Polymerizable lipid bilayers

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. 

Domain formation in binary mixtures of a polymerizable lipid and non-polymerizable lipid is well established for diacetylenic lipids. The rigid diacetylenic unit facilitates the formation of enriched domains in the condensed phase of monolayers or the solid-analogous phase of bilayers. Since diacetylenes polymerize most readily in solid-like states, most studies have focused on conditions that favor domain formation. Only in the case of a mixture of a charged diacetylenic lipid and a zwitterionic PC was phase separation not observed. Ringsdorf and coworkers first reported the polymerization of a phase-separated two-dimensional assembly in 1981 [33], Monolayer films were prepared from mixtures consisting of a diacetylenicPC (6) (Fig. 5) and a nonpolymerizable distearoyl PE (DSPE). 

In 1985 Tyminski etal. [55, 56] reported that two-component lipid vesicles of a neutral phospholipid, e.g. DOPC, and a neutral polymerizable PC, bis-DenPC (15), formed stable homogeneous bilayer vesicles prior to photopolymerization. After photopolymerization of a homogeneous 1 1 molar lipid mixture, the lipid vesicles were titrated with bovine rhodopsin-octyl glucoside micelles in a manner that maintained the octyl glucoside concentration below the surfactant critical micelle concentration. Consequently there was insufficient surfactant to keep the membrane protein, rhodopsin, soluble in the aqueous buffer. These conditions favor the insertion of transmembrane proteins into lipid bilayers. After addition and incubation, the bilayer vesicles were purified on a... 

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. 

Out of a variety of polymerizable lipids tested for possible use of bilayer formation, only three systems exhibited BLM lifetimes of more than a few minutes (Table 2 26)). These BLMs were characterized by measuring their resistance and capacitance (Table 2., see26> for details). The data obtained were comparable with values obtained with egg lecithin the most frequently used material for preparing BLMs. 

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). 

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... 

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

Fabricating a supported lipid bilayer in which both monolayers are composed of polymerizable lipids results in formation of a polymeric network in each monolayer. Furthermore, if the reactive groups are located at the termini of the acyl chains, the monolayers can be covalently linked, which is inherently more stable than a HBM in which lipid polymerization occurs in only one monolayer. 

Overall, these studies were the first to demonstrate that the activity of a TMP can be maintained in a highly cross-linked poly(PSLB). The location of the polymerizable moiety is clearly an important consideration. These findings should provide guidance for designing robust poly(lipid) bilayers functionalized with TMPs for use in membrane-based biochips and biosensors. 

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. 

OmpF has also been reconstituted into the membrane of PMOX-PDMS-PMOX triblock copolymer vesicles carrying polymerizable end groups (236). The triblock copolymer membrane had a thickness of 10 nm, which is two to three times thicker than a conventional lipid bilayer (237). Nevertheless, the channelforming OmpF could be reconstituted and remained functional, showing similar activities as in lipid membranes, despite the extreme thickness of the membranes. The functionality even remained after polymerization of the reactive end groups, which was demonstrated by monitoring the ampicillin hydrolysis of encapsulated /3-lactamase (156,218,237-240). 

Diacetylenes in phospholipid bilayers have been the subject of extensive studies in our laboratory, not only because of the highly conjugated polymers they form, but also because of their ability to transform bilayers into interesting microstructures. Consequent to our synthesis and characterization of several isomeric diacetylenic phospholipids, we have found that the polymerization in diacetylenic bilayers is not complete. In order to achieve participation of all diacetylenic lipid monomer in the polymerization process, diacetylenic phospholipid was mixed with a spacer lipid, which contained similar number of methylenes as were between the ester linkage and the diacetylene of the polymerizable lipid. Depending upon the composition of the mixtures different morphologies, ranging from tubules to liposomes, have been observed. Polymerization efficiency has been found to be dependent on the composition of the two lipids and in all cases the polymerization was more rapid and efficient than the pure diacetylenic system. We present the results on the polymerization properties of the diacetylenic phosphatidylcholines in the presence of a spacer lipid which is an acetylene-terminated phosphatidylcholine. 

A similar study by O Brien and coworkers utilized bilayers composed of a shorter chain diacetylenicPC (9) and DSPC or DOPC [37]. Phase separation was demonstrated in bilayers by calorimetry and photopolymerization behavior. DSC of the 9/DSPC (1 1) bilayers exhibited transitions at 40 °C and 55 °C, which were attributed to domains of the individual lipids. Polymerization at 20 °C proceeded at similar rates in the mixed bilayers and pure 9 bilayers. A dramatic hysteresis effect was observed for this system, if the bilayers were first incubated at T > 55 °C then cooled back to 20 °C, the DSC peak for the diacetylenicPC at 40 °C disappeared and the bilayers could no longer be photopolymerized. The phase transition and polymerizability of the vesicles could be restored simply by cooling to ca. 10 °C. A similar hysteretic behavior was also observed for pure diacetylenicPC bilayers. Mixtures of 9 and DOPC exhibited phase transitions for both lipids (T = — 18 °C and 39 °C) plus a small peak at intermediate temperatures. Photopolymerization at 20 °C initially proceeded at a similar rate as observed for pure 9 but slowed after 10% conversion. These results were attributed to the presence of mixed lipid domains... 

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-... 

It is worto noting that all toe amino acid-diacefylene lipid microstructures studied here could be polymerizable to form blue colored PDAs. However, only hydrophilic amino acid lipids can readily form bilayer vesicles and allow polymerization (77). The intensity of toe initial blue color, however, varies with headgroups. Amino acids with hydrophilic segments give toe darkest blue appearance, while hydrophobic amino acids (lie-) produce barely noticeable blue appearance. 

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