Pre-added vesicles

Figure 10.19 Effect of pre-added vesicles on the formation of oleic acid/oleate vesicles. Turbidity measured at 500 nm (1 cm path length) is plotted as a function of time, T=2°C. (a) 62 p,l of 80 mM aqueous sodium oleate was added to 2.438 ml of0.2Mbicine buffer, pH 8.8 ([oleic acid/oleate] = 2 mM). (b)62 p,l of 80mM aqueous sodium oleate was added to 2.438 ml of a 2 mM oleic acid/oleate 100 nm vesicle suspension (0.2 M bicine buffer, pH 8.8 ([oleic acid/oleate] = 4 mM). (c) Turbidity of 2 mM oleic acid/oleate 100 nm vesicles , (d) the same as (b), but using a 50 nm vesicle suspension, (e) Turbidity of 2 mM oleic acid/oleate 50 nm vesicles . (Modified from Blochiger etal, 1998.)...

Note also that the kinetic progress depends on the size of the pre-added vesicles. This catalytic effect is present also in a ratio 1 100, or less, between the pre-added and added surfactant, reinforcing the analogy with a catalytic effect. 

The uptake of oleate by pre-added vesicles, and in particular the matrix effect, permits regulation of the growth of the size and the number of particles, and in this way it is possible to tackle a series of novel questions. One such question is is there a difference in the rate of uptake of fresh surfactant between two vesicle populations of different sizes ... 

Evidence was found for a POPC matrix effect, in that the pre-added vesicles influenced the size distribution of the newly produced vesicles. The matrix effect leads to rapid formation of vesicle aggregates and control over the final size distribution, such that the mixed systems are more monodisperse than in the absence of the matrix, where the oleic acid vesicles grow in an uncontrolled way. This effect was more obvious at lower concentrations of oleic acid/oleate than at higher concentrations. 

The time course of an actual experiment is shown in Figure 7.17, which shows the hydrolysis of oleic anhydride catalyzed by spontaneously formed oleate vesicles. Note the sigmoid behavior, typical of an autocatalytic process. The lag phase is due to the preliminary formation of vesicles, and in fact the length of the lag phase is shortened when already formed vesicles are pre-added, as shown in the hg-ure. Some mechanistic details of these processes will be discussed in Chapter 10. In this work, an analysis of the number and size distribution of vesicles at the beginning and the end of the reaction was also performed by electron microscopy. 

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 10.11 The use of ferritin as a label for the mechanism of growth of vesicles (adapted from Berclaz et al, 2001a b). Schematic representation of the possible vesicle formation and transformation processes when oleate, and oleic acid, are added to pre-formed vesicles which have been labelled, (a) The situation if only de novo vesicle formation occurs, (b) Growth in size of the pre-formed and labeled vesicles which may lead to division, either yielding vesicles that all contain marker molecules (case i, a statistical redistribution of the ferritin molecules) or also yielding vesicles that do not contain markers (case ii). Compare all this with Figure 10.9.

Consider the experiment illustrated in Figure 10.19, which shows oleate vesicle formation when an aliquot of concentrated surfactant is added to water compared to the situation in which the same amount of surfactant is added to a solution containing pre-formed vesicles. In the second case, the formation of vesicles is remarkably accelerated, as if in the presence of a strong catalytic effect whereas over one hour is needed to reach the turbidity plateau for oleate addition to water, the plateau is reached in less than ten minutes, curve (b), in the second case. 

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.)... 

Figure 3. Freeze-fracture electron microscopy analysis of the vesicle size distribution in the case of the spontaneous vesiculation of oleic acid/oleate. (A) Vesicles formed from the hydrolysis of 25 mM oleic anhydride (overall concentration) at 30 °C, yielding 50 mM oleic acid/oleate. (B) Vesicles extruded throughout 50 nm diameter filters. (C) Vesicles formed upon hydrolyzing 20 mM oleic anhydride (same conditions as in A) in the presence of pre-added extruded vesicles B—all in 0.2 M bicine buffer pH 8.5. For details see ref. 8.

It appears, therefore, that the presence of vesicles accelerates the formation of new vesicles. It is not easy to rationalize how and why. What comes to mind is a general observation from the field of surfactants, that pre-organization makes the organization of further material easier. For example, there is no spontaneous vesiculation when POPC is simply added to water, and no significant amount of... 

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