On measuring the acoustic state changes in lipid membranes using fluorescent probes

Abstract

Ultrasound is increasingly being used to modulate the properties of biological membranes for applications in drug delivery and neuromodulation. While various studies have investigated the mechanical aspect of the interaction such as acoustic absorption and membrane deformation, it is not clear how these effects transduce into biological functions, for example, changes in the permeability or the enzymatic activity of the membrane. A critical aspect of the activity of an enzyme is the thermal fluctuations of its solvation or hydration shell. Thermal fluctuations are also known to be directly related to membrane permeability. Here solvation shell changes of lipid membranes subject to an acoustic impulse were investigated using a fluorescence probe, Laurdan.Laurdan was embedded in multi-lamellar lipid vesicles in water, which were exposed to broadband pressure impulses of the order of 1MPa peak amplitude and 10μs pulse duration. An instrument was developed to monitor changes in the emission spectrum of the dye at two wavelengths with sub-microsecond temporal resolution. The experiments show that changes in the emission spectrum, and hence the fluctuations of the solvation shell, are related to the changes in the thermodynamic state of the membrane and correlated with the compression and rarefaction of the incident sound wave. The results suggest that acoustic fields affect the state of a lipid membrane and therefore can potentially modulate the kinetics of channels and proteins embedded in the membrane.

Thermodynamic state of the interface during acoustic cavitation in lipid suspensions

“…Evidence for the phase transition in lipids is being captured by ∆ / 0 (1→2) which the experiments show is proportional to corresponding enthalpies of transitions (7,31,36), i.e. ∆ / 0 (1→2) ∝ ∆ℎ (1→2) (3) Table 2 shows the ratio of the peak of the ∆ / 0 and the ratio of the enthalpy of transition (from Table 1).…”

“…A custom built upright epifluorescent microscope, with an objective with 1.3 cm working distance (LMPLFLN 20X Objective, Olympus, Tokyo, Japan), was placed above the free surface of the test chamber and the focus of the objective was aligned with the focus of the shock waves ( Fig 1A). The which due to normalization is insensitive to the amplitude of the light intensity (7). For small perturbations ∆ 0 ∝ ∆ℎ , i.e.…”

“…The experimental setup and the MLV preparation, have been described in detail previously (7). The phase state of each lipid was characterized in terms of a "reduced temperature", * = − , where is the temperature of the main phase transition, equivalent to the melting point of of the lipid system.…”

“…Molecules which co-localize at the interface, such as fluorescent probes, can be added to the system and used to indicate the thermodynamic state of the interface. For example, we recently showed that the wavelength shift of the emission spectrum of the fluorescent probe Laurdan can be used to dynamically measure the thermodynamic state of lipid interfaces (7), when subjected to microseconds width acoustic impulses.…”

“…Here we investigate cavitation in a suspension of lipid vesicles by observing the spectral shift in the fluorescence emission of a hydrophobic probe Laurdan, co-localized at the interface (7) with submicrosecond resolution. The lipid composition and the temperature of the suspension were varied to change the initial state of the lipid interfaces.…”