Physical And Chemical Characterization Of Liposomes

In the introduction the need for proper evaluation of liposomes to be administered has been stressed. Physical parameters affecting the behavior of liposomes in vivo are type (multilamellar, oligolamellar, unilamellar, multivesicular), size, charge, bilayer rigidity, and "surface hydrophobicity" (Senior, 1987). In the following paragraphs the pros and cons of the different techniques used to characterize liposomes will be dealt with briefly. More detailed information can be found, for example, in the books of Knight (1981) and Gregoriadis (1984) and in a review article by Lichtenberg and Barenholz (1988).

Liposome size can range from around 20 nm to around 50 ym. To a certain extent, the mean diameter and distribution of the diameters can be controlled by sizing procedures after the formation of the initial liposome dispersion or by a careful selection of the preparation conditions (cf. Sec. II). Several techniques can be used to determine mean particle size and particle size distribution (Groves, 1984).

Large liposomes, those with diameters over 1 ym, can be adequately measured by light microscopy and the Coulter counter. Light microscopy offers the possibility of collecting information on particle shape. This technique cannot be used for particles with diameters smaller than 1 ym because of lack of resolution. With a Coulter counter the volume distribution of liposomes (>1 ym) in dispersions can be determined. For particles in the submicrometer range, particle size can be assessed by electron microscopic analysis. Both negative staining and freeze-fracture techniques have been described. Sample preparation procedures should be properly validated. In particular with the negative staining technique, artifacts occur easily (Szoka and Papahadjopoulos, 1981). With the freeze-fracture technique, artifacts occur easily (Szoka and Papahadjopoulos, 1981). With the freeze-fracture technique, the fracture plane passes through liposomes which are randomly positioned in the frozen sample. Some liposomes will be cut far from their midplane sections, others through their midplane section. Therefore, the analysis of freeze-fracture pictures requires corrections for nonequatorial fracture. Besides, corrections have to be made for the size-dependent probability of a vesicle being in the fracture plane (Jousma et al., 1987; Guiot et al., 1980). Recently, results with a new technique based on electron microscopy was discussed; this technique allows analysis not only of liposome size, but also of the number of bilayers (Lauten-schlager et al., 1988).

Light-scattering techniques and turbidity measurements have been used extensively for liposome size analysis (Groves, 1984; Lichtenberg and Barenholz, 1988). Dynamic light scattering is rapidly growing in popularity for size analysis of liposome dispersions. This relatively new technique has proven to be an excellent tool to determine the mean diameter of dispersions with a relatively narrow particle size distribution. It can be used routinely, readings can be taken in minutes, 1 ml of material (in dilute form) is needed, and it is not destructive. The distribution analyses obtained for hetero-disperse colloidal systems (e.g., bimodal or skewed) need critical evaluation as the technique is biased toward the large particles in a dispersion.

Size exclusion chromatography has been used to analyse the size distribution of liposomes. For example, SUV can be separated from MLV, which elute in the void volume, by using a Sepharose 4B gel.

Vesicles with diameters over 1 ym tend to stick to the top of the column. This method has been used to monitor aggregation processes of liposomes.

Techniques which seem less suitable for routine size analysis are (1) analytical ultracentrifugation combined with a Schlieren optical system (Mason and Huang, 1978; Weder and Zumbuehl, 1984); (2) the sedimentation field flow fractionation (SFFF) technique to separate heterogeneous dispersions (e.g., Kirkland et al., 1982).

B. Charge

The surface potential can play an important role in the behavior of liposomes in vivo and in vitro (e.g., Senior, 1987). In general, charged liposomes are more stable against aggregation and fusion than uncharged vesicles. However, physically stable neutral liposomes have been described (e.g., Van Dalen et al., 1988). They are sufficiently stabilized by repulsive hydration forces, which counteract the attractive van der Waals forces.

In order to obtain an estimate of the surface potential, the ç potential of individual liposomes can be measured (>0.2 ym) by microelectrophoresis (e.g., Crommelin, 1984). This technique also offers the opportunity to detect the presence of structures with deviating electrophoretic mobility and, therefore, deviating composition.

An alternative approach is the use of pH-sensitive fluorophores (Lichtenberg and Barenholz, 1988"). These probes are located at the lipid-water interface and their fluorescence behavior reflects the local surface pH, which is a function of the surface potential at the interface. This indirect approach allows the use of vesicles independent of their particle size. Recently, techniques to measure the Ç potential of liposome dispersions on the basis of dynamic light scattering became commercially available (Millier et al., 1986).

The presence of impurities like free fatty acids in egg or soybean phosphatidylcholine, or in the (semi)synthetic phosphatidylcholines (e.g., DMPC, DPPC, DSPC) can be detected by monitoring the electrophoretic behavior of liposome dispersions of these phospholipids in aqueous media with low ionic strength: a negative charge will be found on these liposomes when free fatty acids are present in the bilayers.

C. Bilayer Rigidity and Homogeneity of Liposome

Dispersions

Bilayer rigidity is a parameter which influences biodistribution and biodégradation of liposomes. In vitro a hydrophilic marker molecule (carboxyfluorescein) leaked much faster from the vesicles with bilayers in a fluid state than from bilayers in a gel state (Crommelin and Van Bommel, 1984). An indication of the bilayer rigidity can be obtained by using fluorescence techniques. Fluorescence polarization of a probe which interacts with the liposome bilayer is determined; 1,6-diphenyl-l ,3,5,-hexatriene (DPH) is often used as a probe. Data obtained with these fluorescing probes have to be interpreted with care as the probe may locally disturb the bilayer or accumulate in certain domains of a nonhomogeneous dispersion.

Inhomogeneity can occur both within liposome structures themselves (asymmetrical distribution of bilayer components) and between liposomes in one dispersion (different liposomes), even when the liposomes are prepared via the same preparation procedure. For unilamellar vesicles the distribution of phospholipids over the inner and outer "monolayer" of the bilayer can be studied by chemical labeling. Aminolipids like PE react with trinitrobenzenesulfonate (TNBS) or fluorescamine; in general, these two compounds are incapable of penetrating lipid bilayers (Lichtenberg and Barenholz, 1988). Alternatively, enzymatic approaches (for membrane proteins) or 31p-NMR techniques are available (Van Dalen et al., 1988). Phase separation of lipid components in the bilayers can be detected by differential scanning calorimetry (DSC) (Mabrey-Gaud, 1981). Different liposome structures in one dispersion can be identified on the basis of density differences (gradient centrifugation, SFFF) or size differences (gel chromatography, dynamic light scattering).

D. Number of Lamellae

The number of lamellae can be determined by ^lp-NMR. The 31p_ NMR spectrum of egg phosphatidylcholine in liposomal form consists of an asymmetrical peak. Upon addition of Mn2+ (impermeable to the membrane) to the external phase of the liposome dispersion the peak surface is quenched, and only the contribution of the phospholipid head groups inside the vesicles is left (Browning, 1981; Jousma et al., 1987). From the change in spectrum the average number of bilayers can be calculated. The assays with TNBS or fluorescamine, mentioned in Section III.C, also offer the opportunity to determine the mean number of bilayers. Then aminolipids have to be present in the bilayers to couple the reagents; it is assumed that these aminolipids are evenly distributed over the bilayers. In addition, small-angle X-ray analysis of liposomes can give an indication of the number of bilayers (Jousma et al., 1987). Finally, electron microscopy can shed light on the question of the number of lamellae. Electron microscopy can be particularly helpful in discriminating between multilamellar and multivesicular liposomes (Cullis et al., 1987; Lautenschlager et al., 1988).

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