Probing Rigidity and Fluidity of the Hydrophobic Region of Liposomes Using FTIR and 2D IR Spectroscopies

Abstract Rigidity and fluidity of a lipid bilayer’s hydrophobic interior in unilamellar liposomes were evaluated using alkyl-azide-type probes for linear infrared (FTIR) and two-dimensional infrared (2D IR) spectroscopies. When embedded into a bilayer of DPPC liposomes, the probes, azido-(CH2)n-COOH with n = 6, 8, 10, 13, 15 (denoted as aznAC), position their azido moieties at specific depths in the bilayer, defined by the alkyl tether length (n). The N3 moiety’s asymmetric stretching mode, νas, at ca. 2100 cm–1 shows abrupt changes in its peak width when the DPPC bilayer undergoes an Lβ–Lα phase transition, thus demonstrating probe sensitivity to the rigidity of the bilayer. Temperature dependences for the νas peak width show a sharp change at the phase transition temperature, Tph, for every depth (n), except for n = 6, although the magnitude of the sharp change at Tph differs for different depths, indicating that the packing order depends greatly on the depth in the bilayer. The range of the νas peak width change for az10AC is remarkable, with widths of 9.5 cm–1 at 25 °C and 22.7 cm–1 at 45 °C, which is a 2.4-fold change. A detailed DFT computational analysis was performed to understand the origin of the high sensitivity of the probes to the rigidity of the environment. It was established that the softness of the CNN angle (θ) and the presence of the θ-dependent multiple strong Fermi resonances with interaction energy exceeding 20 cm–1 result in severe broadening of νas. Furthermore, we showed that specific restrictions in the θ-angle distribution, occurring, for example, in bilayers in the gel phase reduce the νas peak width greatly. Spectral diffusion 2D IR measurements revealed the time scales of the conformational relaxation of the bilayer for az10AC in liposomes, which were found to be ca. 6 ps with a significant static component in the gel phase (25 °C) and ca. 2 ps in the liquid-crystal phase (45 °C). Such a large difference in decay times between the two limiting phases makes the aznAC probes sensitive reporters of bilayer fluidity. An isotopically edited compound, 15(N3)-(CH2)10-COOH (15az10AC), was also tested as a probe for the rigidity of the environment. While the width of the νas,15 peak at 2028 cm–1 is narrower than that of 14az10AC in the same environment, it still shows high sensitivity to the rigidity of the environment. DFT-based modeling revealed that a smaller number of Fermi resonances in 15aznAC is the reason for its narrower peak width. Simultaneous use of the two probes, 14aznAC and 15aznAC, was shown to be practical for simultaneously probing two different membrane regions.