POPC liposomes

In order to verify that the adsorbed lipid membrane indeed forms a bilayer film, another experiment is conducted with an aim to detect the formation of a monolayer lipid. It starts with a piranha-cleaned micro-tube treated with silane to render its inner surface hydrophobic. POPC liposome is then injected into the microtube. It is known that POPC lipid will form a monolayer to such a surface by orienting their hydrophobic tails toward the hydrophobic wall. The experimental results using a mode with similar sensitivity as the previous experiment are shown in Fig. 8.39. The resonance shift in this case is 22 pm, which is about half of that observed for the adsorption of a lipid bilayer. These two experiments suggest that the microtube resonator is capable of accurately determining an adsorbed biomolecular layer down to a few nm thicknesses. 

Figure 4.11 Relative abundance for D L -stereoisomer groups of the oligo-tryptophan n-mers (n = 7 and 10, respectively), obtained after two racemic NCA-Trp feedings (a) in the absence (n = 7) and (b) in the presence of POPC liposomes (n = 10). The relative abundances of the stereoisomer subgroups (dark-gray columns) are mean values of three measurements. Standard deviations are given as error bars. The white columns correspond to the theoretical distribution, assuming a statistical oligomerization. (From Blocher et al., 2001.)...

Figure 9.22 Size distribution of POPC liposomes prepared by injecting 50 pil alcoholic solution of 25 irtM POPC into a 0.1 M borate buffer solution, pH 8.5 [POPCjfinai 0.5 irtM, 2% (v/v) alcohol measuring angle 90°. (FromDomazou and Luisi, 2002.)...

Table 9.2. Geometrical properties of unilamellar POPC liposomes (from Walde, 2000, personal communication)...

Table 9.2 shows some of these values for POPC liposomes of various radius, calculated assuming monolamellarity. 

NCA-activated aminoacids (NCA = A-carboxyanhydride) to the lipophylic surface of palmitoyl-oleyl-phosphatidyl-choline (POPC) liposomes, has permitted the condensation of Trp-oligomers up to a polymerisation degree of eighteen, which is considerable, given that in water the synthesis by way of the same reaction is limited to oligomers of five to six due to their insolubility. The hydrophobic character of the liposome shell can also operate the chemical selection from a mixture of solutes, as illustrated qualitatively in the Figure 10.4 the most hydrophobic solute can in principle be selected out followed by selective polycondensafion. 

In contrast to this is the addition of oleate surfactant - in the form of micelles or free monomer - to oleate or to POPC vesicles. In this case, the ratio of the two competitive rates is such that a considerable binding of the added fresh surfactant to the pre-existing vesicles takes place. The efficient uptake of oleate molecules by POPC liposomes (Lonchin et al., 1999) as well as to oleate vesicles (Blochiger et al., 1998) is well documented in the literature. 

Figure 10.10 Transmission electron micrograph of ferritin entrapped in POPC liposomes (palmitoyloleoylphosphatidylcholine). Cryo-TEM micrographs of (a) ferritin-containing POPC liposomes prepared using the reverse-phase evaporation method, followed by a sizing down by extrusion through polycarbonate membranes with 100 nm pore diameters ([POPC] = 6.1 mM) and (b) the vesicle suspension obtained after addition of oleate to pre-formed POPC liposomes ([POPC] = 3 mM, [oleic acid - - oleate] = 3 mM). (Adapted from Berclaz et al, 2001a, b.)...

The process opposite to vesicle division is that of fusion, when two or more vesicles come together and merge with each other, yielding a larger vesicle. As outlined in the previous chapter, vesicle fusion is generally not a spontaneous process. If two populations of POPC liposomes with different average dimensions are mixed with each other, they do not fuse to produce a most stable intermediate structure - they stay in the same solution as stable, distinct species. This is connected to the notion of kinetic traps, as discussed previously, and is supported by theoretical and experimental data from the literature (for example, Hubbard etal, 1998 Olsson and Wennerstrom, 2002 Silin et al, 2002). 

Figure 10.20 (a) Matrix effect for oleate addition to pre-formed POPC liposomes. In this case, mixed oleate/POPC vesicles are finally formed. Note the extraordinary similarity between the size distribution of the pre-formed liposomes and the final mixed ones. By contrast, the size distribution of the control (no pre-existing liposomes) is very broad, (i) Sodium oleate added to POPC liposomes, radius = 44.13, P-index = 0.06 (ii) POPC liposomes, radius = 49.63, P-index = 0.05 (iii) sodium oleate in buffer, radius = 199.43, P-index = 0.26. (b) matrix effect for the addition of fresh oleate to pre-existing extruded oleate vesicles. In this case, the average radius of the final vesicles is c. 10% greater than the pre-added ones, and again the difference with respect to the control experiment (no pre-added extruded vesicles) is striking, (i) Oleate vesicles extruded 100 nm, radius = 59.77, P-index = 0.06 (ii) oleate added to oleate vesicles, extended 100 nm, radius = 64.82, P-index 0.09 (iii) sodium oleate in buffer, radius = 285.88, P-index = 0.260. (Modified from Rasi et al, 2003.)... 

It is likely that early cells were more permissive, and perhaps an early step in the transition to life is the transition from permeable, simple protocells, to hard and impermeable structures, like our present POPC liposomes. In fact, the common stand of chemists to work with pure compounds may not be the best to model prebi-otic systems. In aprebiofic scenario, most probably, mixtures of several surfactants and CO surfactants were dominating the scene. It is known that the permeability of vesicles increases when co surfactants - like long-chain alcohols - are added. This observation about the importance of mixtures would in principle open the way to a vast area of research (see Sidebox 7.1). 

Figure 11.9 Schematic view of the experimental strategy for carrying out poly(Phe) synthesis in POPC liposomes, (i) Freeze-thaw (x 7) solution containing t-RNAP , poly(U), Phe, ATP, GTP, Mg(OAc)2, NH4CI, spermine, spermidine, phos-phoenolpyruvate. (ii) Soution containing pyruvate kinase, 100 000 g supernatant enzymes, 308 and SOS ribosomal subunits, (iii) 1. Free-thaw (x3) 2. Brief extrusion 3. Addition of EDTA (final concentration = 35 mM. (iv) Withdrawl of aliquots at indicated time and cold TCA precipitation. Analysis of the radioactivity remaining on the glass filter by p-scintillation counting. (Modified from Oberholzer et al, 1999.)...

POPC liposomes containing all different reagents necessary to carry out a PCR reaction. 

POPC liposomes incorporating the ribosomal complex together with the other components necessary for protein expression. 

Reaction initiated by mixing all reagents in water, then transferring to POPC liposomes. 

For that, let us consider the well-known liposome forming agent 1-palmytoyl, 2 oleyl-jn-phosphatidylcholine (POPC), a zwitterionic compound which forms highly hydrophobic liposomes. Suppose that a library of dipeptides is present in the aqueous solution containing the POPC liposomes, of the type H-Trp-X-OH, where X is another amino acid residue. With X = Trp we will have a rather hydrophobic dipeptide, whereas with X = Glu or Lys we will have charged, water-soluble compounds. 

It is clear that POPC liposomes will select out and bind the hydrophobic dipeptide(s), ignoring the hydrophilic ones. If now a hydrophobic condensing agent is present on the liposome membrane, an oligomerization of the bound hydrophobic peptide, e.g. H-Trp-Trp-OH, can take place. This mechanism of selection is illustrated in Figure 5. This is indeed what happens, as shown in the experimental data illustrated in Table 1. 

Table 1. POPC Liposomes-Aided Produa Selectivity for the Cooligomerization of Dipeptides ...

Figure 6. Membrane assisted condensation of amino acids. HPLC analysis (at 289 nm) of the products of the oligomerization of NCA-Trp assisted by POPC liposomes. There are several products (for reaction conditions and other details see ref. 21). It is important to notice the difference between the liposome-assisted oligomerization (A) and the control experiment, in the absence of liposomes (B). In this second case, the highest product has a oligomerization degree n = 7 in the case of liposomes we reach n = 29, although in minimal amounts.

This effect has been studied in our group by Kenichi Morigaki in his dissertation. He utilized the hydrophobic tripeptide Z-Phe-His-Leu-NHj (Z = carboben-zyloxy). This compound has been shown before in the literature to display catalytic properties towards the hydrolysis of certain esters. The authors were particularly interested in the stereoselectivity of the process of L- towards D-amino acids and less in the enhancement of catalysis operated by the micelles. The substrate chosen was a very lipophylic ester, nitrophenylpalmitate. Morigaki used oleate vesicles, and later POPC liposomes, obtaining qualitatively similar results. ... 

Case Study of the Polymerase Chain Reaction inside Conventional POPC Liposomes... 

PNPase has been entrapped inside extruded POPC liposomes, and fed by externally added ADP. In order to increase the entrance of ADP, liposomes were treated with cholate. Optimal permeability was achieved at cholate/POPC = 4/5 mol/mol. 

DNA, coding for the GFP, was introduced in POPC liposomes, together with the whole T T machinery (T7 RNA polymerase and E. coli cell extract, or PURESYSTEM). GFP was synthesized inside LUVs (radius lOOnm). 

Figure 23 Schematic cartoon illustrating the experimental strategy for carrying out poly(Phe) synthesis in POPC liposomes. (Reproduced from Ref. 75. Elsevier, 1999.)...

Once established the chemical stability of the urea-urease reaction in the presence of lipids and of the fluorescent probe, the next step towards a successful oscillating system was the encapsulation of the enzyme into POPC liposomes through the droplet transfer method. This innovative method first takes advantage of the facile compartmentalization of water-soluble solutes (enzyme in this case) in water-in-oil (w/o) droplets, and then convert the solute-filled w/o droplets into vesicles that can be dispersed in an acidic solution of urea. 

---