Nonsteroidal anti-inflammatory drugs (NSAIDs) are effective antipyretic and analgesic pharmacologic agents [1]. Diclofenac is a NSAID of the phenylacetic acid class which inhibits cyclooxygenase (COX)-2 enzyme [1]. NSAIDs also treat other diseases including cancer [2], arthritis [3,4] and neurodegenerative diseases [5].

However, side effects of NSAIDs are also known [6]. Many of these effects are related to the membrane activity of these drugs [7–10]. For diclofenac, this activity may contribute to its gastrointestinal toxicity [6,11,12]. Diclofenac can interact with erythrocyte membranes inducing a disordering of the acyl chains of the lipids and changing the erythrocyte morphology [13]. To understand the therapeutic effects of NSAIDs and to develop ways to limit their side effects, understanding the mechanisms at the molecular level of its interaction with the plasma membrane can be extremely useful [7, 14–20].

Previously, in our laboratory spin-labeled diclofenac (diclofenac-SL) was synthesized [21]. This new substance allows using electron paramagnetic resonance (EPR) spectroscopy for studying interaction of diclofenac with biological membranes. Here we study diclofenac-SL in model membranes of three different types: palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), equimolar mixture of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), and this mixture with added 20 mol% of cholesterol. These types of model membranes are often used to mimic real biological membranes. POPC includes in its structure a fully saturated acyl chain and a single-unsaturated acyl chain, DOPC includes two single-unsaturated acyl chains, and DPPC includes two fully saturated acyl chains. DOPC/DPPC/cholesterol bilayers are known to form nanoscale liquid disordered and liquid ordered lateral structures, the latter called lipid rafts [22–26].

Biological membranes and their various sub-structures (like lipid rafts) are nanoscale objects. Therefore, it is reasonable to use experimental methods that allow studying the nanoscale structure. Such a method is double electron-electron resonance (DEER, also known as PELDOR) [27–31].

DEER spectroscopy is based on the electron spin echo (ESE) phenomenon. The DEER signal appears due to modulation by microwave (mw) pulses of magnetic dipole-dipole (d-d) interaction between spins. For some selected pair of spins A and B, the DEER signal is [27–31]

$${v_A}(t)=1 - {p_B}(1 - \cos {\omega _{AB}}t)$$

1

,

where

$${\omega _{AB}}=\frac{{{g_A}{g_B}\mu _{{Bohr}}^{2}}}{\hbar }\frac{{(1 - 3{{\cos }^2}{\theta _{AB}})}}{{r_{{AB}}^{3}}}$$

.

Here, *µ**Bohr* is the Bohr magneton, *g**A* and *g**B* are the effective *g-*factors of two interacting spins, A and B, *θ**AB* is the angle between the vector **r***AB* connecting two spins and the magnetic field of the spectrometer. The dimensionless parameter *p**B* *<* 1 is the fraction of electron spins excited by the pumping mw pulse (excitation efficiency).

The important property of ESE spectroscopy is that the *ω**d−d* in organic and biological solids is the order of 106 rad/s. Therefore, DEER experiment probes the distances *r**AB* of the order of several nanometers.

To take into account all spins in a sample, Eq. (1) is to be multiplied for all pairs of spins:

$$V(t)=\prod\limits_{{j>i}}^{{}} {\left\{ {1 - {p_B}(1 - \cos {\omega _{ij}}t)} \right\}}$$

2

.

For mono-spin-labeled molecules randomly distributed in a three-dimensional (3-D) space, theoretical expression for the DEER signal states that [27–31]

$${V_{3D}}(t)=\exp ( - \frac{{8{\pi ^2}{g^2}\mu _{{Bohr}}^{2}}}{{9\sqrt 3 \hbar }}{p_B}{C_{local}}t)$$

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where *C**local* is the local spin concentration taken in the cm− 3 units.

For the two-dimensional (2-D) model of the spatial distribution which could be more appropriate for biological membranes, the analogous expression is [32]

$${V_{2D}}(t)=\exp ( - 3.37{(\frac{{{g^2}\mu _{{Bohr}}^{2}}}{\hbar })^{2/3}}{p_B}{\sigma _{local}}{t^{2/3}})$$

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where *σ**local* is the local surface concentration taken in the cm− 2 units.