Drug binding and mobility relating to the thermal fluctuation in fluid lipid membranes

Abstract

Drug binding and mobility in fluid lipid bilayer membranes are quantified in situ by using the multinuclear solution NMR combined with the pulsed-field-gradient technique. One-dimensional and pulsed-field-gradient (19)F and (1)H NMR signals of an anticancer drug, 5-fluorouracil (5FU) are analyzed at 283-313 K in the presence of large unilamellar vesicles (LUVs) of egg phosphatidylcholine (EPC) as model cell membranes. The simultaneous observation of the membrane-bound and free 5FU signals enables to quantify in what amount of 5FU is bound to the membrane and how fast 5FU is moving within the membrane in relation to the thermal fluctuation of the soft, fluid environment. It is shown that the mobility of membrane-bound 5FU is slowed down by almost two orders of magnitude and similar to the lipid movement in the membrane, the movement closely related to the intramembrane fluidity. The mobility of 5FU and EPC is, however, not similar at 313 K; the 5FU movement is enhanced in the membrane as a result of the loose binding of 5FU in the lipid matrices. The membrane-bound fraction of 5FU is approximately 0.1 and almost unaltered over the temperature range examined. It is also independent of the 5FU concentration from 2 to 30 mM with respect to the 40-50 mM LUV. The free energy of the 5FU binding is estimated at -4 to -2 kJ/mol, the magnitude always close to the thermal fluctuation, 2.4-2.6 kJ/mol.

Kinetics of Membrane Binding and Mobility of Drugs by Multinuclear Dynamic NMR <i>in Situ</i>

“…As illustrated in panel A of Figure 2, one-dimensional (1D) 19 F NMR spectrum of 5FU shows a new broad component in the presence of large unilamellar vesicles (LUVs) of egg phosphatidylcholine (EPC) as model cell membranes, where lipids are in the liquid-crystalline phase. The signal is assigned to the bound component of 5FU [8]. The assignment is confirmed by the PFG technique [10] in accordance with the criterion that the NMR signal of the bound component is insensitive to the the PFG application because of the motional slowdown in the membrane environment, whereas the free component preferentially decays with increasing the field gradient (FG) strength [8].…”

“…The presence of two fast components are the result of signal splitting due to the vicinal spin-spin coupling between 5-F and 6-H nuclei of 5FU. It was found that the bound fraction f B of 5FU was 0.1 and also independent of the 5FU concentration from 2 to 30 mM with respect to 40 50 mM LUV [8]. The binding free energy ΔG was estimated at 4 to 2 kJ/mol at 283 313 K, the magnitude always close to the thermal fluctuation, 3 kJ/mol.…”

“…Okamura and Yoshii have succeeded in distinguishing both bound and free states of the drug in situ, to quantify how much of the drug is bound to the membrane without any separation procedures of the bound and the free species. An example has been reported by applying high-resolution 19 F NMR to rather hydrophilic anticancer drug, 5-fluorouracil (5FU, 5-fluoro-1H-pyrimidine-2,4-dione; MW 130) as a target [8]. Fluorine-19 NMR is advantageous to observe the drug side because 19 F nucleus is absent in natural and model cell membranes.…”

“…Okamura and Yoshii have proposed a strategy to quantify drug binding and mobility in membrane in situ by applying high-resolution solution-state NMR in combination with the pulsed-field-gradient (PFG) technique [8]. It is an extension of the high-field-gradient NMR study on the mobility of lipids and trapped drugs in fluid lipid membranes [9].…”

“…The signal is assigned to the bound component of 5FU [8]. The assignment is confirmed by the PFG technique [10] in accordance with the criterion that the NMR signal of the bound component is insensitive to the the PFG application because of the motional slowdown in the membrane environment, whereas the free component preferentially decays with increasing the field gradient (FG) strength [8]. For example, the 19 F NMR signal of 5FU consists of three components, two sharp (in red and blue) and one broad peak (in green), as illustrated in Figure 3.…”