(368bx) Three Questions for Laurdan

Phospholipid bilayers, which constitute biological membranes as well as those of many drug delivery systems, exist in a variety of 2-dimensional phases, varying in fluidity and permeability. Evaluation and control of the phase behavior of these membranes is necessary to manipulate leakage and stability of drug delivery vehicles. Laurdan is a widely employed fluorescent probe for the investigation of these membranes. Most often, membranes containing Laurdan are investigated using steady-state fluorescence spectroscopy to evaluate its polarity-induced spectral shift, quantified as Generalized Polarization (GP) and understood as a measure of a bilayer’s hydration. Additionally, fluorescence anisotropy and lifetime measurements of Laurdan can yield insight into the fluidity of its environment. Anisotropy, lifetime, and GP are generally taken to provide the same information. Lower anisotropy, shorter lifetime, and lower GP are all associated with the liquid disordered () phase, while the opposites are associated with both the gel/solid ordered () and cholesterol-induced liquid ordered () phases. Each of these metrics, however, ask a different question:

• GP: How exposed to the aqueous environment are you?
• Anisotropy: How much are you able to rotate?
• Lifetime: How long do you remain in your excited state?

Our study explores some interesting insights that arise from discrepancies between Laurdan’s answers to these questions. We are able to detect gel/liquid-disordered phase separation by evaluating anisotropy as a function of emission wavelength. Additionally, we find that the supercritical transition between liquid-disordered and liquid-ordered phases is marked by an outsized disagreement between membrane polarity and fluidity. Membrane property analysis using Laurdan is a powerful tool that can be of use in development and assessment of drug delivery vehicles in response to the various perturbations that are used to trigger release.