pullulan binding to liposomes was detected by fluorescence, this binding was too weak and the polymer was extracted from the liposomes during gel filtration. Analogous results were obtained for the binding to dimyristoylphosphatidylcholine (DMPC)-containing liposomes of poly-(N-isopropylamides) modified by the same hydrophobic substituent, either at random sites along the macromolecular chain or specifically at one chain end (Fig. 7) [48]. Conventional SEC (Table 2, line 4) has shown

Fig. 6. Incorporation of polymers into liposome bilayers. Conventional SEC on Sephacryl S400 of CholHS-PEG polymer (curve 1), SPC/Chol/SPG liposomes (curve 2), liposome/polymer mixtures (curve 3) and liposomes with 5 mol% of CholHS-PEG (curve 4) prepared by the extrusion procedure (see text). Adapted from Ref. [45].

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Fig. 7. Influence of the polymer architecture on binding to liposome bilayers. Conventional SEC on Sephacryl S1000 of: (A) DMPC liposomes (.), b-PNIPAM-Py (z) end-labeled polymer and (B) of DMPC liposomes (.) with b-PNIPAM-Py (z) indicating polymer anchoring into liposomes; (C) of DMPC/Chol liposomes (.), PNIPAM-Py (z) randomly labeled polymer and (D) DMPC/Chol liposomes (.) with PNIPAM-Py (z) indicating no polymer/liposome binding. Redrawn from Ref. [48].

a strong anchoring of the end labeled polymer into liposomes as they coelute (Fig. 7A and B). In contrast, the randomly labeled polymer liposomes were completely dissociated during the elution through the column and no polymer was detected in the lipid fraction (Fig. 7C and D). The difference in elution behavior was attributed to the higher hydrophobicity of the end-labeled polymer. Steric stabilization of non-ionic surfactant liposomes (diglycerol hexadecyl ether (C16G2)/cholesterol) was obtained by means of monomethoxypoly(ethylene glycol) cholesteryl carbonates (M-PEG-cholesterol) [49]. Two different chain lengths (MPEG1000-cholesterol and M-PEG2000-cholesterol) were tested and M-PEG-cholesterol NSV were prepared by sonication from hydrated mixed films of C16G2, cholesterol and M-PEG-cholesterol. Sizing analysis by HPSEC with a TSK-G6000 PW column (Table 2, line 5) coupled with turbidity on-line detection showed (Fig. 8A) that M-PEG-cholesterol was entirely incorporated into the C16G2/cholesterol membrane. However the integrity of the M-PEG-cholesterol1000 and M-PEG-cholesterol2000NSV were preserved only for a limited polymer amount, which depends on the polymer chain length (10 – 20 mol% for MPEG1000-cholesterol and 5– 10 mol% for M-PEG2000-cholesterol). Beyond this threshold, elution profiles were shifted toward smaller particles, which were attributed to the formation of disk-shaped bilayer fragments on the basis of cryo-transmission electron microscopy observations. The stability and impermeability properties of classical NSV

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Fig. 8. PEG coating of non-ionic surfactant liposomes. (A) HPSEC on a TSK-G6000 PW column of NSV (C16G2/ Chol/DCP, 48.0/48.0/3.6 mol%) and (C16G2/Chol/M-PEG2000, 50/50 – x/x mol%) mixed aggregates prepared by ultrasonication. Percentages refer to polymer molar ratios x. Inset: evolution of the mean hydrodynamic diameter deduced from the chromatograms versus polymer content. The integrity of PEG-coated liposomes are preserved up to 10 M-PEG2000 mol% and beyond smaller particles are formed (see text). Redrawn from Ref. [49]. (B) HPSEC on TSK-G6000 and -G4000 PW connected in series of NSV and M-PEG-Chol-NSV loaded with calcein. Percentage of calcein released from liposomes versus time for NSV (thick +), and M-PEG-Chol-NSV containing 2 (D), 5 (o), 7.5 (w), 10 (q) mol% M-PEG1000-Chol or 2 (5), 5 (+) mol% M-PEG2000-chol. Curves 1 (NSV) and 2 (data of the two M-PEG-Chol-NSV systems) correspond to calcein released versus time according to a firstorder kinetic law. Inset: enlarged view of the first 4-day period. Redrawn from Ref. [50].

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(cholesterol/C16G2/dicetylphosphate) and M-PEG-cholesterol-NSV containing low amount of M-PEG-cholesterol were tested by using encapsulation of calcein within the internal aqueous volume of the liposomes [50]. The analysis was performed on the TSKG6000 PW and -G4000 PW two-column system (Table 2, line 5) coupled with fluorescence and 90j light scattering on-line detections as described above [32]. NSV and M-PEG-cholesterol-NSV partially released calcein. The efflux from the liposomes obeyed a first order law over 2 weeks of storage at room temperature whereas the diffusion coefficient of the calcein through the lipidic membrane was three times higher in the presence of the polymer chain grafted cholesterol, whatever the polymer chain length. (Fig. 8B). The physical stability of the M-PEG-cholesterol-NSV was assessed both by 90j light scattering and fluorescence detections. 3.3.4. Liposome permeability induced by surfactants For the use of liposomes as drug carriers, their permeability properties are of primordial importance as they govern the efficiency of drug retention, the delivery of entrapped molecules and the physical stability of the liposomes under different biological media. Understanding the behavior of liposomes with respect to the lipid solubilizing properties of surfactants have also implications for liposome drug loading by detergent removal from a mixed micellar solution, the fate of liposomes in vivo, for membrane protein reconstitution. Lipid dissolution from liposome into lipid – surfactant mixed micelles as added surfactant molecules partition into the membrane and inversely liposome formation by surfactant removal from lipid –surfactant micelles occurs via a succession of aggregation states [51 – 53]. Conventional SEC and HPSEC among other methods (light scattering, fluorescence, small-angle X-ray scattering) have provided information on size evolution of the vesicular surfactant – lipid intermediates [37,43,54], liposome stability [37,43], mechanisms of permeabilization [29,37,38,55,56], the threshold of the liposome closure in liposome formation process [54] and liposome reconstitution [57 –59]. The cholate-induced membrane solubilization of EPC, EPC/Sph and EPC/cholesterol liposomes was followed with [14C]DPPC as lipid marker (Table 3, line 1). The liposome stability against sodium cholate was checked from the release of entrapped 3H-inulin. Conventional SEC of liposome/cholate mixtures was performed on Sepharose 4B-Cl for 70-nm liposomes and on Sepharose 2B-Cl for 220-nm liposomes. The addition of cholate on 220-nm liposomes led to mixed lipid/cholate liposomes smaller in size, which is not the case with the 70-nm liposomes. The chromatography results also show that the gradual release of the entrapped carbohydrate observed at low cholate concentrations is not due to liposome solubilization and may result from transient intramembrane pores [37]. In the presence of calcium, cholate induces liposome growth via a fusion mechanism [55]. This mechanism was deduced from the retention of FITC-dextran (MW: 70,000) in 30-nm EPC liposomes in a Ca2 + (10 mM CaCl2) containing medium, following addition of cholate (Table 3, line 2). Prior and at varying times after addition of cholate, aliquots were eluted through small (8  8 mm) concanavalin A (Con A) – Sepharose columns, which retained the unentrapped fluorescent dextran and not the fraction encapsulated in the liposomes. The eluent was the Ca2 + buffer used for liposome preparation. The liposomes recovered at the void volume were solubilized

208

Table 3 Synopsis of conventional SEC and HPSEC studies: insertion/interaction with solubilizing surfactants Vesicles nm

Markers

Surfactant

1

70, 200

14

Na cholate

30

FITCdextran 70,000

EPC, EPC/Sph. 7/3, EPC/Chol 7/3 vesicles extruded EPC vesicles

2

3

EPC SUV

C-DPPC, H-inulin

3

23

4

EPC SUV

34

FITCdextran 70,000

5

C16G2/Chol/DCP (47.5/47.5/5 wt.%) sonicated NSV

72 (66)a

6

EPC/EPA 9/1 extruded vesicles

136 – 147 FITCdextrans 4400 to 148,000

7

EPC/OG mixed micelles

14

C-glucose, H-inulin

3

Column gel

Eluent

Applications

Sepharose CL-4B, 10 mM phosphate, vesicle/free probe Sepharose CL-2B 150 mM NaCl, separation pH 7.35 size analysis Na cholate Con A – Sepharose 135 mM NaCl, vesicle/free 10 mM CaCl2, probe separation, pH 7.4 free probe removal TSK-G6000 PW 145 mM NaCl, on-line size Sephacryl S1000 10 mM Hepes, pH 7.4 analysis OG TSK-G6000 PW 145 mM NaCl, on-line size 10 mM Hepes, analysis with OG, pH 7.4 OG Con A – Sepharose 135 mM NaCl, vesicle/free probe pH 7.4 separation, free probe removal TSK-G6000 PW, 145 mM NaCl, on-line size 10 mM Hepes, pH 7.4 analysis 10 mM OG TSK-G6000 PW same buffer with 10 mM OG OG TSK-G6000 PW + vesicle medium, on-line analysis: NaTC G4000 PW pH 7.4 encapsulated probe, probe release, size analysis Sephadex G25 (PD10), Sephadex G150

145 mM NaCl, 10 mM Hepes, 14 C-glucose, pH 7.4 free buffer

OG removal

Information from chromatography

References

vesicle size growth probe release via transient pores probe release via vesicle fusion

[37]

vesicle size, vesicle size growth, size reversibility after OG removal probe release via a lipid transfer mechanism vesicle impermeability

[55]

[54]

[56]

[43]

release kinetic [29,38] mechanism via transient pores (OG) and permanent pores (NaTC) vesicle closure [54] from micelle dilution

external inulin C-glucose removal Sephacryl S1000, 145 mM NaCl, on-line size vesicle formation TSK-G6000 PW + 10 mM Hepes, pH 7.4 analysis G4000 PW 14

8

enzymatically formed DPPC vesicles DPPC/DG vesicles a

calcein 1 mM

Calculated from the selectivity curve of the TSK G6000 PW column [12].

[58,59]

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No. Lipids

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209

by excess cholate and the fraction of dextran retained in them was determined by fluorescence measurements. Addition of subsolubilizing octylglucoside (n-octyl-h-D-glucopyranoside) (OG) concentrations to EPC SUV also results in liposome size growth [54]. Liposomes in the presence or absence of 10 mM OG were chromatographed on a gel exclusion column (TSK-G6000 PW) equilibrated with or without 10 mM OG, respectively (Table 3, line 3). EPC SUV with an initial diameter of 23 nm eluted at a position equivalent to 25.6 nm in OG. This liposome swelling due to OG insertion in the lipid bilayer was reversible as liposomes exposed to V 10 mM OG and rapidly returned to low surfactant concentration, were eluted on a Sephacryl S1000 gel column at the same position as the initial SUV (23nm diameter). The elution volumes of the EPC-OG aggregates in 20 mM OG from the TSK-G6000 column corresponded to smaller structures ( < 20 nm). However, exposure of SUV to 20 mM OG and more followed by rapid dilution to low OG concentration, resulted in significantly larger liposomes (61 nm in diameter) than SUV. Above a ratio of OG to phospholipid in the liposome bilayer of 1, a mechanism of liposome growth via lipid transfer was proposed by Almog et al. [56] from FITC-dextran (MW: 70,000) retention experiments and chromatography on Con A – Sepharose column (Table 3, line 4). According to Ollivon et al. [54], leakage of 3H-inulin would mark the appearance of large OG stabilized pores. The presence of cholesterol strongly reduces the kinetic of OG insertion in membrane. NSV, which are formed with 50 mol% of cholesterol, are impermeable to OG up to a surfactant concentration of 10 mM. HPSEC (Table 3, line 5) has shown that pure sonicated NSV elute at the same elution volume over a TSK-G6000 PW column either with OG-free buffer or 10 mM OG containing buffer [43]. Once OG is capable to penetrate the bilayers, a liposome growth is observed as for the EPC – OG system. Fig. 9 shows the elution profiles of pure NSV and mixed aggregates at two steps of the NSV solubilization (E and D, Fig. 9 inset), analyzed with a TSK-G6000 PW column pre-saturated with a 10 mM OG containing buffer. Partitioning of surfactants between lipidic membrane and aqueous medium leads to a progressive permeabilization of the liposomes without disrupting the liposome bilayers. Solubilization occurs after saturation of the membrane by the surfactant molecules [51,53]. The processes of permeabilization have been examined with extruded EPC/EPA (90/10 mol/mol) liposomes entrapping FITC-dextrans of increasing average molecular masses [29,38]. The effect was analyzed with the HPSEC system described above (see Section 3.2, Fig. 4) from the leakage of the markers induced by OG and sodium taurocholate surfactants. An example of the release kinetic obtained with the FITC-dextran 4400 loaded liposomes and OG is presented in Fig. 10 (Table 3, line 6). Briefly, the methodology consists of continuously adding under stirring, a 100 mM OG solution to the liposomes at a constant rate, using a syringe pump. The dextran leakage was analyzed by injecting the surfactant-treated suspensions on the TSK-G6000 and -G4000 columns in series. The eluent was the continuous medium in equilibrium with the mixed OG – liposomes. Indeed, it is very important to maintain during the elution process the medium in equilibrium with the mixed aggregates obtained at the end of OG addition. Elution with free buffer would dilute the surfactant in the external aqueous phase of the mixed aggregates and necessarily remove surfactant from the liposome membrane. The release content was quantitatively

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Fig. 9. Size growth of liposomes induced by surfactant during the liposome-micelle transition. HPSEC on a TSKG6000 PW column of sonicated NSV (mean diameter MD = 66 nm, initial lipid concentration [lip]init = 2.5 mM, elution profile NSV) and mixed aggregates at points E (MD = 131 nm, [lip]init = 2.3 mM, elution profile E) and D (MD = 196 nm, [lip]init = 2.1 mM, elution profile D) of the solubilization curve reported in the inset. This curve was obtained by a continuous addition of OG at 6.94  10-5 mmol/min. The column was pre-equilibrated with buffer containing 10 mM OG, used also as eluent. Flow rate: 1 ml/min, sample loading: 50 Al. Redrawn from Ref. [43].

Fig. 10. Liposome permeability to surfactant. Permeability to OG of the extruded EPC/EPA (9/1, w/w) liposomes loaded with FITC-dextran 4400. The kinetic of release was monitored by HPSEC on TSK-G6000 and -G4000 PW columns connected in series. The amount of the marker released from the liposomes as a function of the OG concentration in the sample ([OG]tot) was determined from the calibration curve reported in the inset. Inset: fluorescence intensity at the maximum of the elution peak of dextran in buffer solution versus the concentration of FITC-dextran 4400. HPSEC conditions as described in Fig. 5. Redrawn from Ref. [29].

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determined from a calibration curve (FITC fluorescence intensity versus polymer concentration (Fig. 10 inset) obtained by elution of solutions of FITC-dextran 4400 in the conditions used for the permeability experiments. The 90j light scattering allows to follow both the integrity of the liposomes and changes in their size. The HPSEC data shows that no release of FITC-4400 from the liposomes is detected below a specific OG concentration and thereafter the leakage is governed by the amount of surfactant molecules in the liposome membrane. Two distinct mechanisms of efflux of FITC-dextrans from the loaded liposomes have been evidenced independently of the average molecular masses of the FITC-dextrans: the release of the entrapped markers occurs via the formation of transient intramembrane pores with OG and permanent ones with sodium taurocholate [38]. 3.3.5. Liposome formation and reconstitution A classical strategy for liposome reconstitution is based on surfactant removal from mixed micelles [53]. By using the appropriate surfactant, it represents the most efficient way to encapsulate labile drugs or biological substances such as proteins, peptides and nucleic acids while maintaining intact their biological functions. Surfactant removal can be performed by dialysis [8,19,23], dilution [54,55,57,60], SEC [10,61,62] or adsorption onto hydrophobic polymer beads (Sephadex, Biobeads SM2,) [63 – 66]. SEC offers the major advantage to form liposomes and remove the unencapsulated material in a single step. Liposome formation, during elution through the gel beads with buffer free of surfactant, results from surfactant removal from the mixed micelles by a dilution process which is accelerated by the gel sieving between the monomers and mixed micelles accessible to the gel pores and liposomes which are excluded from them [53]. In the liposome reconstitution process, the surfactant depletion from the mixed micelles generates bilayer curvature, which ends in liposome closure. This particular point can be estimated from entrapment of large molecules at the time of the closure. This was done with inulin for the transformation of EPC – OG mixed micelles into liposomes by surfactant dilution [54] (Table 3, line 7). A mixture of EPC (12.8 mM), OG (40 mM), 3 H-inulin and 14C-glucose was diluted 11-fold with OG-free buffer containing equivalent concentrations of 14C-glucose and unlabeled inulin. The inulin, EPC and OG are diluted equally until the liposomes close, whereas after closure, the inulin within the liposomes is not diluted further. At each dilution step, the residual OG was removed by a passage of the sample successively on a Sephadex G25 column prewashed with buffer containing 14Cglucose and EPC liposomes and a Sephadex G150 column to remove the remaining OG, the external inulin and glucose. Aliquots of the initial mixture and washed liposome fractions were taken for liquid scintillation counting. The concentration of OG at closureto-inulin was determined from the dilution of inulin relative to the entrapped volume marker 14C-glucose. An enzymatic procedure for liposome preparation through micelle to liposome transition has been developed. Formation of DPPC liposomes from dodecyl h-D-maltoside (DM) –DPPC mixed micelles was achieved in the presence of 1 mM calcein by using two successive enzymatic reactions with amyloglucosidase and h-D-glucosidase enzymes, respectively [58]. Calcein was used as a marker of bilayer closure. Conventional SEC, which can separate calcein molecules encapsulated in the aqueous cavity of aggregates

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from those free in the external aqueous medium, is a suitable technique to assess the formation of close structures. Thus, formation of DPPC liposomes was first evidenced by chromatography of the aggregates obtained, on a Sephacryl S1000 coupled with on-line fluorescence [58]. Then, HPSEC with the set-up of Fig. 4 (Table 3, line 8) was used to follow the kinetics of the transformation during the enzymatic process from the elution profile observed by fluorescence for the fraction of the encapsulated calcein (Fig. 11A, curves a to c) compared to that detected by 90j light scattering (Fig. 11B, curves a to c) for the aggregate turbidity [59]. In parallel, the feasibility of forming DPPC –dodecyl-h-Dmaltoside (DG) liposomes by sonication from a DPPC –DG mixed film hydrated with

a

Fig. 11. Formation of DPPC liposomes via an enzymatic process. (A, B) HPSEC on TSK-G6000 and -G4000 PW columns of the aggregates formed during the enzymatic process from DM – DPPC mixed micelles in the presence of 1 mM calcein, as a function of the concentration of the enzyme solution ([Enz] = 0.3 (a), 0.6 (b) and 1.2 (c) AM, respectively; total [DPPC] = 5 mM, initial [DM/DPPC] = 1.8). Chromatography analysis (same system as above) was made at 5 days of the enzymatic reaction. Total [DM] of the samples are 1.67 (a), 5.2 (b) and 6.7 (c) mM, respectively. Curve d corresponds to sonicated DPPC liposomes loaded with calcein ([DPPC] = 10 mM). Prior to analysis, the columns were saturated with DPPC – DG liposomes prepared without calcein. The chromatograms in A are shifted along the axis for clarity. (C, D) Formation of DPPC – DG mixed liposomes by ultrasonication: HPSEC analysis (as above) of 1 mM calcein ultrasonicated pure DPPC ( ) and DPPC – DG mixed liposomes (total DPPC/DG molar ratios equal to 0.1 (— - - —), 1.0 (- - -) and 1.8 (. . .). On-line detections: (A, C) fluorescence (excitation at 367 nm, emission at 419 nm), (B, D) 90j light scattering at 367 nm. Eluent: 145 mM NaCl, 10 mM Hepes, pH 7.4; flow rate 1 ml/min. Void (Vo) and total (VT) volumes of the columns are indicated by arrows. Redrawn from Ref. [67].

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213

isotonic buffer containing 1 mM calcein was investigated for different DG/lipid molar ratios [67] with the same HPSEC system (Table 3, line 8). As above, entrapment of calcein is indicative of the existence of closed liposomes (Fig. 11C and D). As already mentioned, the interest in using a gel column in the size range of the liposomes to be analyzed is to get information on their physical characteristics. It can be seen that the elution behavior of the enzymatically formed liposomes is similar to that of sonicated unilamellar DPPC liposomes taken as reference (Fig. 11, curve d). 3.3.6. Interaction and insertion of biological amphiphilic molecules Application of size exclusion chromatography in the analysis of drug entrapped liposomes and protein –liposome association has been reviewed by Lundahl et al. [16]. Conventional SEC or HPSEC are powerful techniques of investigation that has provided information on: solubility limit of triglycerides (3.8%) into EPC liposomes [68]; distribution of drug [69] and cytokine protein [28] between the liposome membrane and the internal and external aqueous compartments; the conformational behavior of gramicidin A inserted in lipid dispersion, liposomes and micelles [70]; pore formation and sizing in liposomes by melittin [71,72]; non-association of proinsulin C-peptide with liposome bilayers [73]; formation of proteoliposomes and characterization of the size distribution, protein and lipid content of the liposomes [74]; liposome –polyelectrolyte-interactions [75,76]; biotin peroxydase activity in targeted streptavidin –liposomes [77]; interaction of charge isomers of myelin basis protein with negatively charged liposomes [78]; association of interleukin with liposomes [79].

4. Conclusion Conventional SEC and HPSEC provide a variety of information in different liposome fields as shown by the data presented here. In addition, a major interest of this technique is its ability to separate liposomes from solutes and to fractionate heterogeneous suspensions into monodisperse subpopulations. Provided the elution buffer is isoosmotic with the aqueous medium of the liposomes, the analysis practically presents no stress for the liposome structure. It can be used simply as a routine method or as much more elaborated one with minimum SEC equipment as well as fully automated and sophisticated HPSEC apparatus. Conventional SEC and HPSEC can be scaled both on analytical and preparative scales. However, it is recommended to test first the conditions appropriate for preparative separations on an analytical scale. Studies based on conventional SEC show that, due to the gel bed generally used, the liposomes systematically elute at the void volume of the columns. This favors the separation gap between liposomes and unloaded solute but offers a major drawback, i.e., liposomes cannot be fractionated and characterized in size and size distribution. This is also verified when using HPLC exclusion gels of low porosity, which elute also the liposomes at the void volume. Consequently, to analyze loaded liposomes, it is necessary to connect in series two columns, one covering the size range of liposomes (e.g., TSKG6000 PW or Sephacryl S1000 columns) and the other that of smaller particles. Precise description of the elution profiles requires on-line detection, while multi-mode ones

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provide simultaneous information obtained from the same sample in the same conditions. Furthermore, this avoids the consuming task of fraction collection and subsequent analysis. There are obvious advantages in using HPSEC with TSK PW series columns, particularly for kinetic analysis (stability, material release, interaction/insertion of exogenous substances, etc.), as the rapid run time allows serial time sampling and time resolved analysis, in addition to reproducibility, separation efficiency and quantitative determination. With fluorescent markers, as often used in permeability studies, it is sufficient to encapsulate a small amount since labels entrapped into liposomes are separated from the unentrapped ones onto the gel column, so that quenching effect is no longer requisite. Moreover, when the encapsulated marker does not present extinction of fluorescence, separation by chromatography is the only way to monitor liposome leakage. From a general viewpoint, the SEC technique offers the advantage of being a modular tool, highly relevant for the control and understanding of any loaded liposome system, whatever the nature of the exogenous substances: charged or uncharged, water-soluble, amphiphilic or hydrophobic.

Acknowledgements We are grateful to Maud Seras (PhD 1992), Sophie Beugin-Deroo (PhD 1997), Brigitte Carion-Taravella (PhD 1998) and Karine Andrieux (PhD 2000) for their contribution to a number of works cited in this review.

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