High Pressure Experiments
In an attempt to artificially reproduce the vesicle formation process described in Fig. 1, the key ingredients for the vesicles (water and the lipid) were combined with CO2 in a high pressure cell. Initially, the cell was filled with 5 mL of double-distilled water. In order to avoid direct vesicle formation in the aqueous phase, the phospholipid (40 mg) was dissolved in ethanol (2 mL) and introduced into the high pressure cell in a separate glass container which was positioned in the center of the pressure vessel (Fig. 2, step 1). With this setup, the cell was flooded with CO2 and brought to pressure and temperature conditions where a supercritical state was reached and which corresponded to the situation in the continental crust slightly below a depth of 1 km (P = 120 bar, T 333 K). Under these conditions, the water as well as the lipid from the ethanolic solution in the insert is expected to partially dissolve in the scCO2-phase (Fig. 2, step 2). Subsequently, the pressure was reduced (simulating e.g. a high tide situation) until the CO2 turns subcritical again (P = 72 bar). At this stage, the dissolved components precipitated in small droplets and the gas phase appears opaque (Fig. 2, step 3). Over time, the droplets settled onto the water surface outside the insert, saturated the interface with lipid and then interacted with this lipid layer to form vesicles. After that, the pressure was periodically cycled between 120 bar and 72 bar in order to repeatedly induce the conditions for vesicle formation. Altogether, four pressure cycles were applied.
During all these steps, all possible sources of shear were avoided in the bulk water phase. As vesicles are thermodynamically unstable, they cannot form spontaneously by diffusion of the phospholipid into the water phase. Therefore, we believe that the only possible phenomenon leading to vesicle formation in the water outside of the insert is the proposed mechanism of droplets crossing the phase boundary.
After the four cycles, the pressure was carefully released and the insert was separated. A sample of the bulk aqueous phase was studied by optical microscopy under ambient conditions. The micrograph (Fig. 3) shows spherical objects with diameters smaller than 5 μm which have the appearance of unilamellar vesicles.
In order to further identify the structure of the particles, the dispersion was studied by pulsed field gradient nuclear magnetic resonance spectroscopy (PFG-NMR). This technique allows for an estimation of the self-diffusion behavior of system components as a function of time. The intensity of the NMR echo signal I of water molecules was observed as a function of the gradient strength G and the time interval ∆ and referenced against the standard echo intensity I0 for G = 0. Commonly, the logarithm of the relative echo intensity (ln I / I0) is plotted vs. the parameter γ2δ2G2(∆-δ/3) in the so called Stejskal-Tanner-plot (with γ being the gyromagnetic ratio and δ being the duration of the gradient). In case of free diffusion of the water molecules in a bulk water system, such a plot would follow a straight line with a slope identical to the negative self-diffusion coefficient, and it would be independent on the time interval ∆. However, if water molecules are encapsulated in hollow spheres such as vesicles, they would show a completely different behavior. In this case, the slope of the plot (corresponding to the apparent diffusion constant) strongly depends on the duration of the time interval: for very short intervals ∆, it is similar to bulk water as the dislocation of the water molecules is too short to be significantly influenced by the presence of the vesicle membranes. For longer periods ∆, the average number of collisions with the membrane gradually increases, leading to a decreasing apparent diffusion constant. Finally, for very large values of ∆, the position of the water molecules averages out to the center of the vesicle and the slope of the plot represents the Brownian motion of the vesicles themselves. In this part, the intersection with the vertical axis indicates the fraction of the encapsulated molecules on a logarithmic scale.
Figure 4 shows Stejskal-Tanner-plots obtained on the dispersion of the pressure cell experiment for different time intervals (∆ = 15 ms, 25 ms, 50 ms, 100 ms, 200 ms). All plots clearly show the presence of bulk water represented by a steep initial part of each plot, its slope indicating the self-diffusion coefficient of water (D = 2.3∙10−9 m2/s). However, each plot also includes a second, shallow part. Its level (and partially its residual slope) strongly depends on the time interval ∆. The initial slopes (particularly between 1∙1010 and 5∙1010 s/m2) clearly reflect the expected decrease of the apparent diffusion constant with increasing ∆. The terminal slopes (near and beyond 2∙1011 s/m2) are in accordance with Brownian motion based on a particle diameter of 0.5 μm. This means that the vast majority of the vesicles are smaller than the ones which could actually be observed by optical microscopy (Fig. 2). Based on the intersection with the vertical axis (which is near −5.5 for the longest ∆), the volume contribution of the vesicles can be estimated as 0.4 % of the overall bulk water phase.
Altogether, the PFG-NMR results clearly prove the presence of small dispersed compartments which are filled by liquid water. With the given system components, this leaves only one possible interpretation: the lipid has formed spherical vesicles with a bilayer structure, encapsulating a corresponding fraction of the bulk water. Vesicles are thermodynamically unstable, therefore they cannot form spontaneously. As shear was practically avoided, the only mechanism leading to vesicle formation is the interaction of lipid-coated droplets with a lipid monolayer on the water surface, as described above. Hence, we consider these data as a strong indication for the relevance of the proposed mechanism. Further, we also believe that it took place in the crust of the early Earth and presently. With the constant accumulation process linked to the initial droplet formation and the generation of a bilayer membrane, it offers a favorable scenario for early chemical evolution and the development of protocells.