**3. Liposomes**

### **3.1 General characterisation of liposomes**

Liposomes, membrane-like spherical structures consisting of one or more concentric lipid bilayers enclosing aqueous compartments were first formulated and described by Alex Bangham in 1965 (Bangham et al., 1965), and have become a useful tool and model in various areas of science. Liposomes represent the oldest and the most explored nano- and micro systems for biological studies on model membranes and for medical applications, especially for drug formulations, because they eliminate or suppress organ-specific toxic side-effects of various drugs (Allen, 1997). Through their 46-year history, liposomes have been approved as suitable delivery systems for applications ranging from cosmetics and dermatology, anti-infection and anticancer therapy and diagnostics up to human as well as veterinary vaccines (Gregoriadis, 1995).

Liposomes are classified in terms of number of bilayers enclosing the sequestered aqueous volume as follows: unilamellar, oligolamellar, and multilamellar. Unilamellar vesicles can be further divided into small unilamellar vesicles (SUVs) with a large curvature, and large unilamellar vesicles (LUVs) with a low curvature and hence, with properties similar to those of a flat surface. Multilamellar vesicles (MLVs) are liposomes that represent a heterogenous group in terms of size and morphology (Cullis et al., 1987). Lipid composition, size and morphology are variables determining the fate of liposomes in biological milieu; therefore, the selection of suitable method for the preparation of liposomal drugs and vaccines is of importance in respect to subsequent animal experiments and future successful marketing of the product. Schematic structures of various types of liposomes as well as realistic picture obtained by cryoelectron microscopy are presented in Fig. 4.

### **3.2 Liposome-based vaccines**

The use of liposomes as vaccine adjuvants was first described by Allison and Gregoriadis in 1974 (Allison & Gregoriadis, 1974). Since that time, numerous studies were performed and proved that liposomes can be used to enhance the immune response towards a large variety of peptide and protein antigens derived from various microbial pathogens as well as tumours.

Liposomes, membrane-like spherical structures consisting of one or more concentric lipid bilayers enclosing aqueous compartments were first formulated and described by Alex Bangham in 1965 (Bangham et al., 1965), and have become a useful tool and model in various areas of science. Liposomes represent the oldest and the most explored nano- and micro systems for biological studies on model membranes and for medical applications, especially for drug formulations, because they eliminate or suppress organ-specific toxic side-effects of various drugs (Allen, 1997). Through their 46-year history, liposomes have been approved as suitable delivery systems for applications ranging from cosmetics and dermatology, anti-infection and anticancer therapy and diagnostics up to human as well as

Liposomes are classified in terms of number of bilayers enclosing the sequestered aqueous volume as follows: unilamellar, oligolamellar, and multilamellar. Unilamellar vesicles can be further divided into small unilamellar vesicles (SUVs) with a large curvature, and large unilamellar vesicles (LUVs) with a low curvature and hence, with properties similar to those of a flat surface. Multilamellar vesicles (MLVs) are liposomes that represent a heterogenous group in terms of size and morphology (Cullis et al., 1987). Lipid composition, size and morphology are variables determining the fate of liposomes in biological milieu; therefore, the selection of suitable method for the preparation of liposomal drugs and vaccines is of importance in respect to subsequent animal experiments and future successful marketing of the product. Schematic structures of various types of liposomes as well as realistic picture

The use of liposomes as vaccine adjuvants was first described by Allison and Gregoriadis in 1974 (Allison & Gregoriadis, 1974). Since that time, numerous studies were performed and proved that liposomes can be used to enhance the immune response towards a large variety of peptide and protein antigens derived from various microbial pathogens as well as tumours.

Fig. 3. Structural differences between Romurtide and MT05.

obtained by cryoelectron microscopy are presented in Fig. 4.

**3.1 General characterisation of liposomes** 

veterinary vaccines (Gregoriadis, 1995).

**3.2 Liposome-based vaccines** 

**3. Liposomes** 

A) Photograph of various liposomal structures by cryoelectron microscopy. B) Schema of types of liposomes: SUV – small unilamellar vesicle, LUV - large unilamellar vesicle,, MLV – multilamellar vesicle, MVV – multivesicular vesicle

Fig. 4. Schematic representation of various morphological classes of liposomes and their real image obtained by cryoelectron microscopy.

The potential for the participation of liposome-based recombinant vaccines on the human and veterinary vaccine market is very promising (Adu-Bobie et al., 2003). Liposomal vaccines have been around for about 30 years and plenty of liposome variants have been developed; some of them with evident immune-stimulating properties and an attractive safety profile which resulted in registered products on the market or preparations in advanced stages of clinical testing. Liposomal hepatitis A vaccine is the first formulation of liposomes to become licensed for clinical use in humans (Gluck et al., 1992) (Hepatitis A - HepA, Epaxal http://www.crucell.com/Products-Epaxal). Epaxal liposomes contain influenza hemagglutinin protein which facilitates their binding and endocytosis by specific receptor on antigen presenting cells. Such forms of liposomes are called virosomes. Liposomes represent almost ideal carrier system for the preparation of synthetic vaccines due to their biodegradability and versatility as regards the incorporation of quite a number of various molecules having different physico-chemical properties (the size of the molecule, hydrophilicity or hydrophobicity, the electric charge).

The molecules and antigens can be either sterically entrapped into the liposomes (the internal aqueous space), or embedded into the lipid membrane (e.g., membrane-associated proteins/antigens) by hydrophobic interactions. Further, they can be attached to either the external or the internal membrane by electrostatic, covalent or metallo-chelating interactions. It is possible to encapsulate simultaneously various compounds into the liposomes: hydrophilised/lipophilised adjuvants (e.g., MPL A, CpG oligonucleotides, MDP and its analogues), soluble or membrane protein antigens, and ligands for the targeting to specific receptors on the antigen-presenting cells. Further, liposomes can be coated with mucoadhesive biopolymers, or undergo surface-charge modifications (e.g., by cationic lipids) (Altin & Parish, 2006).

As a great advantage, liposomes can be used for the preparation of self-assembling hybrid supramolecular nanosystems such as proteoliposomes, which can combine liposomal nanoparticles with suitable immunopotentiating/adjuvant molecules (e.g., MPL A, CpG oligonucleotides, MDP, and its analogues).

### **3.3 Liposomes as antigen carriers**

Structural diversity of liposomes permits tailoring of liposome-based vaccines to obtain an optimal adjuvant effect for a particular antigen. Their safety proles and ability to induce an immune response makes them likely to be included in vaccine formulations. Liposomal formulations offer several major advantages. They can (1) prevent degradation of the delivered antigens and adjuvants; (2) allow membrane proteins to reconstitute and preserve their antigen structure; (3) increase the antigenic effect of weak immunogens; (4) target antigen-presenting cells (APCs) and direct the antigen to MHCI or MHCII presentation; (5) contain the antigen and adjuvant molecules in one particle, thus functioning simultaneously as a delivery system and a vaccine adjuvant; and (6) reduce the antigen and adjuvant doses required for an immune response and controlled release. Therefore, liposomes provide a safe and effective platform for construction of subunit vaccines.

With respect to the physico-chemical nature of liposomes and phospholipid bilayers, the liposomes represent one of the most versatile structures for the preparation of drug delivery systems. Both hydrophobic and hydrophilic protein or peptide antigens can be associated with liposomes. Generally, antigens can be associated with liposomes in two ways and it is known that the encapsulated liposomal antigens induce a different immune response than the surface-linked antigens in both humoral (Shahum & Therien, 1988) and cell-mediated immunity (Fortin et al., 1996). If entrapped into the internal aqueous space of a liposome, the protein or peptide antigen is protected against proteolytic degradation and the antigen clearance is decreased. On the other hand, the liposomal membrane represents a barrier restricting the interaction of the antigen with and its recognition by B-cells. Especially, the stable multilamellar liposomes were found to be low immunogenic (Shek & Heath, 1983) and the antibody response reached is low or absent when the liposomes are made of lipids with a high transition temperature; in other words, when they are composed of saturated phospholipids (Gregoriadis et al., 1987). These liposomes are very stable in body fluids as well as in digestive tract and prevent a release of the entrapped antigen. Also the interaction of the encapsulated antigens with B-cells is limited. The fluidity of liposomes was found to be an important parameter also for the immune response towards a surface-linked antigen. Again, more fluidic liposomes composed of unsaturated phospholipids are more efficiently phagocytosed by APC and induce a one order of magnitude higher immune response than rigid liposomes composed of saturated phospholipids (Uchida & Taneichi, 2008).

Liposomes are potentially very useful for the construction of vaccination systems given their facile biodegradability and versatility as carriers for varieties of molecules having different physico-chemical properties (such as size, hydrophilicity, hydrophobicity, or net electrical charge). Liposomes also offer the possibility to associate or entrap simultaneously more than one type of molecules. Of particular interest to us has been the co-association of hydrophilic or lipophilic adjuvants (e.g., monophosphoryl lipid A [MPL A], CpG oligonucleotides, muramyl dipeptide (MDP), and/or MDP lipophilic analogues) with soluble or membrane protein antigens or ligands for the targeting of specific receptors on antigen-presenting cells. The molecules and antigens can be either sterically entrapped into the liposomes (the internal aqueous space), or embedded into the lipid membrane (e.g., membrane-associated proteins/antigens) by hydrophobic interactions (Fig. 5). The ligands for the targeting to specific receptors on the antigen-presenting cells can significantly enhance the intensity of

nanoparticles with suitable immunopotentiating/adjuvant molecules (e.g., MPL A, CpG

Structural diversity of liposomes permits tailoring of liposome-based vaccines to obtain an optimal adjuvant effect for a particular antigen. Their safety proles and ability to induce an immune response makes them likely to be included in vaccine formulations. Liposomal formulations offer several major advantages. They can (1) prevent degradation of the delivered antigens and adjuvants; (2) allow membrane proteins to reconstitute and preserve their antigen structure; (3) increase the antigenic effect of weak immunogens; (4) target antigen-presenting cells (APCs) and direct the antigen to MHCI or MHCII presentation; (5) contain the antigen and adjuvant molecules in one particle, thus functioning simultaneously as a delivery system and a vaccine adjuvant; and (6) reduce the antigen and adjuvant doses required for an immune response and controlled release. Therefore, liposomes provide a

With respect to the physico-chemical nature of liposomes and phospholipid bilayers, the liposomes represent one of the most versatile structures for the preparation of drug delivery systems. Both hydrophobic and hydrophilic protein or peptide antigens can be associated with liposomes. Generally, antigens can be associated with liposomes in two ways and it is known that the encapsulated liposomal antigens induce a different immune response than the surface-linked antigens in both humoral (Shahum & Therien, 1988) and cell-mediated immunity (Fortin et al., 1996). If entrapped into the internal aqueous space of a liposome, the protein or peptide antigen is protected against proteolytic degradation and the antigen clearance is decreased. On the other hand, the liposomal membrane represents a barrier restricting the interaction of the antigen with and its recognition by B-cells. Especially, the stable multilamellar liposomes were found to be low immunogenic (Shek & Heath, 1983) and the antibody response reached is low or absent when the liposomes are made of lipids with a high transition temperature; in other words, when they are composed of saturated phospholipids (Gregoriadis et al., 1987). These liposomes are very stable in body fluids as well as in digestive tract and prevent a release of the entrapped antigen. Also the interaction of the encapsulated antigens with B-cells is limited. The fluidity of liposomes was found to be an important parameter also for the immune response towards a surface-linked antigen. Again, more fluidic liposomes composed of unsaturated phospholipids are more efficiently phagocytosed by APC and induce a one order of magnitude higher immune response than

rigid liposomes composed of saturated phospholipids (Uchida & Taneichi, 2008).

Liposomes are potentially very useful for the construction of vaccination systems given their facile biodegradability and versatility as carriers for varieties of molecules having different physico-chemical properties (such as size, hydrophilicity, hydrophobicity, or net electrical charge). Liposomes also offer the possibility to associate or entrap simultaneously more than one type of molecules. Of particular interest to us has been the co-association of hydrophilic or lipophilic adjuvants (e.g., monophosphoryl lipid A [MPL A], CpG oligonucleotides, muramyl dipeptide (MDP), and/or MDP lipophilic analogues) with soluble or membrane protein antigens or ligands for the targeting of specific receptors on antigen-presenting cells. The molecules and antigens can be either sterically entrapped into the liposomes (the internal aqueous space), or embedded into the lipid membrane (e.g., membrane-associated proteins/antigens) by hydrophobic interactions (Fig. 5). The ligands for the targeting to specific receptors on the antigen-presenting cells can significantly enhance the intensity of

oligonucleotides, MDP, and its analogues).

safe and effective platform for construction of subunit vaccines.

**3.3 Liposomes as antigen carriers** 

the immune response (Altin & Parish, 2006). For the mucosal application, the liposomes can be coated with mucoadhesive biopolymers or modified with surface-charge modifiers (e.g., cationic lipids). In this way, liposomes become a versatile platform that represents a real multifunctional vaccination carrier.

The importance of liposomes for the effective co-administration of adjuvants could be demonstrated using MDP as an example. MDP has a weak immunoadjuvant activity in aqueous solution due to its rapid excretion into urine. Appropriate formulations of hydrophilic MDP in "water in oil" emulsions (Parant et al., 1979) or liposomes were used to harness its full adjuvant potenital (Tsujimoto et al., 1986). Some lipophilic derivatives of MDP like B30-MDP and MDP-Lys (L18) were synthesised and tested as adjuvants for recombinant hepatitis B surface antigen (Tamura et al., 1995) or influenza surface antigens hemagglutinin and neuraminidase (Nerome et al., 1990). We used new synthetic nonpyrogenic lipophilic analogues of norAbu-MDP modified at a peptide part by hydrophobic ligands (Fig. 2) and these well defined synthetic molecules were used for the first time in combination with metallochelating liposomes to construct an experimental recombinant vaccine. Surprisingly, we have found that at certain surface density of lipophilic analogues of norAbu-MDP (about 5 mol % of total liposomal lipid), the liposomes are promptly recognised and phagocytosed by human dendritic cells. The phagocytosis is about one order of magnitude higher than that of proteoliposomes or liposomes lacking norAbu-MDP adjuvant. This finding implicates an existence of receptors on dendritic cells, which can recognise some molecular pattern formed by the hydrophilic part of norAbu-MDP exposed on the liposomal surface (illustration of this phenomenon is in Fig. 12B).

a) physical entrapment inside the liposome (blue protein); b) reconstitution of membrane protein in lipid bilayer via hydrophobic transmembrane domain (yellow protein); c) anchoring of lipidised protein onto liposomal surface or attachment of recombinant protein onto the liposomal surface by covalent bond using activated lipids (red protein); d) attachment of recombinant protein onto the liposomal surface by non-covalent bond using metallochelating lipids

Fig. 5. Association of protein antigen with liposome.

### **3.4 Methods of liposome preparation**

The laboratory and industrial procedures for the liposome preparation have been established and liposomes have been approved by FDA for biomedical applications. A variety of procedures for the preparation of various types of liposomes has been developed and reported in several reviews and monographs (Gregoriadis, 1992; Woodle & Papahadjopoulos, 1988). To classify these methods, they were arranged in three categories: 1) mechanical dispersion methods such as hand shaking or vortexing, sonication, and high pressure homogenisation; 2) detergent-solubilizing dispersion methods including solubilized lecithin dispersion with sodium cholate or octylglucoside; and 3) solvent dispersion methods such as ethanol injection, ether infusion, and reverse-phase evaporation. These primary processes can be linked with secondary processes such as high-pressure homogenisation or extrusion through polycarbonate filters of various pore size, which are easy ways to prepare liposomes of a desired size and morphology (Barnadas-Rodriguez & Sabes, 2001; Berger et al., 2001; Cullis, 1987; Hope et al., 1985; Perrett et al., 1991; Turanek, 1994; Woodle & Papahadjopoulos, 1988; Schneider et al., 1995).

The stability of proteins is limited and not all the methods are useful for the preparation of proteoliposomes, especially if the protein is to be entrapped inside the liposome. The detergent dilution method is characterized by very mild conditions during the process and is suitable for the reconstitution of membrane proteins. Because many recombinant proteins tend to precipitate, this method is also useful to work with these protein antigens. Next paragraph describes in detail a modified detergent dilution method for the preparation of proteoliposomes.

### **3.5 Preparation and characterization of metallochelating liposomes**

Because the preparation of metallochelating liposomes represents a post-forming modification of liposomes, it avoids a denaturation of proteins owing to the process used for the liposome production. Therefore, nearly all the methods mentioned above could be used to prepare more or less monodisperse liposomes. Here we describe in brief a modification of the detergent removal method, which is suitable for the preparation of very monodisperse unilamellar liposomes that are useful for structural studies by various techniques (e.g., TEM, dynamic light scattering, and gel permeation chromatography).

When essentially unilamellar monodisperse liposomes of spherical shape are needed (which is a prerequisite for a precise monitoring of the proteoliposome formation by dynamic light scattering), the detergent removal method is preferred to the other methods. The method is based on the transformation of phospholipid micelles stabilized by detergent with high critical micellar concentration (CMC) (e.g., cholate) to desk micelles and finally to vesicles during the process of detergent removal (Zumbuehl & Weder, 1981) (Fig. 6). The mild conditions provided by this method are advantageous for the preparation of proteoliposomes, especially for the reconstruction of membrane proteins (Rigaud & Levy, 2003) like viral or bacterial antigens or recombinant his-tagged proteins that are often prone to precipitation.

There are many variants of the detergent removal method, e.g., the dilution of the solution of mixed micelles, gel permeation chromatography, a simple dialysis or the controlled one in a special apparatus, cross-flow filtration, adsorption on beads, etc. (Schubert, 2003). The application of the flow-through ultrafiltration cell represents a new approach to the detergent removal method (Masek et al., 2011a). The linkage of the cell with systems like FPLC facilitates automation of the whole procedure and manipulation with the sample. The full control over the dialysis rate and the removal of the undesired residua (e.g., detergent, organic solvents, protein solubilizers) is ensured and various steps like an addition of required components through an injection valve during various stages of the liposome formation are easy to perform without breaching the sterile conditions. In this case, the sterile filter inserted in front of the cell inlet ensures that the sterility is kept during the whole process (Fig. 7). The low dead volume of the cell is of great importance for the preparation of liposomes and proteoliposomes in small laboratory scale. However, this arrangement enables also very easy up-scaling of the whole technology. A precise control

Papahadjopoulos, 1988). To classify these methods, they were arranged in three categories: 1) mechanical dispersion methods such as hand shaking or vortexing, sonication, and high pressure homogenisation; 2) detergent-solubilizing dispersion methods including solubilized lecithin dispersion with sodium cholate or octylglucoside; and 3) solvent dispersion methods such as ethanol injection, ether infusion, and reverse-phase evaporation. These primary processes can be linked with secondary processes such as high-pressure homogenisation or extrusion through polycarbonate filters of various pore size, which are easy ways to prepare liposomes of a desired size and morphology (Barnadas-Rodriguez & Sabes, 2001; Berger et al., 2001; Cullis, 1987; Hope et al., 1985; Perrett et al., 1991; Turanek,

The stability of proteins is limited and not all the methods are useful for the preparation of proteoliposomes, especially if the protein is to be entrapped inside the liposome. The detergent dilution method is characterized by very mild conditions during the process and is suitable for the reconstitution of membrane proteins. Because many recombinant proteins tend to precipitate, this method is also useful to work with these protein antigens. Next paragraph describes in detail a modified detergent dilution method for the preparation of

Because the preparation of metallochelating liposomes represents a post-forming modification of liposomes, it avoids a denaturation of proteins owing to the process used for the liposome production. Therefore, nearly all the methods mentioned above could be used to prepare more or less monodisperse liposomes. Here we describe in brief a modification of the detergent removal method, which is suitable for the preparation of very monodisperse unilamellar liposomes that are useful for structural studies by various techniques (e.g., TEM,

When essentially unilamellar monodisperse liposomes of spherical shape are needed (which is a prerequisite for a precise monitoring of the proteoliposome formation by dynamic light scattering), the detergent removal method is preferred to the other methods. The method is based on the transformation of phospholipid micelles stabilized by detergent with high critical micellar concentration (CMC) (e.g., cholate) to desk micelles and finally to vesicles during the process of detergent removal (Zumbuehl & Weder, 1981) (Fig. 6). The mild conditions provided by this method are advantageous for the preparation of proteoliposomes, especially for the reconstruction of membrane proteins (Rigaud & Levy, 2003) like viral or bacterial

There are many variants of the detergent removal method, e.g., the dilution of the solution of mixed micelles, gel permeation chromatography, a simple dialysis or the controlled one in a special apparatus, cross-flow filtration, adsorption on beads, etc. (Schubert, 2003). The application of the flow-through ultrafiltration cell represents a new approach to the detergent removal method (Masek et al., 2011a). The linkage of the cell with systems like FPLC facilitates automation of the whole procedure and manipulation with the sample. The full control over the dialysis rate and the removal of the undesired residua (e.g., detergent, organic solvents, protein solubilizers) is ensured and various steps like an addition of required components through an injection valve during various stages of the liposome formation are easy to perform without breaching the sterile conditions. In this case, the sterile filter inserted in front of the cell inlet ensures that the sterility is kept during the whole process (Fig. 7). The low dead volume of the cell is of great importance for the preparation of liposomes and proteoliposomes in small laboratory scale. However, this arrangement enables also very easy up-scaling of the whole technology. A precise control

antigens or recombinant his-tagged proteins that are often prone to precipitation.

1994; Woodle & Papahadjopoulos, 1988; Schneider et al., 1995).

**3.5 Preparation and characterization of metallochelating liposomes** 

dynamic light scattering, and gel permeation chromatography).

proteoliposomes.

over the rate of the detergent removal yields a final liposomal preparation of high monodispersity (PDI within the range of 0.05 - 0.06), which is shown to be reached routinely (Fig. 8). This monodispersity is better than those obtained by the dialysis method performed in the dialysis bags or slides (produced by Pierce) (PDI ≈ 0.08-0.12).

The size of the mixed micelles (≈5-6 nm; see Fig. 8) used by us for the preparation of liposomes is in good correlation with the Small's mixed micellar model proposing the structure of a small phospholipid bilayer disc stabilised at its hydrophobic edges by the molecules of cholate (Small, 1971; Schubert, 2003).

The process of the formation of the monodisperse liposomes is in good accordance with the proposed kinetic model of the micelle-vesicle transition based on a rapid formation of disklike intermediate micelles followed by a growth of these micelles up to their critical size and their subsequent closure to form vesicles. The final size of the liposomal preparation could be controlled by ionic strength of the buffer used for the preparation of the micelles ( Fig. 9). An increase of the NaCl concentration reduces CMC of cholate and shields the negative charge of the mixed micelles. These two factors are responsible for the formation and stabilisation of the large discoid bilayer micelles that are transformed into the larger liposomes (Schubert, 2003).

Various additives like bilayer stabilising sugars (e.g., sucrose) or recombinant protein solubilizers (e.g., urea, guanidine) are compatible with this method and can shift the size of the liposomes into the required range (Walter et al., 2000). Some recombinant proteins (e.g., circovirus envelope protein), which tend to precipitate in the absence of stabilizing buffers (imidazole and urea stabilizing buffer) were successfully linked onto metallochelating liposomes by one-step procedure based on the addition of the protein into the mixed micelle solution prepared in protein stabilizing buffer and transforming into proteoliposomes during the ultrafiltration procedure (Turánek, unpublished results).

Adapted: R. Schubert, Methods Enzymol. 367 (2003) 46–70.

Fig. 6. Principle of detergent removal method and formation of liposomes from mixed bile salt-phospholipid micelles.

Small mixed micelles are fused in disc phospholipid micelles stabilized at edges by detergent. Further removal of detergent induces formation of large disc micelles which spontaneously vesiculate after reaching a critical size.

The transformation of micelles into liposomes during the ultraltration removal of cholate is a critical step affecting the final quality of liposomes. This process is easy to be monitored by DLS. The removal of cholate induced a formation of disc micelles, which was reflected by an increase of the micelle size and eventually by a formation of liposomes (Fig. 10). The process of liposome formation had been completed before the CMC of cholate was reached, as shown by the dashed vertical line. This line divides the ow-through volume axis into the left part, where micelles do predominantly exist and are transformed into liposomes, and the right part, where liposomes represent the main lipid form, while the residual detergent and other low molecular weight contaminants (e.g., traces of ethanol or tetrahydrofuran used to solubilize the lipids) are continuously removed by the process of ultraltration*.* 

(A) Schematic illustration of the ultraltration cell. (B) Photograph of the ultraltration cell in detail (pink: LR-PE-labelled liposomes inside the cell). (C) Schematic illustration of the linkage of the ultraltration cell with the FPLC system. (D) Photograph of the system

Fig. 7. System for preparation of liposomes by removal of detergent using ultraltration.

left part, where micelles do predominantly exist and are transformed into liposomes, and the right part, where liposomes represent the main lipid form, while the residual detergent and other low molecular weight contaminants (e.g., traces of ethanol or tetrahydrofuran used to solubilize the lipids) are continuously removed by the process of ultraltration*.* 

(A) Schematic illustration of the ultraltration cell. (B) Photograph of the ultraltration cell in detail (pink: LR-PE-labelled liposomes inside the cell). (C) Schematic illustration of the linkage of the

Fig. 7. System for preparation of liposomes by removal of detergent using ultraltration.

ultraltration cell with the FPLC system. (D) Photograph of the system

A) Size distribution of micelles and liposomes. The hydrodynamic diameters of the micelles and liposomes were determined by dynamic light scattering instrument NanoSizer NS (Malvern, UK) at 25 °C. Silica cuvette of 45-µl volume (Hellma, Germany) was used. (B) TEM micrograph of monodisperse liposomal preparation. (C) AFM micrograph of monodisperse liposomal preparation

Fig. 8. Size distribution of micelles and metallochelating liposomes analysed by dynamic light scattering and visualization of liposomes by TEM and AFM.

Fig. 9. Effect of ionic strength on the size distribution of liposomes prepared by detergent removal method.

Fig. 10. Transformation of micelles into liposomes during ultraltration monitored by DLS. The dashed vertical line indicates the ultraltrate volume, when the CMC of sodium cholate was reached. This line divides the ow-through volume axis into the leftpart, where micelles do predominantly exist and are transformed into liposomes, and the right part, where liposomes represent the main lipid form, while residual detergent and ethanol/THF are continuously removed by the process of ultraltration.
