Effects of NSAIDs on the nanoscopic dynamics of lipid membrane

Effects of NSAIDs on the nanoscopic dynamics of lipid membrane

Journal Pre-proof Effects of NSAIDs on the nanoscopic dynamics of lipid membrane V.K. Sharma, E. Mamontov, M. Tyagi PII: S0005-2736(19)30246-9 DOI:...

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Journal Pre-proof Effects of NSAIDs on the nanoscopic dynamics of lipid membrane

V.K. Sharma, E. Mamontov, M. Tyagi PII:

S0005-2736(19)30246-9

DOI:

https://doi.org/10.1016/j.bbamem.2019.183100

Reference:

BBAMEM 183100

To appear in:

BBA - Biomembranes

Received date:

30 May 2019

Revised date:

16 August 2019

Accepted date:

19 September 2019

Please cite this article as: V.K. Sharma, E. Mamontov and M. Tyagi, Effects of NSAIDs on the nanoscopic dynamics of lipid membrane, BBA - Biomembranes(2019), https://doi.org/ 10.1016/j.bbamem.2019.183100

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© 2019 Published by Elsevier.

Journal Pre-proof

Effects of NSAIDs on the Nanoscopic Dynamics of Lipid Membrane V. K. Sharma1*, E. Mamontov2 and M. Tyagi3,4 1

Solid State Physics Division, Bhabha Atomic Research Centre, Mumbai 400085, India Neutron Scattering Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37830, United States 3 National Institute of Standards and Technology Center for Neutron Research, Gaithersburg, Maryland 20899, United States 4 Department of Materials Science and Engineering, University of Maryland, College Park, Maryland 20742, United States

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Abstract

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Nonsteroidal anti-inflammatory drugs (NSAIDs) are widely prescribed for their antipyretic, anti-inflammatory, and painkiller actions despite a wide spectrum of side effects. The mechanisms involved in their therapeutic actions and side-effects have not been clearly understood yet. The assertion that effects of NSAIDs are related to their action at cellular membrane level has stimulated the investigation of interaction between NSAIDs and membranes. Here, we report effects of two different NSAIDs, ibuprofen and indomethacin, on the thermotropic phase behaviour and the dynamics of DMPC membrane as studied using neutron scattering techniques. Elastic intensity scan measurements showed that both drugs substantially alter the phase behaviour of the membrane. However, the effects of these drugs are found to differ quantitatively, which is correlated with their respective interactions with phospholipid membrane. Quasielastic neutron scattering measurements showed that in the ordered phase, both drugs enhance the dynamics of the membrane, but the drugs’ effects on the membrane dynamics differ in the fluid phase. Indomethacin suppresses the dynamics of the membrane, whereas ibuprofen does not show significant effect at the same molar concentration. We have also investigated the effect of concentration of ibuprofen on the dynamics of the membrane. Our measurements provide clear evidence that interaction of NSAID with the membrane depends on both the physical state of the membrane and the nature and concentration of NSAID. Hence, one must investigate each NSAID independently while analysing its action mechanism. Better understanding of NSAID-membrane interaction can pave the way for designing more effective NSAID with reduced side effects. Keywords: Indomethacin, Ibuprofen, DMPC, Lateral motion, Internal motion, Neutron scattering

*Corresponding Author: [email protected]; [email protected]; Phone:+91-22-25594604

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Journal Pre-proof 1. INTRODUCTION Nonsteroidal

anti-inflammatory drugs

(NSAIDs)

are

well

known

pharmaceutical

formulations prescribed globally for their anti-inflammatory, analgesic, anti-platelet, and antipyretic characteristics. In addition to these therapeutic actions, NSAIDs have also demonstrated beneficial effects in treating cancer [1], Alzheimer’s [2], and arthritis disease [3-4]. Although NSAIDs are among the most commonly utilized drugs, their use is associated with a broad spectrum of side effects, which may include gastrointestinal and cardiovascular toxicity, among others [4-6]. The mechanisms involved in the therapeutic actions and sideeffects of NSAID have been investigated by various methods in the last few decades [7-16].

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The primary therapeutic action of NSAIDs is through the inhibition of the cyclooxygenase

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(COX) enzyme activity to disrupt synthesis of the prostaglandin, a principal mediator of pain and inflammation [16]. In order to reach their primary target, NSAIDs have first to pass

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through the plasma membranes. Hence, interaction of drugs with plasma membrane is inevitable. Beside COX pathway, there is increasing evidence of alternative action

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mechanism of NSAIDs through membrane-NSAIDs interaction [7,17,18]. NSAID-induced

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changes to the membrane can influence various physiological processes including the function of the embedded membrane proteins. Hence, it is of immense interest to understand the molecular-level mechanisms by which NSAID interact with the plasma membranes. This

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is critically important not only for understanding of the therapeutic actions of these drugs, but also the rational development of new ways to limit NSAIDs side effects. Plasma membrane is

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a highly complex heterogeneous dynamic system, which is a mixture of phospholipids, membrane proteins, and other small molecules, such as sterols. Model membrane systems have been widely employed to study membrane-drug interactions because they bypass the complexity of the plasma membranes and provide experimental conditions conducive to elucidating the molecular mechanisms that governs such interactions. We have used unilamellar vesicle based on a saturated phosphatidylcholine, 1,2-dimyristoyl-sn-glycero-3phosphocholine (DMPC) as a model membrane system. Phosphatidylcholines are selected because they are one of the principal structural lipids of the plasma membranes [19]. A schematic of a lipid bilayer and chemical structure of DMPC is shown in Fig. 1 It has been shown that incorporation of drugs disrupts the membrane and modulates its various biophysical properties, such as phase behaviour, thickness, bending modulus, permeability, etc. [20-25]. The disturbing effects of NSAIDs on membrane appear to be

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Fig. 1 Schematic of a lipid bilayer. Chemical structures of DMPC lipid, ibuprofen, and

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indomethacin are also shown.

related to their beneficial actions, such as antioxidant properties and anti-tumoral activity [7].

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It has also been suggested that direct interaction of NSAIDs with zwitterionic phospholipids is primarily responsible for gastrointestinal toxicity [26]. Effects of NSAIDs on the structure

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of the lipid membrane have been studied extensively [10,13,14,20,21,23,27-30]. It has been shown that NSAIDs interact strongly with the lipid bilayer and significantly perturb the

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membrane structure. For example, NSAID-induced bilayer thinning [21], membrane fusion [13-14], membrane defects, and pores [27] have been observed. In the case of hydrated lipid stacks [28], addition of 20 mol % ibuprofen disrupted the bilayer structure, and a transition from lamellar to cubic phase was observed. However, no such transition to cubic phase was observed in the case of lipid vesicles, even up to 62 mol % ibuprofen [21]. Small angle neutron scattering data [21] demonstrated bilayer thinning by about 15 % due to incorporation of ibuprofen in the DMPC vesicles. Recently, interaction of aescin and ibuprofen with DMPC membrane was studied using various biophysical methods including differential scanning calorimetry (DSC), small-angle, and wide-angle X-ray scattering [30]. It was found that both the additives alter the cooperativity of the phase transition and structure of DMPC membrane in a concentration-dependent manner. At the same time, there is not much information available on how NSAIDs influence the dynamics of lipid membranes. Such influence could play a major role in diverse processes such as membrane-protein interactions, cell signalling, drug delivery, membrane trafficking, bilayer permeability, cell 3

Journal Pre-proof division and fusion, etc. The alteration of membrane dynamics can dictate fluidity and permeability, which in turn may considerably modulate the transport properties of the cell membrane. The goal of the present paper is to analyse effects of NSAIDs on the nanoscopic dynamics of phospholipid membrane. Lipid membrane shows complex dynamical behaviour, which can be described by a superposition of different motions of individual lipid (such as lateral, flip flop, rotational) and the entire bilayer (e.g., bending motion and thickness fluctuations) [31-38]. These motions cover a broad range of time and length scales. Quasielastic neutron scattering (QENS) is one of the most powerful techniques for studying

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nanoscopic dynamics of lipid-based self-assembled molecular aggregates on a pico- to

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nanosecond time scale and on a length scale from Angstroms to nanometers [39-53]. Interactions between drugs and phospholipid membrane depend on various parameters,

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including chemical structure of the drug and lipid, physical state of the lipid bilayer, concentration of drugs, and ionic strength [7,20,21,24,25]. Here, we have investigated the

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role played by some of these parameters in the effects exerted by NSAIDs on the dynamical and phase behaviour of phospholipid membrane. To this end, we have selected two NSAIDs

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of different chemical origins, ibuprofen and indomethacin, which are profens and indole derivatives, respectively. Chemical structures of these two drugs are shown in Fig. 1. Here,

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we report the effects of ibuprofen and indomethacin on the dynamical and phase behaviour of DMPC membrane as investigated using incoherent elastic and quasielastic neutron scattering.

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QENS measurements have been carried out at different temperatures to investigate the role of physical state of the membrane. To examine the effect of the drugs concentration, QENS experiments have also been performed at two different concentrations of ibuprofen. The results showed that NSAIDs significantly alter the nanoscopic dynamic and phase behaviour of the membrane, with the effects strongly dependent on the physical state of the lipid bilayer and the chemical structure and concentration of NSAID.

2. MATERIALS AND METHODS 2.1 Materials and Sample Preparation DMPC lipids (powder) and D2O (99.9%) were procured from Avanti Polar Lipids (Alabaster, AL) and Cambridge Isotope Laboratories (Andover, MA), respectively. Ibuprofen and indomethacin were purchased from Sigma Aldrich (St. Louis, MO). Large unilamellar vesicles (LUV) were prepared using the extrusion method as described in supporting

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Journal Pre-proof information (SI). LUV solutions of 100mM DMPC, (a) neat, (b) with 25 mol % ibuprofen, (c) with 25 mol % indomethacin, and (d) with 50 mol % ibuprofen, were prepared using the extrusion method as described in SI. It should be noted that concentration of NSAID reported here is a drug-to-lipid mole ratio. For example, 25 mol % NSAID is defined with respect to the lipid concentration used (x =nNSAID/nDMPC; where nNSAID and nDMPC are the number of molecules of NSAID and DMPC, respectively. This means 25 mol % ibuprofen is 0.25/1 mol/mol drug-to lipid mole ratio. For direct comparison between ibuprofen and indomethacin, the number of drug molecules with respect to lipid molecules was kept the same for the 25 mol % LUV solutions. A number of studies [6,11,54] indicate that NSAIDs

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have high partition coefficients for phosphatidylcholine lipid bilayer. At neutral pH, partition

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coefficients (log10K) for ibuprofen and indomethacin in phosphatidylcholine lipid bilayer are found to be ~ 6 [11] and 3 [54], respectively. Hence, most of the NSAIDs are incorporated

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into the lipid bilayer. 2.2 Dynamic Light Scattering

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Dynamic light scattering (DLS) technique has been used to obtain the size of DMPC LUV

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with and without NSAIDs. DLS experiments were carried on DMPC LUV with and without 25 mol % ibuprofen at 310 and 280 K using a Zetasizer nano zs system from Malvern Instruments. The light source used was a 633 nm He-Ne laser, and scattered light was

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measured at an angle of 173o.

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2.3 Elastic Fixed Window Scan

Elastic Fixed Window Scans (EFWS), or elastic intensity scans, have been carried out on 100mM DMPC, neat and with 25 mol % ibuprofen or indomethacin, using high energyresolution (E~0.8 eV) HFBS spectrometer [55] at the NIST Center for Neutron Research (NCNR), Gaithersburg, Maryland. Reactor-based backscattering spectrometer are best suited for the elastic intensity scan measurements, in which Doppler-driven monochromator kept at rest. A standard spectrometer configuration with Si(111) as the monochromator and analyser crystals was used, corresponding to the incident and analysed neutron wave length of 6.271Å. The elastic intensity is averaged over scattering angles corresponding to a Q range of 0.36 to 1.75 Å-1 and normalised by the monitor counts. For EFWS experiments, the vesicles samples were placed in annular aluminium sample containers with 0.5 mm internal spacing, the outer diameter of 29.0 mm, and the inner diameter of 28.0 mm. The effective height of the sample holder was 54 mm. All elastic intensity scan measurements have been carried out in heating

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Journal Pre-proof cycles with a heating rate of 0.1 K/min. Measurement time at each temperature was set for 2 min per data point. 2.4 QENS Experiment QENS measurements were carried out on 100 mM DMPC with 25 mol % ibuprofen or indomethacin using a high energy-resolution backscattering spectrometer, BASIS [56] at the Spallation Neutron Source (SNS), Oak Ridge National Laboratory (ORNL). BASIS is an inverted geometry spectrometer which uses a wide angular coverage of large Si (111) crystal arrays to Bragg-select the final energy (2.08 meV) of the neutrons scattered off the sample out of a broad wavelength band of incoming neutrons. BASIS has an excellent energy

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resolution of 3.4 μeV (Q-averaged, full width at half-maximum). For the chosen experimental

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setup, the spectrometer had accessible energy transfers of ±100 μeV, and the available Q range was 0.3 Å-1 to 1.9 Å-1. For QENS experiments, the vesicles samples were placed in the

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same annular sample containers as used in the EFWS measurements. These containers were selected as to have no more than 10% scattering from the samples in order to minimize

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multiple scattering effects. To investigate the effect of NSAID concentration, QENS

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measurements have also been performed on 100mM DMPC with 50 mol % ibuprofen. QENS measurements were carried out at three temperatures, 280, 293, and 310 K where neat

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membrane is in the gel, ripple and fluid phases. QENS data were also recorded from pure D2O, as a reference sample, at the same temperatures. To obtain the instrument resolution data, QENS measurement was carried out on a vanadium standard. MANTID software [57]

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was used to execute standard data reduction, which included subtraction of background and correction for detector efficiency.

3. QENS DATA ANALYSIS

QENS spectra representing the signal from the DMPC membrane can be obtained by removing the signal originating from the solvent (D2O) from the LUV solution spectra. The scattering intensity from the DMPC membrane, Imem(Q,), was obtained using the following relationship

I mem  Q,    I solution  Q,     I D 2O  Q,  

(1)

where  is the volume fraction of D2O in vesicle solution. BASIS spectrometer (E~ 3.4 eV) is well-suited to study lateral (on the nano-second time scale) and internal (on the pico-second time scale) motions of the lipid [35-36]. Assuming both motions are uncoupled, the scattering law for the membrane can be written as 6

Journal Pre-proof S mem  Q,    Slat  Q,    Sint  Q,  

(2)

where Slat (Q,) and Sint (Q,) are the scattering functions corresponding to lateral and internal motions of the lipid molecules, respectively. The lateral motion of lipids is expected to exhibit continuous diffusion, at least at distances greater than the lipid’s diameter [35,46]. Hence, Slat(Q,) can be written as Slat  Q,    Llat (lat ,  ) 

lat    2 1

2 lat

(3)

where lat is the half width at half maximum (HWHM) of the Lorentzian, Llat (lat ,  )

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associated with the lateral motion of the lipid molecule. The internal motion of lipid is restricted by the chemical structure of the molecule and

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hence is locally confined. A finite probability exists for finding the scattering entity within the chain volume even after an observation time exceeding the energy resolution of the

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spectrometer, which gives rise to the scattering perceived as elastic. Hence, Sint(Q,) can be

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written as [41]

Sint  Q,    A  Q      1  A  Q   Lint ( int ,  )

(4)

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Here the first term represents the elastic part, whereas the second represents the quasielastic component, which is approximated by a single Lorentzian function. A(Q) is Elastic

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Incoherent Structure Factor (EISF) and Γint is the HWHM of the Lorentzian describing the internal motion. Hence, the total scattering law for the membrane becomes

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S mem  Q,    A  Q  Llat  lat ,    1  A  Q   Ltot (lat   int ,  )

(5)

For data fitting, Eq. (5) was convolved with the resolution function (as measured with a vanadium standard), and A(Q), lat, and int were determined by the least squares fitting of the spectra. DAVE software [58] was used for QENS data analysis.

4. RESULTS AND DISCUSSION 4.1 NSAIDs Enhances Fluidity of the Membrane in Ordered Phase In dynamic light scattering, intensity autocorrelation function, g2() of the scattered light is measured as a function of correlation delay time (). The observed intensity autocorrelation functions for DMPC LUV with and without 25 mol % ibuprofen at 310 and 280 K are shown in Fig.2. It is evident that addition of ibuprofen does not affect the decay of the autocorrelation function. Intensity autocorrelation function, g2(), is related to the first order 1

autocorrelation function of the electric field g () by the Siegert relation [59], 7

Journal Pre-proof g 2 ( )  1  C g1 ( )

2

(6)

where C is a spatial coherence factor determined by the instrument optics. For an ideal solution of monodisperse spherical particles, the electric field auto correlation function is represented by a single exponential. However, in the case of a polydisperse continuous distribution, the field autocorrelation function is given by a weighted sum of exponentials. For narrow polydispersity, it can be simplified to the well-known cumulant expansion [60].

ln( g ( ))  b   DLS  1

2 2

(7)

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where b is a constant. Here, the first and second cumulant,  DLS and 2 give the mean and variance, respectively. The ratio of the variance to the square of the mean is a measure of the

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polydispersity in the diffusion coefficient, represented by polydispersity index (PI). From the

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average relaxation rate  DLS , the diffusion coefficient of the particle DDLS, can be obtained using:

(8)

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 DLS  DDLS Q 2

Fig. 2 DLS data of DMPC LUV, neat (black square) and with 25 mol % ibuprofen (red triangle) at 310 and 280 K. Solid lines are fits as per cumulant analysis using eqs. 6 and 7.

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Journal Pre-proof Measured DLS data are well described using Eqs. 6 and (7) and are shown by solid lines in Fig. 2. Polydispersity index is found to be in the range of 0.07 to 0.11.Translational diffusion coefficients of whole vesicles, DDLS are obtained from the relaxation rate using Eq. (8) and for DMPC vesicles in the absence and presence of ibuprofen at 310 K are found to be 5.00.110-8 cm2/s and 5.10.110-8 cm2/s, respectively. At 280 K, DDLS for DMPC vesicles without and with ibuprofen are found to be 2.10.110-8 cm2/s and 2.20.110-8 cm2/s, respectively. The effective hydrodynamic radius (RH) of the LUVs is calculated from DDLS using the Stokes-Einstein relation, RH 

k BT , where kB is Boltzmann’s constant, T is the 6 DDLS

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temperature in Kelvin and  is the viscosity of the solvent (i.e. D2O). The calculated average

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diameter of DMPC LUV in the absence and presence of ibuprofen at 310 K are found to be

significantly affect the size of the vesicles.

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110 nm and 106 nm, respectively. It is evident that incorporation of ibuprofen does not

Elastic neutron scattering intensity scan is a highly sensitive technique for probing

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temperature-activated microscopic mobility and associated phase transitions. This technique

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monitors the thermal evolution of the microscopic dynamics in the system, which is sensitive to the mobility of atoms or molecules. An abrupt or discontinuous decrease in the elastic intensity is a strong indicator of a phase change associated with changing dynamics. We have

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systematically studied the effects of ibuprofen and indomethacin on the phase behaviour of the membrane using elastic intensity scan measurements. Q-averaged elastic intensity scans

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for DMPC membrane, neat and with 25 mol % ibuprofen and indomethacin, are shown in Fig. 3. It is evident that for neat membrane there is a step like decrease at 297 K corresponding to the main phase transition (gel-fluid phase). Dissimilar to the main phase transition, a small kink (change in slope) is observed at ~288 K corresponding to a relatively weak pre-transition (gel-ripple phase) [61]. Ripple phase is a special kind of gel phase in which membrane surface is not planar and has periodic ripples, usually with order of a few tens of nanometers. It is evident from Fig. 3 that incorporation of ibuprofen and indomethacin significantly affects the phase behaviour of the membrane. In a broad sense, both of the drugs shift the main phase transition towards lower temperatures, indicating that incorporation of NSAID enhances fluidity of the membrane. It is also found that the main phase transition gets broadened, which indicates that cooperatively of the transition is also affected due to the addition of NSAID. These results are consistent with the earlier differential scanning calorimetry (DSC) studies [62,63], which indicated that incorporation of indomethacin or 9

Journal Pre-proof ibuprofen shifts the main phase transition towards lower temperature, and the transition gets broadened. Furthermore, it has been shown that no significant change in the enthalpy (H) is observed due to addition of indomethacin or ibuprofen. Information about the microscopic dynamical behaviour of the membrane can be obtained by quantitative comparison of relative amplitude of elastic intensity scan. For all elastic intensity scan measurements, the same sample cell was used, and the concentration of DMPC was kept identical (100 mM). All elastic intensity scan measurements were normalised to beam monitor to take into account

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possible reactor power fluctuation. Hence, direct comparison of elastic intensities can provide

Fig. 3 Q averaged elastic intensity measured for 100 mM DMPC membrane, neat (black

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square) and with 25 mol % ibuprofen (red triangle) or indomethacin (blue circle) in heating cycle. Solid lines are the guide to the eyes.

us with qualitative information about the change in the dynamical behaviour of the membrane system. Below the main phase transition temperature, elastic intensity for DMPC membrane with ibuprofen or indomethacin is found to be always lower compared to the neat membrane, indicating that incorporation of drugs induces disorder in the membrane. Elastic intensity for the membrane with ibuprofen is much lower compared to the membrane with indomethacin, indicating that ibuprofen induces more disorder, probably due to the stronger interactions with and higher penetration into the bilayer. However, this is not the case in the fluid phase of the membrane, where effects of indomethacin on the membrane are stronger compared to those of ibuprofen. Moreover, elastic intensity for DMPC membrane with indomethacin in the fluid phase is higher compared to neat membrane, indicating suppression of the 10

Journal Pre-proof membrane dynamics. This is in contrast with the ibuprofen-loaded membrane, where no such suppression is observed. It is likely that indomethacin, being a larger molecule, can get embedded within the membrane core, thereby suppressing the membrane dynamics due to the steric hindrance, only in the more disordered fluid phase. Thus, our elastic intensity scan data showed that interactions of NSAID strongly depend on the physical state of the membrane and nature of drugs. in the This conclusion can be examined quantitatively, using QENS experiments, which are discussed in the next section.

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4.2 NSAIDs Significantly Alters the Dynamics of Lipid: Role of Phase of the Membrane and Nature of NSAID

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QENS spectra representing the DMPC membrane were obtained via subtracting the signal originating from the solvent (D2O) from that of the LUV solution. The spectra for the

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membrane, with and without ibuprofen or indomethacin, at a typical Q=1.1 Å-1 are shown in Fig. 4 at 280, 293, and 310 K. Instrument resolution (from a vanadium standard) is also

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presented in Fig 4. For direct comparison, spectra are normalized to the peak amplitude

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observed for DMPC with indomethacin. Sizable quasielastic (QE) broadening is observed for the DMPC membrane with and without NSAIDs, indicating stochastic motion of the lipid

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molecules within the spectrometer range. It is evident that incorporation of NSAIDs significantly affects the QE broadening, indicating that NSAIDs alter the dynamics of the lipid bilayers. In the ordered phase (280 and 293 K), incorporation of both NSAIDs leads to

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increase in QE broadening, indicating enhancement in the membrane dynamics. Largest QE broadening is observed for DMPC membrane with ibuprofen at 280 and 293 K, indicating maximum fluidity in the membrane. This is consistent with the elastic intensity measurement, which shows that below the main phase transition temperature, elastic intensity for DMPC membrane with ibuprofen is found to be lowest. However, in the fluid phase, indomethacin is found to act stronger compared to ibuprofen. Inclusion of indomethacin in the fluid phase of membrane leads to significant reduction in the QE broadening. This is in a stark contrast with the ibuprofen, which has little visible effect (slight increase) on the QE broadening. QENS measurements show that interaction of NSAIDs strongly depends on the physical state of the membrane and nature of the drugs. In the ordered phase, both drugs act as plasticizers, which enhance the dynamics of the membrane. Ibuprofen is found to act stronger compared to indomethacin. On the other hand, in the fluid phase, ibuprofen does not affect much (or

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Fig. 4 Typical observed QENS Spectra at Q=1.1 Å-1 for DMPC membrane, neat (black

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square) and with 25 mol % ibuprofen (red triangle) or indomethacin (blue circle) at 280, 293 and 310 K. The instrument resolution as measured from a vanadium standard is shown by

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solid line in the upper left panel. For quantitative comparison, the spectra are normalised to the peak amplitude. Individual components of the QENS fit for DMPC membrane with 25

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mol % indomethacin at Q=1.1 Å-1 obtained with the model scattering function given by Eq.

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(5) are shown in the right bottom panel.

slightly enhances) the dynamics of the membrane, but indomethacin acts oppositely and suppresses the dynamics of membrane at the same molar concentration. It was found that scattering law represented by Eq.(5) could describe the QENS spectra well for the membrane in the absence and presence of ibuprofen or indomethacin at all the measured temperatures and Q values. Typical fitted spectra for DMPC membrane with indomethacin at 310 K at Q=1.1 Å-1 are shown in the right bottom panel of Fig. 4. For a detailed insight into the lateral and internal motions, the parameters associated with these motions are analyzed as a function of Q in the following sections.

4.2.1 Lateral Motion of Lipid is Altered Significantly: The lateral motion of a lipid within the membrane leaflet is of principal interest due to its vital role in various physiologically relevant processes, including cell signaling, membrane trafficking, activity of membrane protein, and energy transduction pathways. Variations of HWHM of the Lorentzian 12

Journal Pre-proof corresponding to the lateral motion of lipid, lat with Q2 for DMPC membrane in the absence and presence of 25 mol % ibuprofen or indomethacin are shown in Fig.5 at different temperatures. It is clear that for all the membranes, lat increases linearly with Q2 at each of the measured temperatures. This indicates that lateral motion of lipid follows continuous diffusion and can be described using Fick’s law, lat  Dlat Q 2 as indicated by the lines. Here Dlat is the diffusion coefficient for lateral motion of DMPC lipid that can be derived from the slope of the linear fit, as shown in Fig. 6. Lateral diffusion coefficient for DMPC membrane in the fluid phase (310K) is found to be 6.010-7 cm2/s, which is an order of magnitude faster

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than the Dlat observed in the gel phase (0.710-7 cm2/s) of the membrane. Results are compared with the early QENS studies on the phosphatidylcholine membranes [46, 49-51].

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Tabony et al [49] have carried out QENS measurements on hydrated powder of DPPC membrane which showed that Dlat of DPPC in the fluid phase was 1210-7 cm2/s, which was

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almost an order of magnitude faster than in gel phase (1.410-7 cm2/s). QENS measurements

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on DMPC membrane have been carried out on systems with different morphologies, such as supported lipid bilayer, hydrated powder, multilamellar vesicles, etc [46, 50, 51]. Lateral cm2/s.

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diffusion coefficient of DMPC in the fluid phase is found to be in the range of 5.2 to 1210-7

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In the ordered phase (280 and 293 K), both ibuprofen and indomethacin accelerate the lateral motion of the lipid, indicating enhancement in the fluidity of the membrane. For

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example, at 293 K, Dlat for neat DMPC membrane is found to be 1.510-7 cm2/s, but increases to 2.610-7 cm2/s and 2.210-7 cm2/s due to addition of 25 mol % ibuprofen and indomethacin, respectively. In the fluid phase, addition of 25 mol % ibuprofen does not affect much (except for a slight increase) the lateral diffusion coefficient, but indomethacin acts strongly and in opposite direction. Indomethacin is found to act as a stiffening agent and suppresses the lateral motion of the lipids. At 310 K, Dlat for DMPC membrane is found to be 6.0 10-7 cm2/s, which reduces to 5.2 10-7 cm2/s due to addition of 25 mol % indomethacin. For a direct comparison between ibuprofen and indomethacin, we have kept all the measurement conditions the same and used identical molar concentrations of these two drugs. We have found that in the ordered phase, ibuprofen, which is a relatively smaller molecule, is more effective in comparison to indomethacin, as evident from the relatively larger enhancement in the lateral motion due to ibuprofen. However, in the fluid phase, indomethacin acts stronger, and in the opposite direction, compared to ibuprofen, suppressing the lateral motion of the lipids. This leads to decrease in the permeability of the bilayer, 13

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Fig. 5 Variation of half width at half maximum (HWHM) of the Lorentzian corresponding to lateral motion of lipid, lat with Q2 for DMPC membrane, neat (black square) and with 25

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mol % ibuprofen (red triangle) or indomethacin (blue circle) at 280 K (filled), 293 K( halffilled) and 310 K (open). lat values obtained for DMPC membrane with 50 mol % ibuprofen

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text.

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are shown in the inset. The lines represent the Fickian diffusion description as discussed in

which could be the prime reason behind the reduced leakage of the inner content of DMPC vesicles in the presence of indomethacin [63]. When another small NSAID, aspirin [25], is added into consideration, we find that indomethacin is a remarkable exception, which suppresses the lateral motion of the lipid in the fluid phase, whereas the other drugs, aspirin and ibuprofen, do not affect much the lateral motion of the lipids in the fluid phase at the same 25 mol % concentration. The variations in the effect of these drugs might be correlated with the difference in their size, hydrophobicity, and interaction with DMPC membrane. Aspirin, ibuprofen, and indomethacin have pKa values ~3 to 5 in the aqueous solution, which indicates that at neutral pH all these drugs can be considered anionic. NSAID interacts with the lipid membrane mainly via electrostatics interaction with the polar head group and hydrophobic interactions with the nonpolar alkyl tails. Aspirin and ibuprofen, being smaller molecules with polar O atoms, are more strongly governed by its electrostatic interactions with the headgroup. However, due to a larger nonpolar part, indomethcain, even in the 14

Journal Pre-proof charged form, is primarily governed by its interactions with the hydrophobic tails. In the ordered phase, lipid molecules are well ordered, and incorporation of NSAID in the membrane core would introduce a disorder in the membrane, which would enhance the membrane dynamics. Because of the higher order of tails in the ordered phase, penetration into the membrane core is not as easy for indomethacin as for ibuprofen, due to the larger size of the former drug molecule. This can explain the observed stronger effects of ibuprofen on the ordered phase of the membrane. However, in the fluid phase, lipid molecules are disordered, with large area per molecule. In that case, indomethacin can easily penetrate within the membrane core and, being a large molecule, creates steric hindrance that results in

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suppression in the membrane dynamics. From the molecular interaction standpoint, due to

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smaller nonpolar part, aspirin and ibuprofen prefer to stay near the head group of the phosphatidylcholine membrane [11, 23], whereas indomethacin, with its larger hydrophobic

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part, is located deeper inside the membrane, interacting primarily with the hydrophobic tails. Therefore, the effect of indomethacin on the membrane dynamics is expected to be stronger,

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from both the steric and interaction standpoints, provided that indomethacin can penetrate into the membrane core, as is the case for the fluid phase of the membrane. Thus, our results

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indicate that effects of different NSAID on the lateral motion of the lipids are not identical and strongly depend on the size and hydrophobicity of NSAID, which might be correlated

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with the location of NSAID in the membrane.

As pointed out in [6], both electrostatic and hydrophobic interactions, as well as the

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location of the NSAID in the membrane, may have a distinct pH dependence, by affecting the state of the drugs (protonated or unprotonated), with the uncharged species inserting deeper into the bilayer. Besides, the charged state of drugs can be associated with the additional interaction with water molecules, resulting in the completely hydrated polar groups, which is lacking in the uncharged state. Thus, the comprehensive picture of the NSAID/membrane interactions can be nuanced and rather complex. The prime reason for the significant difference in the effects on the membrane lipids between ibuprofen and indomethacin, found in our study despite possible variation in the drug locations due to their charged/uncharged states, is the large difference in the drugs’ molecular sizes, which must overshadow the charged/uncharged state variations. For example, the difference in the peak location of charged and uncharged forms of ibuprofen in DOPC is only ca. 2 Å [64], which is smaller than the difference in the molecular size between ibuprofen and indomethacin.

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Fig. 6 Lateral diffusion coefficient, Dlat, for the DMPC membrane, neat and with 25 mol %

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ibuprofen or indomethacin at different temperatures. It is evident that in the ordered phase

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(280 K, 293 K) both NSAIDs enhance the lateral motion (ibuprofen effect being stronger). However, in the fluid phase (310 K), indomethacin acts oppositely and suppresses the lateral

is shown in the inset.

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motion. Lateral diffusion coefficient obtained for DMPC membrane with 50 mol % ibuprofen

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4.2.2 Internal Motion of the Lipid also Gets Modified: Elastic incoherent structure factor (EISF), and HWHM, int corresponding to the internal motion of lipid in the absence and presence of 25 mol % ibuprofen or indomethacin are shown in the Figs. 7 and 8, respectively. It is evident that incorporation of NSAID also affects the internal motion of the lipid, depending on the phase of the membrane. In the ordered phase (280 and 293 K), addition of ibuprofen or indomethacin results in a decrease in EISF, indicating that incorporation of NSAID enhances the flexibility of the lipid molecule. In the fluid phase, no significant change in EISF is observed from addition of ibuprofen. However, EISF increases systematically due to incorporation of indomethacin, indicating that in the fluid phase, indomethacin reduces the flexibility of the lipid. These results are consistent with the behaviour of int as shown in Fig. 8. In the ordered phase, int increases due to addition of both drugs, indicating faster internal motion. However, in the fluid phase, no significant

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Fig. 7 Variation with Q of the elastic incoherent structure factor (EISF) for the DMPC

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membrane, neat (black square) and with 25 mol % ibuprofen (red triangle) or indomethacin

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(blue circle) at 280 K (filled), 293 K (half-filled) and 310 K (open). EISF obtained for DMPC

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described in text.

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membrane with 50 mol % ibuprofen is shown in the inset. The lines show the model fits as

Fig. 8 Variation of half width at half maximum of the Lorentzian corresponding to the internal motion of lipid, int with Q for DMPC membrane, neat (square) and with 25 mol % ibuprofen (triangle) or indomethacin (circle) at 280 K (filled), 293 K( half-filled) and 310 K (open). int values obtained for DMPC membrane with 50 mol % ibuprofen are shown in the inset. The lines show the model fits as described in text. 17

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change is observed in int due to addition of ibuprofen or indomethacin (except for a slight decrease). Internal motion of lipid is complex and actually comprises various motions, including rotation of lipid molecules, bending, stretching modes of chemical bonds, large amplitude oscillations, out of plane fluctuations, etc. The superposition of all these motions can be effectively described by assuming that the hydrogen atoms of each CH2 unit undergo localised translational diffusion (LTD) within a spherical volume. This is supported by the behaviour of the EISF and HWHM (int) corresponding to internal motion. It is evident that, in the zero-Q limit, int flattens towards a finite nonzero value and increases with increasing

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Q, which is a typical signature of the LTD [65]. Due to flexibility of alkyl chain, hydrogen

Fig. 9 Maximum radius of confining sphere (Rmax) and associated diffusion coefficient (Dmax) corresponding to the internal motion of lipid for DMPC membrane, neat and with 25 mol % ibuprofen or indomethacin, as obtained from least squares fitting of EISF and HWHM assuming localized translational diffusion model. Rmax and Dmax obtained for the internal motion of lipid for DMPC membrane with 50 mol % ibuprofen are shown in the insets. 18

Journal Pre-proof atoms at different positions along the chain may move in the spheres having different radii and diffusivity. We have assumed a simple linear distribution similar to that used to explain the dynamics of lipid bilayer [34,52,53]. LTD model is used to describe the internal motion of the lipids. Details of LTD model are discussed in the SI. In brief, hydrogen atoms are involved in localised translation diffusion within the spheres of varying radii characterized by varying diffusivities. Hydrogen atoms in the first CH2 unit nearest to the head group experience maximally constrained space, hence having the smallest radius of sphere, Rmin and diffusivity Dmin. Sphere size and diffusivity increase linearly with the alkyl chain and attain the maximum value of radius (Rmax) and diffusivity (Dmax) for the last CH3 unit of the alkyl

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chain. Resultant scattering law corresponding to this model is given in Eq. S1-S4 (SI). It can

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be seen that LTD model describes the data well, as shown by the lines in Figs. 7 and 8, and the fitting parameters for the maximum size of confining volume and associated diffusivity

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are shown in Fig.9 (a) and (b), respectively. Small values of Rmin and Dmin are omitted as these values merely reflect the negligible movement of hydrogen atoms that are in the first

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carbon position held by the head group. Rmax for DMPC membrane in this temperature range is found to be in the range of 2.2- 5 Å. Results are compared with the earlier QENS studies

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on the phosphatidylcholine membranes [34,52,53]. For example, König et al [52] have used LTD model to describe internal motion of DPPC membrane in a wide range of

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temperatures, from 275 K to 333 K, and Rmax was found to be in the range of 1.5 to 4.4 Å. Doxastakis et al.[53] have studied internal motion of anhydrous DPPC membrane in

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the temperature range of 310 K to395 K using QENS and molecular dynamics simulation techniques. Internal motion of DPPC membrane is described using LTD model, and Rmax was found to be in the range of 3.2-6.4 Å. Busch et al [34] have studied dynamics of hydrated DMPC powder in the temperature range from 278 K to 313 K and used LTD model to analyse the internal motion. Rmax was found to be in the range of 2.0 to 3.6 Å. It is evident from Fig. 9 that incorporation of NSAIDs significantly affects the internal motion of the lipid, especially in the ordered phase (280 and 293 K). Incorporation of both drugs results in increase in the size of confining volume and associated diffusivity, indicating enhancement in the internal motion of the lipid. For example, at 293K, Rmax for DMPC membrane is found to be 2.9 Å which increases to 3.7 Å in presence of 25 mol % ibuprofen. Similar to the lateral motion, ibuprofen is found to enhance the internal motion to a greater extent compared to indomethacin. In the fluid phase (310 K), ibuprofen does not affect much the internal motion, but indomethacin is found to restrict the internal motion.

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Journal Pre-proof 4.2.3 Dependence on the Concentration of NSAID: So far, we have kept the same molar concentration of both ibuprofen and indomethacin, which enables a direct comparison between these two drugs. To investigate the effect of the concentration of NSAID, we have also carried out QENS experiments on DMPC membrane with 50 mol % of ibuprofen at 280, 293 and 310 K. Scattering law (Eq. (5)) is found to describe the observed QENS spectra well, and the obtained fitting parameters lat , A(Q) and int are shown in the insets of Figs. 5, 7, and 8, respectively. Lateral and internal motion of lipid are described well using Fickian and LTD models, respectively, and the obtained fitting parameters are shown in the insets of Figs.6 and 9. It is found that in the ordered phase, (at 280 K and 293 K), incorporation of ibuprofen

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accelerates the lateral motion of lipid molecules in a concentration-dependent manner. As the

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concentration of ibuprofen is increased, the lateral diffusion coefficient, Dlat increases, indicating enhancement in the fluidity of the membrane. At 280 K, for neat DMPC

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membrane, Dlat is found to be 0.710-7 cm2/s, which increases by about 130 % due to addition of 50 mol % ibuprofen. In the fluid phase (310 K), inclusion of 25 mol % ibuprofen does not

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significantly affect (except for a slight increase) the lateral motion of lipid, but as the

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concentration of ibuprofen is increased to 50 mol %, Dlat decreases, and its value becomes lower than the value for neat DMPC membrane. This indicates that in the fluid phase, the effect of ibuprofen on the dynamics of the membrane is not monotonic, unlike in the ordered

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phase, where an increase in the Dlat is observed with the increasing concentration of ibuprofen. For internal motion, it is clear from Fig. 9 that in the ordered phase (280 and 293

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K), the size of the confining volume and diffusivity corresponding to internal motion increase with the ibuprofen concentration. This indicates that in the ordered phase, as the concentration of ibuprofen is increased, it creates more disorder in the membrane. However, in the fluid phase (310 K), Rmax and Dmax remain about same with the changing concentration of ibuprofen. Our present results indicate that interaction mechanism between NSAID and lipid bilayer is complex and depends on the various factors including physical state of the membrane, chemical structure, and concentration of NSAID. We have shown that incorporation of NSAIDs induces different degrees of perturbations in the membrane and modulates its dynamical, and phase behaviour.

5. CONCLUSIONS Our high energy-resolution neutron scattering measurements reveal that lipid motion and phase behaviour in DMPC membrane are affected significantly by addition of nonsteroidal 20

Journal Pre-proof anti-inflammatory drugs (NSAIDs), ibuprofen and indomethacin. The main membrane phase transition, gel-to-fluid, becomes broader and shifts toward lower temperature, in agreement with the differential scanning calorimetry results available in the literature, indicating that fluidity and cooperatively of the transition are affected by NSAIDs. Furthermore, incorporation of NSAIDs into the membrane affects the dynamics of lipids at the molecular level. Both the lateral and internal lipid motions are affected to various extents, depending on the physical state of the lipid membrane. In the ordered phase, NSAIDs accelerate the lipid dynamics. On the other hand, in the fluid phase, 25 mol % ibuprofen and indomethacin show opposite effects, of a vastly different magnitude, on the lipid motion. While indomethacin

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significantly suppresses the lipid motion, ibuprofen facilitates it, but only to a small extent.

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When adding into consideration another small NSAID, aspirin, we find that indomethacin is outstanding in its suppression effect on the lateral lipid motion in the fluid phase. We

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conclude that, while NSAIDs may share common features in their chemical mechanisms of therapeutic action, their interaction with and influence on the plasma membrane may vary

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greatly, depending on the chemical structure, size, and hydrophobicity, which define their location within the membrane, and, in turn, their effect on the membrane dynamics. This

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result, especially considered together with another interesting observation made in this work, that the effect of NSAIDs on the membrane is not monotonic with concentration, may need to

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be taken into account when considering possible effects of administering these drugs.

21

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steroidal anti-inflammatory drugs. Biophysical Chemistry 2008, 137, 28-34. 64. Boggara, M. B.; Mihailescu, M.; Krishnamoorti, R. Structural Association of

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65. Volino, F.; Dianoux, A. J. Neutron Incoherent Scattering Law for Diffusion in a Potential of Spherical Symmetry: General Formalism and Application to

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Diffusion Inside a Sphere. Molecular Physics 1980, 41 (2), 271-279.

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Effects of ibuprofen and indomethacin on motions of the membrane lipids are probed NSAID-membrane interaction depends on membrane phase state and drug concentration NSAIDs broaden the main phase transition and shift it toward a lower temperature In ordered phase, ibuprofen and indomethacin enhance the dynamics of the membrane In fluid phase, indomethacin, but not ibuprofen, suppresses the membrane dynamics

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