Polymers With Phospholipid Bilayers

The effect of branched polypeptides on phospholipid membranes was further investigated using lipid bilayers with DPPC/PG (95/5, 80/20 mol/mol). Two fluorescent probes of different character were used to analyse the effect of polymers on the outer surface (negatively charged, sodium anilino naphthalene sulfonate, ANS) and on hydrophobic core (hydrophobic, l,6-diphenyl-l,3,5-hexatriene DPH) of bilayers. For these studies small imilamellar vesicles were used. 

Lipids dissolved in chloroform were mixed in a round bottom flask and solvent was eliminated by rotary evaporation from the clear solution at 55°C 

Fluorescence studies were carried out by measuring changes in fluorescence intensity and polarisation of ANS and DPH probes located in the bilayers, using a PE-LS50 spectrofluorometer provided with four cuvettes thermostated bath. Fluorescence intensity changes could be due to variations in the environmental conditions of the fluorophore, while polarisation is mainly related to its motion. 

In the first set of e qreriments liposomes were incubated with fluorescent probes at various concentrations in the dark at 55°C for 60 min and the saturation curves were recorded. The optimal probe/phospholipid molar ratios determined from the saturation curves were 1/26 (mol/mol) for ANS/phospholipid and 1/240 (mol/mol) for DPH/phospholipid. 

At these ratios, liposome preparations were mixed either with polymer solution or with buffer (0.1 M sodium acetate, pH=7.4) and fluorescence intensity was measured as a function of temperature. Excitation and emission wavelengths were X,=380-480 nm for ANS and X,=365-425 nm for DPH, respectively. The degree of fluorescence polarisation was calculated according to Shinitzky and Barenholz applying equation 1.  

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Multiplex CARS microspectroscopy, in conjunction with appropriate spectral analysis tools, was successfully applied to the study of phospholipid bilayer model systems [120, 121, 142, 70, 143], lipids within cells [144, 127, 145-147, 141], a single pollen grain [148], a single bacterial endophore [140], a molecular J-aggregate microcrystal [149], silicon components on a wafer [130], separated phases in polymer blends [123, 135], and concentration profiles in a microreactor [150]. 

Figure 30 Illustration of the MAOSS strategy with peptide in phospholipid bilayers adsorbed on a polymer sheet (PET). B0, the magnetic field otm is the magic angle (54.74°) N, the normal of the bilayer and ZR, the rotor axis. Taken from Ref. [112].

The extreme sensitivity of the visible absorption spectrum to small changes in the surrounding medium has made this betaine dye a useful molecular probe in the study of micellar systems [298, 299, 443-445], mieroemulsions and phospholipid bilayers [299], model liquid membranes [300], polymers [301, 446], organic-inorganie polymer hybrids [447], sol-gel matrices [448], surfaee polarities [449-451], and the retention behaviour in reversed-phase liquid chromatography [302]. Using polymer membranes with embedded betaine dyes, even an optical alcohol sensor has been developed [452]. 

Low-molar mass lipids have been known for more than 30 years for their ability to self-assemble into vesicles or liposomes, however, with limited overall stability. Polymer membranes, on the other hand, are almost one order of magnitude tougher and at least 10 times less permeable to water than common phospholipids bilayers, due to the increased length and conformational freedom of polymer chains compared to lipids [1]. Biohybrid polymer vesicles combine the toughness of polymers and the biocompatibility of peptides or sugars, making them promising candidates... 

Thickness. The thickness of polymersome bilayers is several times greater than that of typical phospholipid bilayers in natural membranes. Lipid bilayers have a hydrophobic core thickness that is in a very narrow range of rf 3-4 nm to be compatible with integral membrane proteins. For self-assembled bilayers of PEE-PEO vesicles, the hydrophobic core thickness increases with increasing molecular weight from d 8-21 nm (see Fig. 18) to more than 100 nm (53,151-153). The observed d scaling is t5q)ical for random coil polymers and agrees... 

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. 

The effect of branched polypeptides on phospholipid bilayers saturated with fluorescent probes located either at the polar surface (ANS) or within the hydrophobic part (DPH) of the liposome indicate that only polymers with high positive charge density are capable to initiate significant changes. 

Many complex systems have been spread on liquid interfaces for a variety of reasons. We begin this chapter with a discussion of the behavior of synthetic polymers at the liquid-air interface. Most of these systems are linear macromolecules however, rigid-rod polymers and more complex structures are of interest for potential optoelectronic applications. Biological macromolecules are spread at the liquid-vapor interface to fabricate sensors and other biomedical devices. In addition, the study of proteins at the air-water interface yields important information on enzymatic recognition, and membrane protein behavior. We touch on other biological systems, namely, phospholipids and cholesterol monolayers. These systems are so widely and routinely studied these days that they were also mentioned in some detail in Chapter IV. The closely related matter of bilayers and vesicles is also briefly addressed. 

A further partihon system based on the use of liposomes, and commercialized under the name Transil [110, 111], has shown its utiUty as a UpophiUcity measure in PBPK modeling [112]. Fluorescent-labeled liposomes, called fluorosomes, are another means of measuring the rate of penetration of small molecules into membrane bilayers [113, 120]. Similarly, a colorimetric assay amenable to HTS for evaluating membrane interactions and penetrahon has been presented [116]. The platform comprises vesicles of phospholipids and the chromahc Upid-mimehc polydiacetylene. The polymer undergoes visible concentrahon-dependent red-blue transformahons induced through interactions of the vesicles with the studied molecules. 

Polymerization in Bilayers. Upon irradiation with UV light the monomer vesicles are transferred to polymer vesicles (Figure 12.). In the case of the diyne monomers (2,5-9,12,13,14) the polyreaction can again be followed by the color change via blue to red except phospholipids (5,6), which turn red without going through the blue intermediate as observed in monolayers. The VIS spectra of these polymer vesicle dispersions are qualitatively identical to those of the polymer monolayers (Figure 13.). 

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