Polymerizable liposomes

The incorporation of a membrane protein into a polymerizable liposome from (22) was demonstrated by R. Pabst n9). The chromoprotein bacteriorhodopsin — a light-driven proton pump from halophilic bacteria — was incorporated into monomeric sulfolipid liposomes by ultrasonication. The resulting proteoliposomes were poly-... 

Bader, H. (1985) Functional amphiphiles in model membranes for surface recognition reactions and as membrane-fonning compounds for polymerizable liposomes, thesis, Mainz. 

While most vesicles are formed from double-tail amphiphiles such as lipids, they can also be made from some single chain fatty acids [73], surfactant-cosurfactant mixtures [71], and bola (two-headed) amphiphiles [74]. In addition to the more common spherical shells, tubular vesicles have been observed in DMPC-alcohol mixtures [70]. Polymerizable lipids allow photo- or chemical polymerization that can sometimes stabilize the vesicle [65] however, the structural change in the bilayer on polymerization can cause giant vesicles to bud into smaller shells [76]. Multivesicular liposomes are collections of hundreds of bilayer enclosed water-filled compartments that are suitable for localized drug delivery [77]. The structures of these water-in-water vesicles resemble those of foams (see Section XIV-7) with the polyhedral structure persisting down to molecular dimensions as shown in Fig. XV-11. 

Mixtures of proteins, natural and polymerizable lipids can be transferred into liposomes and polymerized hereafter. Initial experiments have shown that even very complex proteins such as F F.-ATPase can be incorporated in polymeric liposomes by this method under retention of the activity of the protein (76). 

The scientific community was attracted to the study of liposomes due to the relatively simple procedure of their preparation. Moreover, if prepared from natural phospholipids, they are biocompatible, and possess low cytotoxicity, low immunogenicity, and biodegradability [304], Liposomes, however, have two main disadvantages the structural instability both in vitro and in vivo, and low cell specificity [304], To increase the stability, the structure of the phospholipid layer has been modified to include artificial lipids and/or cholesterol. Polymerizable vesicles have also been prepared [305]. It is obvious that the biocompatibility of these modified systems has to be addressed. 

SUVs have also been stabilized by coating their outer surfaces with chitin [328], polylysine [329-332], polyelectrolytes [333], and polysaccharides [334]. Advantage has also been taken of electrostatic interactions to attract oppositely charged polyelectrolytes to outer SUV surfaces and subsequently polymerize them in situ [335-339]. These systems have been referred to as liposomes in a net [72]. A particularly telling example is the attachment of a two-dimensional polymeric network either to the inner or to the outer surfaces of SUVs by ion exchanging the vesicle counterions with oppositely charged polymerizable short-chain counterions and their subsequent polymerization (Fig. 42) [340-342]. 

All four types of polymerizable lipids shown in Fig. 4 have been realized synthetically. In this context, one need not attempt to reproduce mother nature slavishly (Fendler 8)). Kunitake 9) was able to show that simple molecules like dialkyldimethyl-ammonium salts also form bilayer assemblies. Fuhrhop 10) and Kunitake U) could accomplish the formation of monolayer liposomes with molecules containing only one alkyl chain and two hydrophilic head groups. Acryloylic and methacryloylic groups (type a and d, Table 1), as well as diacetylenic, butadienic, vinylic and maleic acid groups (type b and c), have been used as polymerizable moieties. A compilation of amphiphilic, photopolymerizable molecules is given in Table 1. 

Aqueous dispersions of polymerizable lipids and surfactants can be polymerized by UV irradiation (Fig. 18). In the case of diacetylenic lipids the transition from monomeric to polymeric bilayers can be observed visually and spectroscopically. This was first discussed by Hub, 9) and Chapman 20). As in monomolecular layers (3.2.2) short irradiation brings about the blue conformation of the poly(diacetylene) chain. In contrast, upon prolonged irradiation or upon heating blue vesicles above the phase transition temperature of the monomeric hydrated lipid the red form of the polymer is formed 23,120). The visible spectra of the red form in monolayers and liposomes are qualitatively identical (Fig. 19). 

Fig. 18. Scheme of formation of polymeric liposomes from polymerizable lipids 19-33)... 

Whether polymerized model membrane systems are too rigid for showing a phase transition strongly depends on the type of polymerizable lipid used for the preparation of the membrane. Especially in the case of diacetylenic lipids a loss of phase transi tion can be expected due to the formation of the rigid fully conjugated polymer backbone 20) (Scheme 1). This assumption is confirmed by DSC measurements with the diacetylenic sulfolipid (22). Figure 25 illustrates the phase transition behavior of (22) as a function of the polymerization time. The pure monomeric liposomes show a transition temperature of 53 °C, where they turn from the gel state into the liquid-crystalline state 24). During polymerization a decrease in phase transition enthalpy indicates a restricted mobility of the polymerized hydrocarbon core. Moreover, the phase transition eventually disappears after complete polymerization of the monomer 24). 

First, a mixture of synthetic or natural phospholipids, polymerizable lipids, and proteins can be converted to liposomes and then be polymerized. Second, polymerizable lipids are introduced into e.g. erythrocyte ghost cells by controlled hemolysis and subsequent polymerization as described by Zimmermann et al.61). This hemolysis technique is based on a reversible dielectric breakdown of the cell membrane. Dielectric breakdown provides a third possible path to the production of bi omembrane models. Zimmermann et al. could show that under certain conditions cells can be fused with other cells or liposomes61). Thus, lipids from artificial liposomes could be incorporated into a cell membrane. A fourth approach has been published by Chapman et al.20). Bacterial cells incorporate polymerizable diacetylene fatty acids into their membrane lipids. The diacetylene units can be photopolymerized in vivo. The investigations discussed in more detail below are based on approaches 1. and 3. 

As an example of an asymmetric membrane integrated protein, the ATP synthetase complex (ATPase from Rhodospirillum Rubrum) was incorporated in liposomes of the polymerizable sulfolipid (22)24). The protein consists of a hydrophobic membrane integrated part (F0) and a water soluble moiety (Ft) carrying the catalytic site of the enzyme. The isolated ATP synthetase complex is almost completely inactive. Activity is substantially increased in the presence of a variety of amphiphiles, such as natural phospholipids and detergents. The presence of a bilayer structure is not a necessary condition for enhanced activity. Using soybean lecithin or diacetylenic sulfolipid (22) the maximal enzymatic activity is obtained at 500 lipid molecules/enzyme molecule. With soybean lecithin, the ATPase activity is increased 8-fold compared to a 5-fold increase in the presence of (22). There is a remarkable difference in ATPase activity depending on the liposome preparation technique (Fig. 41). If ATPase is incorporated in-... 

Electric field-induced fusion has been applied to a vast variety of cells including human erythrocytes and liposomes made from asolectin and egg phosphatidylcholine. To what extent this method can be utilized for fusing polymerizable vesicles will be demonstrated in the following. 

Phase-separated monolayers and liposomes were characterized by R. Elbert1011 who synthesized saturated and polymerizable fluorocarbon amphiphiles (59, 60, 61) and investigated their mixing behavior with CH2-analogues and natural lipids. In these systems the fluorocarbon compounds are incompatible with hydrocarbon lipids in a wide range of compositions and tend to form domains of pure fluorocarbon and hydrocarbon amphiphiles. The domains can be visualized by freeze-fracture electron microscopy. 

K. Dorn 105 > polymerized dialkylammonium lipids with the polymerizable methacryloyl moiety either in the head group (29) or at the end of one of the hydrophobic chains (5). GPC revealed Mw 1.9 x 106, Mn 3.5 x 105, Mw/Mn 5.4 for (29) and Mw 1.9 xl06,Mn 3.9 x 105, Mw/Mn 2.4 for (5). It was also found that Mw varies inversely with the time of sonication, i.e. in smaller liposomes lower-molecular-weight polymers are formed. In a following paper, K. Dorn 108 present data for the permeability of monomeric and polymeric vesicles from (29). 

In addition to enzymatic hydrolysis of natural lipids in polymeric membranes as discussed in chapter 4.2.2., other methods have been applied to trigger the release of vesicle-entrapped compounds as depicted in Fig. 37. Based on the investigations of phase-separated and only partially polymerized mixed liposomes 101, methods to uncork polymeric vesicles have been developed. One specific approach makes use of cleavable lipids such as the cystine derivative (63). From this fluorocarbon lipid mixed liposomes with the polymerizable dienoic acid-containing sulfolipid (58) were prepared in a molar ratio of 1 9 101115>. After polymerization of the matrix forming sulfolipids, stable spherically shaped vesicles are obtained as demonstrated in Fig. 54 by scanning electron microscopy 114>. 

Early in the development of liposomes, it was recognized that their plasma instability could be a serious detriment in certain applications. Consequently, there were efforts to Lrst incorporate polymerizable lipids into the liposomal bilayers, and then initiate polymerization by, for example, photolysis, to form interchain crosslinks to stabilize the bilayer. The most commonly used polymerizable lipids have been PCs-containing diacetylene or butadiene moieties in the tailgroups... 

Freeman, F. J., Hayward, J. A., and Chapman, D. (1987). Permeability studies on liposomes formed from polymerizable diactylenic phospholipids and their potential applications as drug delivery systems. Biochim. Biophys. Acta, 924, 341-451. 

Hupfer, B., Rinsdorf, H., and Schupp, H. (1983). Liposomes form polymerizable lipidsm. Phys. Lipids,... 

Because of the stability problems with conventional liposomes, scientists have sought many methods to stabilize them. One important development is the sterically stabilized liposomes (SSLs), which are sometimes called stealth liposomes. 25-28 Synthetic polymers are used for steric stabilization. Another approach involves cross-linking membrane components covalently or by the polymerization of polymerizable lipids.29,30 A third approach utilizes unusually stable archaebacterial membrane lipids mimics.31... 

Keywords Lipid bilayer Liposome Lipo-polymer Planar lipid membrane Poly(lipid) Polymerizable lipid Stabilized membrane... 

In pursuit of enhanced liposomal stability, Ringsdorf, Regen, Chapman, and O Brien pioneered the use of polymerized liposomes. These liposomes were prepared from polymerizable lipid molecules. Polymerized liposomes demonstrated uniform size distribution and are considerably more stable compared to their unpolymerized counterparts. Various polymerizable groups (e.g., butadiene, dia-cetylene, vinyl, or methacryloyl ) have been used to achieve the polymerization of the lipid bilayers. These reactive groups on the lipid may be in the head group region, the hydrocarbon core, or at the hydrocarbon termini. 

A traceless detection of Escherichia coli has been reported by Ahn and co-workers (Figure 10.2). Two polymerizable lipids (one containing biotin. Figure 10.2) and DMPC were used to form the liposomes. The liposomes were spotted on streptavidin... 

The efficiency of energy transfer from the liposomes to the chelated lanthanide ions can be further enhanced by incorporating the aminosalicylic acids in the structure of the polymerizable lipids (Figure 10.10). MalUk and co-workers... 

Figure 10.11. The structures of the polymerizable lipids used in the liposomes for detection of carbonic anhydrase isozymes.

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