Drug binding to cell membranes is crucial as a primary step of the biological action. In relation to the drug activity evaluation, it is of fundamental significance to quantify how much of the drug is bound in situ. In situ quantification requires no procedures for separating bound and unbound (free) drugs that may perturb the drug binding. The method is desirable for the successive assay without any damage of the cell membrane.

In this work, we present a new strategy to quantify the drug binding in situ by applying pulsed-field-gradient (PFG) solution-state NMR. The target drug molecule is 5-fluorouracil (5FU) which is widely used in the treatment of breast and gastrointestinal cancers. The large unilamellar vesicle (LUV) of phospholipids is employed as a model for freestanding natural cell membranes. We quantify in what amount of 5FU is bound to model cell membranes by monitoring ¹⁹F and ¹H NMR signals of 5FU in situ. When LUV is present, a broad peak, assignable to membrane-bound 5FU, appears in the one-dimensional (1D) ¹⁹F NMR spectrum, in addition to the sharp peak which is assigned to free 5FU. The signal assignment is ensured by the PFG application, where the free 5FU component decreases in intensity under the high field gradient to unveil the bound 5FU signal. The assignment is also confirmed by the diffusion rates of the respective components; the rate of the free 5FU component is comparable to the mobility of 5FU in water, whereas the motion of the bound 5FU is restricted and equal to the lipid mobility in the membrane. In the 1D ¹H NMR spectrum, the membrane-bound signal of 5FU is hardly able to distinguish. It is, however, uncovered by the PFG application that is similar to the ¹⁹F NMR observation.

The quantification of the 5FU binding is accomplished by integrating the free and bound components of the ¹⁹F and ¹H NMR signals in situ. The concentration dependence of 5FU on the membrane binding is discussed by the ¹⁹F and ¹H NMR analyses. Both ¹⁹F and ¹H NMR results are coincident with each other and demonstrate the validity of the method in situ. The present work demonstrates the ability of PFG NMR to quantify a wide variety of drug binding in situ both for laboratory and clinical applications.