Light scattering corrections to linear dichroism spectroscopy for liposomes in shear flow using calcein fluorescence and modified Rayleigh-Gans-Debye-Mie scattering

Abstract

The interpretation of data from absorbance spectroscopy experiments of liposomes in flow systems is often complicated by the fact that there is currently no easy way to account for scattering artefacts. This has proved particularly problematic for linear dichroism (LD) spectroscopy, which may be used to determine binding modes of small molecules, peptides and proteins to liposomes if we can extract the absorbance signal from the combined absorbance/scattering experiment. Equations for a modified Rayleigh-Gans-Debye (RGD) approximation to the turbidity (scattering) LD spectrum are available in the literature though have not been implemented. This review summarises the literature and shows how it can be implemented. The implementation proceeds by first determining volume loss that occurs when a spherical liposome is subjected to flow. Calcein fluorescence can be used for this purpose since at high concentrations (> 60 mM) it has low intensity fluorescence with maxima at 525 and 563 nm whereas at low concentrations (<1 mM) the fluorescence intensity is enhanced and the band shifts to 536 nm. The scattering calculation process yields the average axis ratios of the distorted liposome ellipsoids and extent of orientation of the liposomes in flow. The scattering calculations require methods to estimate liposome integrity, volume loss, and orientation when subjected to shear stresses under flow.

Keywords: Mie Scattering; Rayleigh-Gans-Debye; Scattering; calcein; linear dichroism; liposomes.

Fig. 1

a Schematic of a lipid bilayer and examples of lipids (available from Avanti Polar Lipids Inc., Alabaster, AL, USA) which can form bilayers. DMPC, DPPC, POPC and POPS denote, respectively: 1,2-dimyristoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine. b Absorbance and LD spectra of bacteriorhodopsin (0.2 mg/mL) with soybean (0.5 mg/mL) liposomes at pH 7. 0.5 mm pathlength, 3000 rpm. Data from reference (Rajendra et al. 2006)

Fig. 2

Experimental Couette flow schematic and the predominant local orientation of a lipid bilayer in the cell

Fig. 3

Representative LD spectra (black, upper curves) from 100 nm liposome samples containing DPH (1% w/w, see text for preparation methods) collected with a Jasco J-815 circular dichroism spectropolarimeter adapted for linear dichroism spectroscopy using a microvolume Coutette flow cell (Crystal Precision Optics, Rugby, UK) rotated at 0 rpm or 3000 rpm. a POPC (7.5 mM), b POPC/POPS/cholesterol (total concentration 7.1 mM, mixed at a 75:10:15 ratio), c soybean PC (10 mM) and d BTLE (10 mM) spectra. Soy denotes a polar extract from soybeans (mainly phosphoethanolamines (PE), phosphocholines (PC) and phosphoinositols (PI)); and BTLE refers to brain total lipid extract from Avanti Polar Lipids Inc. Scattering curves (dashed lines, see below for calculation methodology) are determined assuming the liposome surface area remains constant with onset of flow. The difference between the experimental data and the scattering curve are shown in blue (lower curves)—making the absorbance LD signal. Pathlength is 0.5 mm

Fig. 4

a Emission spectra for calcein excited at 460 nm as a function of concentration. b Concentration of calcein vs maximum of emitted fluorescence

Fig. 5

Couette shear flow on (3000 rpm)/off time courses. a Fluorescence collected at 180° with a long-pass cut-off filter and b fluorescence + scattering measured at 90°, for calcein (50 mM initial concentration inside liposomes) and soybean PC liposomes (20 mg/mL). Data collected as for this figure

Fig. 6

Fluorescence of calcein leaked from a variety of liposomes preparations (see Figs. 2 and 3) held in the microvolume capillary LD cell (Crystal Precision Optics, Rugby, UK) with a 480-nm cut-off filter and 180° detection, with and without the influence of shear flow as indicated in the figures. a Stationary samples. b Samples in Couette flow. c Traces for pure DMPC liposomes fitted to a curve, since measured values had poor signal:noise. Initial calcein concentration inside liposomes was 50 mM, lipid concentration, ~ 20 mg/mL; path length, 0.5 mm. Data collected on a Bio-Logic MOS-450 spectrometer (Bio-Logic, Claix, France)

Fig. 7

Ovoid model of liposomes used herein: length L, radii of both other axes are identical and denoted r

Fig. 8

Schematic of an LD experiment for a particle P which scatters a photon along vector s together with geometry definitions used in this work. a Particle orientation axis and related polar and azimuthal angles of the particle orientation in space (ϕ, θ). b Scattering vector s with associated angles (β, ε). c Schematic diagram of the scattering plane and τ_LD axis system {X, Y^', Z^'}; the bisectrix of s is given by angle γ bounded by XOB

Fig. 9

Molecular structure of the fluorescent chromophore DPH and its stretched film LD spectrum (DPH was dropped from a concentrated solution in CHCl₃ onto prestretched polyethylene film) (Razmkhah et al. 2014)