Metastability in lipid based particles exhibits temporally deterministic and controllable behavior

Abstract

The metastable-to-stable phase-transition is commonly observed in many fields of science, as an uncontrolled independent process, highly sensitive to microscopic fluctuations. In particular, self-assembled lipid suspensions exhibit phase-transitions, where the underlying driving mechanisms and dynamics are not well understood. Here we describe a study of the phase-transition dynamics of lipid-based particles, consisting of mixtures of dilauroylphosphatidylethanolamine (DLPE) and dilauroylphosphatidylglycerol (DLPG), exhibiting a metastable liquid crystalline-to-stable crystalline phase transition upon cooling from 60°C to 37°C. Surprisingly, unlike classically described metastable-to-stable phase transitions, the manner in which recrystallization is delayed by tens of hours is robust, predetermined and controllable. Our results show that the delay time can be manipulated by changing lipid stoichiometry, changing solvent salinity, adding an ionophore, or performing consecutive phase-transitions. Moreover, the delay time distribution indicates a deterministic nature. We suggest that the non-stochastic physical mechanism responsible for the delayed recrystallization involves several rate-affecting processes, resulting in a controllable, non-independent metastability. A qualitative model is proposed to describe the structural reorganization during the phase transition.

Figure 1. Time-resolved structural analysis of LPs showing delayed crystallization.

(a) Representative SXS data at different stages of the experiment, compared with DLPE and DLPG powders. Crystalline peaks of the lipid aqueous dispersions match those of pure dry DLPE. (b) Initially, at 37°C (black spectrum), the crystalline state shows wide-angle peaks. When heated to 60°C (red spectra) the wide-angle peaks disappear with the loss of in-plane lipid order. After cooling back to 37°C (blue spectra) the L_α phase remains for 16.1 hours and then phase-transitions back into the crystalline state (green spectra). (c–h) Cryo-TEM images of specimens vitrified at different stages. (c) Specimen vitrified from 37°C prior to heat treatment, showing a large DLPE crystal structure along with uni- and multi-lamellar vesicles. (d) Sample vitrified from 37°C prior to heat treatment showing vesicles with sharp facets. (e) Sample vitrified from 60°C showing large MLVs. (f) Sample vitrified from 37°C 44 hours after cooling. Note the sharp facets forming on a vesicle. (g) Sample of a 90:10 DLPE:DLPG (mole %) vitrified from 37°C three days after cooling, showing multiple facets on a vesicle. (h) Detail of the previous micrograph showing the fusion of two membranes, accompanied by the removal of water from between them. Blue arrowhead indicates the fusion point of the two membranes, separating the crystalline phase (straight facets to the left) from the liquid-crystalline phase (disjoined, curved leaflets to the right). Unless specified, data are of 95:5 DLPE:DLPG (mole %) dispersions in 150 mM monovalent salt. Scale bar in (c) corresponds to 200 nm. Scale bars in (d–g) correspond to 100 nm.

Figure 2. Reproducibility and universality of the delayed crystallization.

(a) Intensity of the lamellar scattering peak as a function of time since cooling back down to 37°C. Intensity normalized to be between 1 (metastable state) and 0 (crystalline state after delayed transition), and time normalized by τ. A collapse of the entire data set, containing more than 60 experiments in varying conditions, highlights the robustness of the metastability dynamics and ensuing collective phase-transition. This emphasizes the governing role of τ in the dynamics. (b) Distribution of τ from measurements on 24 different samples. Black curve shows a Gaussian fit with an average delay time of <τ> = 34 hours and a standard deviation of 16.4 hours. The minimal recrystallization time (τ) measured was 9 hours. Experiments performed on samples containing 95:5 DLPE:DLPG at 150 mM monovalent salt.

Figure 3. Manipulation of the delay time for crystallization at 37°C.

(a) Average delay time increases with the addition of DLPG. Lipid dispersions in buffer containing 150 mM monovalent salt. The last point did not recrystallize in the duration of the experiment (200 hours). (b) Non-monotonic effect of salt concentration on the delay time, τ. With the addition of salt, at low concentrations (<150 mM), τ decreases in a linear fashion (red line), while at high concentrations (>300 mM), there is an order of magnitude increase. (c) Effect of the monensin ionophore on the delay time. Control sample recrystallized at τ = 44.7 hours. The addition of monensin accelerates recrystallization; sample containing 1 mole % monensin:sodium recrystallized with τ = 13.6 hours. Experiments performed on samples containing 90:10 DLPE:DLPG at 150 mM monovalent salt. (d) Consecutive heating-cooling cycles display a prolongation of the delay time accompanied by a decrease in the metastable lamellar scattering intensity. The curves represent the intensity of lamellar (001) scattering from the moment the temperature is brought back to 37°C after heating. Roman numbers indicate the measurement sequence. Inset shows qualitative analysis of the two effects. (b and d) represent data of 95:5 DLPE:DLPG (mole %). Error bars represent the average phase transition time <τ*>.

Figure 4. Schematic representation of the morphology during the different stages of the experiment.

(a) At 37°C prior to heating, the system is composed of a population of large crystals and vesicles with facets. (b) After heating to 60°C, the lamellae adopt the L_α phase with water and ions penetrating between the membranes. (c) Cooling the system to 37°C, after heating, the membranes remain in the L_α phase and 2D to 3D facets begin to form and grow. (d) The crystallization phase transition occurs after large enough facets disrupt the morphology of the LPs. The structural reorganization and coexistence of DLPE crystals and mixed DLPE/DLPG LPs result in MLVs with fewer lamellae on average.