5734 DOI: 10.1021/la903854s Langmuir 2010, 26(8), 5734–5745Published on Web 12/16/2009

pubs.acs.org/Langmuir

© 2009 American Chemical Society

Small-Angle Neutron Scattering Studies of Phospholipid-NSAID Adducts

Mohan Babu Boggara and Ramanan Krishnamoorti*

Department of Chemical and Biomolecular Engineering, University of Houston, Houston, Texas 77204

Received October 12, 2009. Revised Manuscript Received November 21, 2009

Nonsteroidal anti-inflammatory drugs (NSAIDs) are known to have strong interactions with lipidmembranes. Usingsmall-angle neutron scattering, the effect of ibuprofen, a prominent NSAID, on the radius of small unilamellar vesiclesof 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC) and their bilayer structure was studied systematically as afunction of pH (ranging from 2 to 8) and drug-to-lipid mole ratio (from 0/1 to 0.62/1 mol/mol). Ibuprofen with a pKa of∼4.6 was found to significantly affect the bilayer structure at all pH values, irrespective of the charge state of the drug.At low pH values, the drug reduces the bilayer thickness, induces fluid-like behavior, and changes headgroup hydration.The incorporation of the drug in the lipid bilayer while affecting the local bilayer structure and hydration of the lipiddoes not affect the overall stability of the vesicle dispersions over the pH range studied.

Introduction

Nonsteroidal anti-inflammatory drugs (NSAIDs) are amongthe most widely used drugs worldwide.1 They are used for theirantipyretic (fever reducing), analgesic (pain reducing), and anti-inflammatory action. Despite significantly different pharmaco-kinetics (e.g., release profile) among the different NSAIDs, oralconsumption of different NSAIDs results in gastrointestinal (GI)toxicity and mild to fatal ulcers. However, the mechanism bywhich NSAIDs cause GI toxicity is not fully understood andremains an active area of research. This GI toxicity led to thediscovery of a range of alternative safer NSAIDs based ondifferentmechanisms1,2 such as cyclooxygenase (COX)-II-inhibit-ing NSAIDs (Celebrex, Vioxx) or nitric oxide releasing NO-NSAIDs, notwithstanding their long-term safety issues.1 One ofthe suggested mechanisms, supported by preclinical and clinicalevidence, indicates that (in oral aswell as systemic administration)it is the direct interaction of NSAIDs with zwitterionic phospho-lipids (GI tract lining) that is primarily responsible for GItoxicity.3 This hypothesis has led to the development of NSAIDspreassociated with phospholipids, especially phosphocholines(PCs), at as much as equimolar ratios as safer alternatives tounmodifiedNSAIDs. SuchPC-NSAIDadducts have been shownto significantly reduce GI toxicity and enhance therapeuticactivity in both animal and human models.4

NSAIDs are amphiphilic, and hence the interactions betweenthe drug and the phospholipid bilayer are critical to the design ofPC-NSAID drug-delivery systems. Previously, Levin and co-workers used surface enhanced Raman and IR spectroscopymethods to demonstrate that ibuprofen interacts with lipids in a

pH-sensitive manner.5 Further, pH, similar to ionic strength, canalso influence the phospholipid’s acyl chain melting transition,6

the phase transformations such as bilayer to bicontinuous cubicphase,7 membrane fusion, protein function, and crystallization,8

and induce structural modification of lipids by changing thesurface charge density (σ) of the headgroup region. For example,a value of σ> 1-2 μC/cm2 (achieved using calcium) can inducecurvature and promote formation of unilamellar vesicles.9

Vesicles are closed objects made of a lipid bilayer membranewith an inner aqueous core that can potentially carry water-soluble drugs, while the bilayer can potentially carry hydrophobicor amphiphilic drugs such as NSAIDs. For example, by pre-associating the drugs with vesicles, increased drug solubility orlower toxicity can be achieved.10 Vesicles not only serve as themost common models of biological membranes but are alsowidely used systems for drug-delivery, owing to the tunability inmorphology, responsiveness (to pH or temperature), and adapt-ability to targeted delivery. However, the success of vesicle-baseddrug delivery depends onmeeting a range of, often contradictory,criteria, e.g., long shelf life (high stability) and drug release byfusion at the site (low stability). Two key aspects of the vesicle aresignificant in addressing the challenges of successful vesicle-baseddrug delivery. First, vesicle size stands out as an importantparameter in the recognition by phagocytes and circulation timein blood (100 nm being most effective), as shown in vesiclescarrying cancer targeting drugs.11 On the other hand, lipidcomposition and fluidity play a minimal role in dictating thecirculation time in blood. Second, bilayer thickness plays asignificant role in the performance of drug-vesicle complexes.For example, changes in bilayer thickness along with surfacetension and lateral pressure can induce highly nonlinear changesin the structure and function of membrane proteins11 and even*To whom correspondence should be addressed.

(1) Donnelly,M. T.; Hawkey, C. J.Aliment. Pharmacol. Ther. 1997, 11, 227–236.(2) Vane, J. R.Nature 1971, 231, 232–235.Wallace, J. L.; Soldato, P. D.Fund. Clin.

Pharmacol. 2003, 17, 11–20. Williams, J. L.; Borgo, S.; Hasan, I.; Castillo, E.;Traganos, F.; Rigas, B. Cancer Res. 2001, 61, 3285–3289.(3) Lichtenberger, L. M. Biochem. Pharmacol. 2001, 61, 631–637.(4) Lichtenberger, L. M.; Wang, Z. M.; Romero, J. J.; Ulloa, C.; Perez, J. C.;

Giraud, M. N.; Barreto, J. C. Nat. Med. 1995, 1, 154–158. Lichtenberger, L. M.;Ulloa, C.; Vanous, A. L.; Romero, J. J.; Dial, E. J.; Illich, P. A.; Walters, E. T.J. Pharmacol. Exp. Ther. 1996, 277, 1221–1227. Lichtenberger, L. M.; Romero, J. J.;de Ruijter, W. M. J.; Behbod, F.; Darling, R.; Ashraf, A. Q.; Sanduja, S. K.J. Pharmacol. Exp. Ther. 2001, 298, 279–287. Anand, B. S.; Romero, J. J.; Sanduja,S. K.; Lichtenberger, L. M. Am. J. Gastroenterol. 1999, 94, 1818–1822.(5) Levin, C. S.; Kundu, J.; Janesko, B. G.; Scuseria, G. E.; Raphael, R. M.;

Halas, N. J. J. Phys. Chem. B 2008, 112, 14168–14175.

(6) Vautrin, C.; Zemb, T.; Schneider, M.; Tanaka, M. J. Phys. Chem. B 2004,108, 7986–7991.

(7) Okamoto, Y.; Masum, S. M.; Miyazawa, H.; Yamazaki, M. Langmuir 2008,24, 3400–3406.

(8) Trivedi, V. D.; Yu, C. Chem. Phys. Lipids 2000, 107, 99–106.(9) Uhrikov�a,D.; Teixeira, J.; Lengyel, A.; Alm�asy, L.; Balgav�y, P.Spectroscopy

2007, 21, 43–52.(10) Salvati, A.; Ristori, S.; Oberdisse, J.; Spalla, O.; Ricciardi, G.; Pietrangeli,

D.; Giustini, M.; Martini, G. J. Phys. Chem. B 2007, 111, 10357–10364. Ristori, S.;Oberdisse, J.; Grillo, I.; Donati, A.; Spalla, O. Biophys. J. 2005, 88, 535–547.

(11) Nagayasu, A.; Uchiyama,K.; Kiwada, H.Adv. DrugDelivery Rev. 1999, 40,75–87.

DOI: 10.1021/la903854s 5735Langmuir 2010, 26(8), 5734–5745

Boggara and Krishnamoorti Article

regulate the gating of ion channels.12 Thus, the study of pertur-bant effect on the bilayer thickness is important, as shown in thecase of local anesthetics that reduce bilayer thickness and inducevesicle-bicelle transition13 or cholesterol and other sterols thatincrease the bilayer thickness.14

The focus of this paper is to understand the effect of ibuprofenon the structural characteristics of phospholipid-based bilayermembranes both as a function of pH as well as drug concentra-tion. In model membrane systems, changing pH provides a facilemechanism to change the surface charge density of the lipidheadgroups without any chemical modification15 and allowsinsight into the electrostatic interactions between lipids andbetween lipid and drug molecules. The pH studies described herewill help provide molecular insight into interactions betweenNSAIDs and phospholipid membranes and form the basis fordeveloping NSAID-liposome complexes as a viable safer alter-native to unmodified NSAIDs. Further, it is important to under-stand the in vitro colloidal stability16 of liposome dispersionscarryingNSAIDs, whichwill help quantify the extent of stabiliza-tion needed in vivo for such PC-NSAID adducts.17

Materials and Methods

Materials. 1,2-Dimyristoyl-sn-glycero-3-phosphatidylcholine(DMPC) and 1,2-dimyristoyl-D54-sn-glycero-3-phosphatidyl-choline (D54-DMPC) in powder form were purchased fromAvanti Polar Lipids, Inc. (Alabaster, AL) and were used withoutfurther purification. The lipids were stored at -20 �C until used.HCl and NaOH were purchased from Mallinckrodt Chemicalsand EM Science (Merck KGaP in Germany), respectively, whileibuprofen, DCl, NaOD, and D2O were purchased from SigmaAldrich (St. Louis, MO). All other chemicals were reagent grade.

Sample Preparation. Calorimetry experiments were per-formed using DMPC vesicles (with and without drug) in pH-adjusted deionized water (resistivity 18.2 MΩ 3 cm) at a heatingrate of 5 �C/min. The pH of the water was adjusted using dilutesolutions of HCl and NaOH. All calorimetric experiments weredone with 1% of DMPC wt/wt with water (∼14.7 mM), and theibuprofen-DMPC samples had a 1:15 mol/mol drug-to-lipidratio. Small-angle neutron scattering (SANS) experiments wereperformed at 30 �C with pD (= pHþ 0.4) adjusted D2O (from 2to 8) as solvent to reduce the incoherent scattering from hydrogenatoms. The pDof the solvent was adjusted using diluted solutionsof NaOD and DCl in D2O. Additionally, in SANS, the DMPCconcentration was 1 wt %/wt with D2O (∼16.2 mM). In SANSexperiments, pD values were changed systematically from 2 to 8for samples containing DMPC in D2O as well as ibupro-fen-DMPC, samples where drug-to-lipid ratio was maintainedat 0.31/1 mol/mol. The effect of drug concentration was exploredby performing experiments at pD values of 2 and 8 with drug-to-lipid ratios of 0/1, 0.06/1, 0.31/1, and 0.62/1mol/mol.Using chaindeuteratedDMPC (D54-DMPC) for twopDvalues of 2 and 8,weexamined the bilayer structure under conditions where chainscattering length density (SLD)matches withD2O and is differentfrom that of DMPC. The ibuprofen concentration was main-tained at 5 mM (resulting in a drug-to-lipid ratio of 0.33/1 mol/mol) in these experiments. Similar methods were used for vesiclepreparation in both calorimetry and SANS experiments.

Small unilamellar vesicles (SUVs) were prepared by dispersingeither pure lipid or premixed drug/lipid mixtures (codissolved inorganic solvent and dried) in pH/pD-adjusted water. Pure lipidsolutions were prepared by first dissolving powder lipid in waterfollowed by vigorous vortexing and hydration at 30 �C (wellabove the DMPC gel-liquid transition of 23 �C) for at least 2 hbefore being extruded. The samples containing lipid and drugwere first codissolved in chloroform and dried under a stream ofN2 to make thin films. These films were further dried in vacuumovernight (at room temperature) to ensure complete removal ofchloroform. Water was added to these dry samples, which werethen vortexed and hydrated in the same manner as pure lipidsolutions before being extruded. The vesicles were prepared byextrusion of well-hydrated solutions through a polycarbonatemembrane of 100 nm diameter pores using a hand-held miniex-truder purchased fromAvanti Polar Lipids, Inc. (Alabaster, AL).A total of 29 passes through the membrane were performed toobtain SUVs.18

Instrumentation. Lipidmelting temperature wasmeasured bydifferential scanning calorimetry (DSC) using a Perkin-ElmerPyris 1 with subambient capability. Samples (1 wt % DMPCvesicles in water) of ∼20 mg (total weight of DMPC and water)were sealed in a liquid sample vial and then scanned from 10 to50 �C at a heating rate of 5 �C/min. The peak gel-fluid meltingtemperature of the lipid bilayer was obtained from the transitionpeak in the heating scan.

SANS experiments were performed at the NG7 beamline 30 mSANS spectrometer at the NIST Center for Neutron Research(NCNR), Gaithersburg, Maryland. Neutrons with a wavelength,λ, of 6 A (Δλ/λ= 14.2%) and three different sample-to-detectordistances of 1, 4, and 13 m, to provide a q range of 0.0026-0.45 A-1 (where q = 4π sin θ/λ, termed the scattering vector ormomentum transfer, and θ is half the scattering angle).19 SANSdata were reduced from two-dimensional (2D) raw data to one-dimensional (1D) intensity (I(q)) on an absolute scale (differentialscattering cross sections/volume) using incident neutron flux andcorrecting for instrumental background and detector efficiencywith empty sample cell scattering, dark current, and isotropicsample scattering. Incoherent backgroundwas estimated fromtheslope of plots of I(q)q4 versus q4 at high q values (0.2-0.3 A-1).Data reduction was performed using Igor Pro Software(WaveMetrics, Tigard, OR) and with the programs developedat NCNR.20

SANS Analysis. The SANS data were analyzed using twomethods: (a) a model-independent method called the modifiedKratky-Porod (MKP) method and (b) a separated form factor(SFF) model. While the MKP method allows for a model-independent extraction of the bilayer thickness, the SFF methodprovides a way to extract detailed structural parameters of bothvesicles and the bilayers. The SFF is based on the fact that theform factors of the vesicle (with radiusR∼ 50 nm) and that of thebilayer (thickness d ∼ 5 nm) can be separated. This allows one totry different types of SLD profiles independently for the bilayers,while leaving the vesicle form factor unmodified. Also, thissimplifies the form factor mathematically and is shown to becomparable to that of the original unseparated form factor.21

MKP Analysis. Pencer and Hallet22 have proposed anMKP23 method that is based on the fact that the membrane canbe approximated by randomly oriented thin sheets if the vesicleradiusR. d, d being the bilayer thickness. Thus, the form factor

(12) Yuan, C.; O’Connell, R. J.; Jacob, R. F.; Mason, R. P.; Treistman, S. N.J. Biol. Chem. 2007, 282, 7276–7286.(13) Uhrıkov�a, D.; Rapp, G.; Yaradaikin, S.; Gordeliy, V.; Balgav�y, P.Biophys.

Chem. 2004, 109, 361–373.(14) Gallov�a, J.; Uhrıkov�a, D.; Ku�cerka, N.; Teixeira, J.; Balgav�y, P. Biochim.

Biophys. Acta, Biomembr. 2008, 1778, 2627–2632. Gallov�a, J.; Uhríkov�a, D.; Islamov,A.; Kuklin, A.; Balgavy, P. Gen. Physiol. Biophys. 2004, 23, 113–128.(15) Zhou, Y.; Raphael, R. M. Biophys. J. 2007, 92, 2451–2462.(16) Chakraborty, H.; Mondal, S.; Sarkar, M. Biophys. Chem. 2008, 137, 28–34.(17) Lamprecht, A.; Saumet, J. L.; Roux, J.; Benoit, J. P. Int. J. Pharm. 2004,

278, 407–414. Fatouros, D. G.; Antimisiaris, S. G. J. Colloid Interface Sci. 2002, 251,271–277. Jablonowska, E.; Bilewicz, R. Thin Solid Films 2007, 515, 3962–3966.

(18) MacDonald, R. I.; Menco, B. P. M.; Takeshita, K.; Subbarao, N. K.; Hu,L. Biochim. Biophys. Acta, Biomembr. 1991, 1061, 297–303.

(19) Glinka, C. J.; Barker, J. G.; Hammouda, B.; Krueger, S.; Moyer, J. J.; Orts,W. J. J. Appl. Crystallogr. 1998, 31, 430–445.

(20) Kline, S. R. J. Appl. Crystallogr. 2006, 39, 895–900.(21) Pencer, J.; Krueger, S.; Adams, C. P.; Katsaras, J. J. Appl. Crystallogr.

2006, 39, 293–303.(22) Pencer, J.; Hallett, F. R. Phys. Rev. E 2000, 61, 3003–3008.(23) Knoll, W.; Haas, J.; Stuhrmann, H. B.; Fuldner, H. H.; Vogel, H.;

Sackmann, E. J. Appl. Crystallogr. 1981, 14, 191–202. Knoll, W.; Ibel, K.; Sackmann,E. Biochemistry 1981, 20, 6379–6383.

5736 DOI: 10.1021/la903854s Langmuir 2010, 26(8), 5734–5745

Article Boggara and Krishnamoorti

of the membrane is given by

PðqÞ ¼ F22

q2sin

qd

2

� �" #ð1Þ

where F is the SLD of the bilayer. A plot of I(q)q4 versus q shouldhave a maximum at a value of q = qmax and satisfies qmaxd= π.The plots of I(q)q4 versus q were fitted with a fourth-orderpolynomial in the q-range of 0.06-0.11 A-1, and the q-value atmaximumwas determined from that which the bilayer thickness dwas obtained. This method gives good fits for both pure lipid andlipid with drug and has the advantage of being model-indepen-dent. The errors in the thicknesses were obtained using a MonteCarlo error analysis.24 In this method, first synthetic data sets(n = 5000) are generated using the experimental data and thecorresponding errors in q. Each of the synthetic data is then fittedto give the qmax (and d), which are then used to estimate therespective mean and variance.

SFF Model. The intensity of the scattered neutrons fromvesicle dispersions can be given by

IðqÞ ¼ NPðqÞSðqÞ ¼ NjFðqÞj2SðqÞ ð2Þwhere F(q) is the form factor of the individual vesicles, and S(q) isthe structure factor signifying the interaction between the vesiclesand is ∼1 in dilute systems as in this study. Further, in the SFFmodel,25,26 the form factorF(q), which is the Fourier transformofthe SLD for spherical shells such as vesicles, is given by21

FðqÞ ¼ 4π

q

Z ¥

0

½FðrÞ-Fm� sinðqrÞdr ð3Þ

where F(r) and Fm are the SLD of the membrane at any radius rand that of the medium, respectively. The form factor for thevesicle of radius R and bilayer thickness d is written as

Fðq,RÞ ¼ 4π

Z d=2

-d=2

½FðxÞ-Fm�sin½qðRþxÞ�ðRþxÞq ðRþxÞ2dx ð4Þ

where x = 0 defines the middle of the bilayer, F(x) is themembrane SLD within -d/2 < x < d/2. When R . d/2, R þ x≈ R, and the above equation becomes

Fðq,RÞ ¼ 4πR2 sinðqRÞqR

Z d=2

-d=2

½FðxÞ-Fm� cosðqxÞdx ð5Þ

This approximation is valid asR∼ 50 nm and d∼ 5 nm. Thus theSFF model gives the form factors for vesicle and the membraneseparately as

FvesðRÞ ¼ 4πR2sinðqRÞqR

ð6Þ

and

FMðqÞ ¼Z d=2

-d=2

½FðxÞ-Fm� cosðqxÞdx ð7Þ

The number density of scatterers (or vesicles) is given byN= C/NDMPC(R,d) (eq 2) withC being the number of DMPCmolecules

per cubic centimeter, and NDMPC(R,d) being the number ofDMPC molecules in a single vesicle of radius R and membranethickness d. A 1wt%dispersionofDMPC inD2O corresponds toC ≈ 16.2 � 10-3 M ≈ 9.8 � 1018 DMPC molecules/cm3. Thevolume of the lipid membrane in a vesicle of mean radius R andthickness d is estimated as

VðR, dÞ ¼ ð4π=3Þ½ðRþ d=2Þ3 -ðR-d=2Þ3� ð8Þwhich further indicates that

NDMPCðR, dÞ ¼ VðR, dÞ=VDMPC ð9ÞThe volume of a DMPC molecule (VDMPC) is estimated as∼1101 A3, and those of the lipid chain and headgroup areestimated as 782 A3 and 319 A3, respectively.27 For the dataanalysis, these volumes are assumed to be constant at all pHvalues and drug concentrations studied since the volume changebetween the fluid and gel phase of the lipid is much less than10%.27

The membrane form factor FM(q) was modeled using a three-shell model, one each for headgroups of two monolayers withthickness dH and one shell for the bilayer hydrophobic core ofthickness 2dC. Within each region, the SLD was assumed to beconstant. The SLD and the resulting membrane form factor forthis case are given by

FH when -dC -dH < x < -dCFC when -dC < x < dCFH when dC < x < ðdC þ dHÞFw when jxj > ðdC þ dHÞ

8>><>>: ð10Þ

and

FMðqÞ ¼ 2ðFC -FHÞsinðqdCÞ

qþ 2ðFC -FwÞ

sinðqðdC þ dHÞÞq

ð11Þ

Data Fitting. A Gaussian distribution was assumed for boththe vesicle size and the instrumental resolution smearing in theSANS data fitting. The final expression for I(q) is

IðqÞ ¼ N

Z Rþ 3σR

R-3σR

Z qþ 3σq

q-3σq

jFvesðq,RÞj2jFMðqÞj2 1

σR

ffiffiffiffiffiffi2π

p exp

-1

2

R-Rav

σR

� �2" #

1

σq

ffiffiffiffiffiffi2π

p exp -1

2

q-q

σq

!224

35dRdqþ Ibkg

ð12Þwhere q and σq are the expected value and associated error in themeasurement of q directly obtained fromSANS experiments. Thefit parameters are the average vesicle radius (Rav), vesicle poly-dispersity (p = σR/Rav), membrane headgroup thickness (dH),membrane tail thickness (dC), the bilayer headgroup SLD (FH),and the incoherent background intensity (Ibkg) that is independentof q. The data used for fitting was first background-subtracted byestimating the background from the slope of a straight line fittedto Iq4 versus q4 of the high-q data (q>0.3). However, in order toaccount for the residual noise in the data due to background, theparameter Ibkg was used. Further, the six free parameters can bereduced to five by using the constraint on the headgroup areaproposed by Kucerka et al.28 The SLD for the headgroup regionand tail region are given as FH= (bHþNwbw)/(VHþNwVw) andFC = bC/VC, respectively, with bi and Vi being the scatteringlength and molecular volume of the respective components. TheparameterNw is the number ofwatermolecules per lipidmolecule

(24) Press, W. H. Numerical Recipes: The Art of Scientific Computing, 3rd ed.;Cambridge University Press: New York, 2007.(25) Kiselev, M. A.; Zemlyanaya, E. V.; Aswal, V. K.; Neubert, R. H. H. Eur.

Biophys. J. 2006, 35, 477–493.(26) Kiselev, M. A.; Lesieur, P.; Kisselev, A. M.; Lombardo, D.; Aksenov, V. L.

Appl. Phys. A: Mater. Sci. Process. 2002, 74, S1654–S1656.

(27) Nagle, J. F.; Tristram-Nagle, S. Biochim. Biophys. Acta, Biomembr. 2000,1469, 159–195.

(28) Ku�cerka, N.; Nagle, J. F.; Feller, S. E.; Balgav�y, P. Phys. Rev. E 2004, 69,51903.

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Boggara and Krishnamoorti Article

assumed to be uniformly distributed exclusively in the headgroupregion. However, the headgroup area per lipid can be obtainedeither by the parameters of the headgroup region or from that oftail region as AL = (VH þ NwVw)/dH = VC/dC, which acts as aconstraint between the parameters dH, dC, and Nw and hencereduces the total free parameters to five (Rav, p, dH, Nw, Ibkg). Inthe case of vesicles with drug, the drug affects the SLD values forboth chains as well as the headgroup, and thus

FH ¼ bH þNwbw þð1-xdrugÞ � dlratio�bdrug

VH þNwVw þð1-xdrugÞ � dlratio�Vdrugð13Þ

FC ¼ bC þxdrug � dlratio�bdrug

VC þxdrug � dlratio�Vdrugð14Þ

and

AL ¼ VH þNwVw þð1-xdrugÞ � dlratio�Vdrug

dH

¼ VC þxdrug � dlratio�Vdrug

dCð15Þ

where xdrug and dlratio are the fraction of drug in the chains andthe number of drug molecules per lipid molecule, respectively.bdrug and Vdrug are the scattering length and molecular volume ofthe drug. On the basis of the molecular dynamics (MD) simula-tions containing dipalmitoylphosphatidylcholine (DPPC) lipidand ibuprofen, Vdrug was estimated to be ∼256.8 A3.

Results

Before describing the structural characterization of the lipidbilayer membranes and bilayers prepared with lipid-ibuprofenadducts at a fixed temperature of 30 �C, we examine the thermalbehavior to ascertain the physical state of the membranes at thetemperature of interest as a function of pH. Specifically, we focuson the melting temperature of the acyl chains of the phospho-lipids, as it would be expected that below themelting temperaturethe chains would adopt an all-trans conformation, while abovethe transition temperature the chains would adopt amore flexibleconformation.Effect of pH and NSAIDs on Membrane Melting Tem-

perature: DSC. The change in the peak temperature (Tm) of themain lipid gel-fluid phase transition as a function of pH is shownin Figure 1 for DMPC (∼15 mM) and the ibuprofen-DMPCsystem (∼1/15 mol/mol). The DSC results show that the intro-duction of ibuprofen lowers the bilayer Tm at all pH values, andsuggests that the drug partitions into the bilayer at all pH valuesor charge states of the drug. We note that the pKa of ibuprofen is∼4.6, 29 and therefore ibuprofen exists in its neutral form below apH of 4.6 and in its anionic form above a pH of 4.6.

While the above demonstrate that ibuprofen affects the lipidTm at all pH values, an important feature to note in Figure 1 isthat pH itself has a strong effect on the bilayerTm. The bilayerTm

increases by ∼8-12 �C at low pH (<3) while remaining nearlyinsensitive to the change in pH (>3) for both the systems. At lowpH, the headgroup phosphate (pKa ∼ 1.530) is partially proto-nated, leading to the headgroup with net positive charge asso-ciatedwith the choline group. It has been shownusing short-chainlipids that, at low pH, the headgroup moieties tend to repel eachother as a result of the protonation.31 However, the favorable

interactions between water and the phosphate group along withincreased hydrogen bonding between phosphate groups ofadjacent lipids tend to lower the repulsive interactions andprovide stronger attractive interactions between headgroups,thereby finally inducing a gel phase in the lipid. The addition ofthe drug, at low pH, results in similar trends as that observed forthe pureDMPC, and thus suggests that the drugdoes not alter theheadgroup structure significantly.Nevertheless, atT=30 �C, thetemperature at which all the structural characterization wasperformed, at low pH the acyl chains are crystalline, irrespectiveof the addition of the drug, and at high pH the acyl chains are intheir liquid-like disordered state.SANS Characterization: Vesicle Size and Polydispersity

Using the SFF Model. For all the samples studied, namelyDMPCand ibuprofen-DMPC (0/1 to 0.62/1mol/mol) over a pHrange from 2 to 8, it was observed that the form factors in theSANS data corresponded to that of spherical vesicles irrespectiveof the pH or the presence of drug. This indicates that vesicles areformed over a wide range of pHbothwith andwithout drug usingthe membrane extrusionmethod. The fits to the SANS data usingthe SFF model26 for the DMPC and ibuprofen-DMPC (0.31/1 mol/mol) are shown for three pH values, viz. ∼2, 5, and 8, inFigure 2, and the structural parameters are summarized in Table1. In the case of pure DMPC, the vesicle radius is∼70 nm at lowpH and ∼52 nm for pH greater than 3, while for the ibupro-fen-DMPC adducts the vesicle radius is∼54 nm at all pH values(Table 1), which correspond well with the pore size of thepolycarbonate membrane used in the extrusion. At low pH, sincethe lipid bilayer is gel-like, the membrane fluctuations areexpected to be reduced and result in larger vesicle radius with alower polydispersity of sizes. At higher pH values, the vesicle sizeis roughly that of the pore size of the membrane filter andsignificantly more polydisperse and thought to occur because ofthe more fluid-like nature of the bilayer under those conditions.This is further supported by the fact that the vesicle size ismaintained close to ∼54 nm at all pH values in the case ofibuprofen-DMPC adducts. As discussed in the previous section,the drugmakes the bilayer more fluid-like at low pH, leading to asofter and more fluctuating membrane as evidenced by the lowervesicle size than the pure lipid. At higher pH values, the drug hasminimal effect on the vesicle size of already fluid-like pureDMPCbilayer. From the above analysis on the vesicle radius and

Figure 1. DSC-based melting transition temperature for the vesi-cles preparedwithDMPC(1wt%), demonstrating the effect of pHas well as ibuprofen on the peak temperature Tm of DMPCmembrane gel-fluid main phase transition. Measurements werecarried out at a heating rate of 5 �C/min. Above pH ∼ 1.5, thetypical errors were ∼0.3 �C and ∼0.4 �C in DMPC and ibupro-fen-DMPC systems, respectively, with a maximum error of∼0.9 �C in both cases. At pH ∼ 1.5, the error was the highest and∼4 in both cases.

(29) Barbato, F.; La Rotonda, M. I.; Quaglia, F. J. Pharm. Sci. 1997, 86, 225–229.(30) Tocanne, J. F.; Teissi�e, J. Biochim. Biophys. Acta, Rev. Biomembr. 1990,

1031, 111–142.(31) Furuike, S.; Levadny, V. G.; Li, S. J.; Yamazaki, M. Biophys. J. 1999, 77,

2015–2023.

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polydispersity as a function of pH and introduction of the drug, itcan be pointedwith caution that the dispersion is stable within thetime scales of the experiments (experiments were performedwithin 4-5 h of vesicle preparations) and the range of parametersstudied such as pH (∼2 to 8) and drug-to-lipid ratio (from 0/1 to0.62/1 mol/mol). Long-term stability of these dispersions, how-ever, needs to be addressed before using lipid-NSAIDadducts asdrug-delivery systems.SANS Characterization: Bilayer Structure. Model-

Independent Evaluation of Bilayer Thickness Using MKP

Analysis. The MKP method provides a direct probe of thebilayer thickness in a model-independent manner. Specificallyon the basis of previous X-ray work,32 the bilayer thicknessobtained from the MKP method is consistent with the thicknessof the hydrophobic region of the bilayer. The bilayer thickness asa function of pH for DMPC in D2O and ibuprofen-DMPCadducts inD2O, evaluated by the application of theMKPmethodis shown in the Figure 3 and tabulated in Table 2. Two keyfeatures are to be noted: (1) the pure DMPC bilayer is relativelythicker at low pH (∼3.6 nm) compared to the values at higher pHvalues (∼ 3.4 nm), and (2) the bilayer thickness of ibupro-fen-DMPC is nearly constant (∼3.4 nm) and shows, at thelowest pH values studied, a slight increase.

The pure DMPC vesicles at low pH are more gel-like (at T =30 �C, the temperature at which SANS measurements werecarried out) as a result of the rise in Tm and consequent thickerand more ordered acyl chains composing the hydrophobic core.However, at higher values of pH, where the Tm is lower than themeasurement temperature, the bilayer chains are fluid-like, lead-ing to a thinner bilayer. On the other hand, the bilayer thicknesstrends of the ibuprofen-DMPC adducts can be explained basedon the MD simulations33 that demonstrated that the druglocates close to the headgroup when charged (i.e., at high pH

Figure 2. SANS data shown at three different pH values (∼2, 5, and 8) for (a) pure DMPC and (b) ibuprofen-DMPC∼ 0.31/1 mol/mol atT=30 �C. Solid lines are fits to the data using the SFFmodel (seeMaterials andMethods). The data at pH∼2, 5, and 8 were displaced fromeach other by multiplying the original data by 1, 5, and 25, respectively.

Table 1. Fitted Parameters for the Vesicle Radius in Nanometers

(Polydispersity) as a Function of pH for Pure DMPC and Ibupro-

fen-DMPC (∼0.31/1 mol/mol) Systems

vesicle size (R) in nanometers (polydispersity, p)

pH DMPC ibuprofen-DMPC (∼0.31/1 mol/mol)

2 69.0 ( 0.4 (0.2) 52.5 ( 0.4 (0.2)3 61.5 ( 0.3 (0.2) 58.1 ( 2.9 (0.3)5 53.4 ( 0.6 (0.2) 54.7 ( 1.3 (0.3)6 53.0 ( 1.1 (0.3) 48.8 ( 2.6 (0.3)7 51.1 ( 0.9 (0.3) 55.6 ( 3.4 (0.3)8 51.9 ( 1.0 (0.3) 54.4 ( 4.8(0.3)

Figure 3. Model-independent evaluation of the bilayer thickness basedon theMKPmethod. (a) Peakq-valueobtained from theplot of Iq4 vsq gives bilayer thickness (Shown for pure DMPC sample at pH ∼ 2 as an example. For other pH values, see Figure S2 in the SupportingInformation). (b) The bilayer thickness as a function of pH shows for pure DMPC that the bilayer is thicker below pH∼ 3 in comparison tothat above pH ∼ 3 and stays insensitive to pH above 3. The bilayer thickness in the presence of drug stays nearly constant at all pHvalues.

(32) Uhrıkov�a,D.; Ku�cerka,N.; Islamov, A.; Kuklin, A.; Gordeliy, V.; Balgav�y,P. Biochim. Biophys. Acta, Biomembr. 2003, 1611, 31–34.

(33) Boggara, M. B.; Krishnamoorti, R. Biophys. J., in press; DOI:10.1016/j.bpj.2009.10.046.

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with pH>drugpKa) and locates deeper into the acyl chainswhenneutral (i.e., at low pH with pH < drug pKa).

Probing Bilayer Structure with D54-DMPC Using theThree-Shell Model. Detailed modeling of the bilayer structurewas performed using a three-shell model with uniform SLD foreach shell. Specific bilayer features at extreme pH values (2 and 8)for pure DMPC and ibuprofen-DMPC (0.31/1 mol/mol)adducts were probed using D54-DMPC (Figure 4), with thebilayer structure studied systematically at all pH values usingDMPC (next section). For the case of D54-DMPC in D2O, thescattering lengths of the acyl chains and the solvent are virtuallymatched and the only species with a significantly differentscattering length is the headgroup of the phospholipid (Figure 5).Further, since the overall contrast between the phospholipid(treated as a single entity) and the solvent is small, the low-qscattering intensity for the vesicle dispersions in D2O is low. Onthe other hand, because of the significant scattering contrastbetween the headgroup and the solvent as well as the acyl chain,the bilayer structure is clearly observed in the high-q scattering.The mid- and high-q scattering data for the DMPC is shown inFigure 4a (and the fitted parameters in Table 3), and those for theD54-DMPC based systems are shown in Figure 4 (b and c).Clearly, for the case of D54-DMPC based systems, the mid-qpeaks corresponding to that of the bilayer thickness are shifted tohigher q values in the presence of the drug at both pH values of2 and 8. This along with the increased q-range between the firstand second minima in the SANS data indicate that the bilayerthickness (headgroup-headgroup distance) decreases in the pre-sence of the drug. Further, the D54-DMPC results unambi-guously show that the drug locates in the bilayer at all pH values,consistent with the calorimetry results.

Probing Bilayer Structure with DMPC Adducts Using theThree-Shell Model. The bilayer structure was characterized bystudying vesicular dispersions of DMPC in D2O and quantifiedby the headgroup thickness (dH), tail thickness (dC), headgrouparea per lipid (AL), and the number of waters per lipid molecule(Nw). These parameters are tabulated in Table S1 (SupportingInformation) and the most interesting ones, namely, bilayerthickness and the headgroup area/lipid, are shown in Figure 6.From these we note that (a) the headgroup thickness decreases bynearly ∼0.4 nm as a result of the presence of ibuprofen at all pHvalues; (b) the tail thickness is comparable for DMPC andibuprofen-DMPC adducts, with the values at low pH beingsomewhat thicker than those at high pH; (c) the bilayer thickness(2dC þ 2dH) for the ibuprofen-DMPC adducts are decreasedcompared to that of the pure DMPC at all pH values; and (d) theheadgroup area/lipid in the case of pure DMPC is reduced at lowvalues of pH. These are consistent with the gel-like nature of thepure DMPC at 30 �C at low values of pH.27 On the other hand,the relatively unchanged value of the headgroup area/lipid for theibuprofen-DMPC adducts (except for the lower values of pH)indicates that the ibuprofen, either by localization near the head-group or by causing defects in the long-range packing of the acylchains, results in headgroup area values that are higher than those

of pure DMPC at low pH and characteristic of fluid-like bilayersat higher pH values.

In order to understand the effect of drug on the headgrouphydration, the number of water molecules per lipid (Nw) for thepure DMPC was first estimated and found to be in the range of5-10 water molecules per lipid, reasonably close to the value of7.2 predicted from X-ray data.27 On the basis of previous MDsimulations, 33 ibuprofen is predicted to be primarily located inthe bilayer region occupied by the acyl chains, irrespective of its

Table 2. Bilayer Thickness (nm) Evaluated from the MKP Method

bilayer thickness (2dH þ 2dC) in nanometers

pH DMPC ibuprofen-DMPC (∼0.31/1 mol/mol)

2 3.6 ( 0.2 3.4 ( 0.13 3.5 ( 0.1 3.4 ( 0.15 3.4 ( 0.1 3.4 ( 0.16 3.3 ( 0.1 3.4 ( 0.17 3.4 ( 0.1 3.4 ( 0.18 3.4 ( 0.1 3.4 ( 0.1

Figure 4. SANS data at intermediate-q to high-q for (a) DMPC(pH∼2 and 8) and for chain-deuteratedD54-DMPCat (b) pH∼ 2and (c) pH∼ 8. Compared toDMPC, use of D54-DMPC changesthe local bilayer SLD significantly and provides sharper features athigh-q as a result of the bilayer structure, further indicatingibuprofen incorporation into and its effect on the bilayer structure.On the other hand, because of differences in SLD (see Figure 5),high-q scattering due to the bilayer structure is smeared out in thecase of DMPC. Arrows indicate how the peak positions thatcorresponds to the bilayer thickness change as a result of thepresence of ibuprofen.

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charge state. However, the specific location of the drug along thechain length depended on the charge state of the drug. SANS datais inherently of low resolution34 and is insensitive to the variationof xdrug (the fraction of drug molecules in the region correspond-ing to the acyl chain) along the bilayer chains. Thus, xdrug wasfixed at 1 (drug is entirely in chains) and only Nw was allowed tovary as a fitted parameter. The assumption that xdrug ∼ 1 isfurther supported by the D54-DMPC data where xdrug was closeto 1whenused as a fittingparameter. The data for the ibuprofen-DMPC systems indicate that the values of Nw are close to zero,

albeit with large associated errors, at almost all pHvalues studied.Although one cannot be conclusive about the specific druglocation along the bilayer region because of the low resolutionof SANS data, the above fitting results suggest that the drugaffects headgroup hydration irrespective of its location in thechains.Effect of Drug Concentration on the Vesicle and Bilayer

Structure. The effect of drug concentration on the bilayerstructure was quantified by studying vesicles with ibuprofen-to-DMPC ratios of 0/1, 0.06/1, 0.19/1, 0.31/1, and 0.62/1mol/mol atpH values of ∼2 and 8 (Figure 7). The summary of the results istabulated in Table 4, and the most notable structural parametersare plotted in Figure 8. The bilayer thickness and the headgrouparea/lipid change significantly with increasing ibuprofen in thebilayer. The bilayer thickness decreases monotonically withincreasing drug concentration. It is noted that the change inbilayer thickness at the highest drug-to-lipid ratio studied is∼0.5 nm, corresponding to a thickness change of 12-15%. Thisis significant considering the fact that the bilayer thickness playsan important role in many membrane mediated processes such asprotein folding and, more importantly, influences the membranebending modulus in a nonlinear fashion (i.e., ∼d2). The decreasein bilayer thickness is somewhat larger at a pH of ∼2, perhapsaccentuated by the loss of fluidity of the pure DMPC (partiallyprotonated at the phosphate) membrane under these conditions.Further, the main contribution of bilayer thinning came from thereduction in the thickness of the headgroup region while the tailthickness was found to be largely unaffected by the increase indrug molecule incorporation into the bilayer.

Discussion

Bilayer Structure. The combination of calorimetric measure-ments, intermediate-q neutron scattering (especially with D54-DMPC), and the overall modeling of the SANS data of theibuprofen-DMPC adducts as a function of pH and ibuprofencontent indicate that the ibuprofen is incorporated in the bilayer.The incorporation of ibuprofen occurs irrespective of the pH (andthus protonation state of the headgroup and the charge state ofthe drug) and intrinsic bilayer fluidity. Further, ibuprofen incor-poration in the lipid bilayer has profound consequences onthe bilayer properties such as acyl chain melting and bilayerthickness while maintaining the overall vesicle formation in suchsystems.

The peak temperature (Tm) of the pure DMPC bilayer gel-fluid phase transition is significantly affected by pH, with anincrease of∼8-12 �C for a pHof∼2 compared to that for a pHof∼8. A similar effect of pH on the gel-to-fluid transition tempera-ture is also observed for the ibuprofen-DMPC adducts,

Figure 5. Difference in SLD profile between bilayers made ofDMPCandD54-DMPC, as indicated at (a) pH∼ 2 and (b) pH∼ 8.

Table 3. Comparison of Fitted Parameters between DMPC and D54-DMPC Systems at pH ∼ 2 and pH ∼ 8

thickness (nm)

pH headgroup (dH) tail (dC) bilayer (2dH þ 2dC) headgroup area/lipid (AL, nm2)

Pure DMPC (D54-DMPC)

2 0.89 ( 0.04 1.45 ( 0.02 4.69 ( 0.11 0.538 ( 0.006(0.87 ( 0.12) (1.47 ( 0.04) (4.68 ( 0.20) (0.532 ( 0.015)

8 0.86 ( 0.04 1.31 ( 0.02 4.34 ( 0.11 0.599 ( 0.007(0.98 ( 0.08) (1.42 ( 0.03) (4.80 ( 0.13) (0.552 ( 0.014)

Ibuprofen-DMPC (0.31/1) (Ibuprofen-D54-DMPC (0.33/1))

2 0.54 ( 0.01 1.45 ( 0.01 3.98 ( 0.04 0.594 ( 0.006(0.55 ( 0.01) (1.50 ( 0.02) (4.12 ( 0.06) (0.577 ( 0.008)

8 0.62 ( 0.04 1.40 ( 0.02 4.03 ( 0.10 0.616 ( 0.008(0.56 ( 0.01) (1.52 ( 0.02) (4.17 ( 0.06) (0.570 ( 0.009)

(34) Rubinson, K. A.; Stanley, C.; Krueger, S. J. Appl. Crystallogr. 2008, 41,456–465.

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although, at any given pH value, the Tm is somewhat suppressedfor the ibuprofen-DMPC adduct as compared to the pureDMPC case. At low pH, the partially protonated phosphategroup results in headgroup moieties with a slight net positivecharge that could cause the headgroups to repel each other. Onthe other hand, the attractive interactions between the phosphategroup and water molecules and the H-bonding interactionsbetween the phosphate groups of adjacent lipids31 overwhelmthe slight repulsion and lead to a densification of the headgroupsand consequently a gel phase for the lipid.

The partitioning of charged and uncharged local anesthetics toDPPC bilayers has been correlated to their pH-dependent effecton the lipid phase transition temperature, i.e., the higher the

incorporation of the drug in the bilayer, the larger the change inTm.

35 Since ibuprofen lowers the transition temperature at all pHvalues, the correlation with partition coefficients suggests that theibuprofen is incorporated into the membranes, irrespective oftheir pH-dependent charge state. However, it is to be noted herethat the effect of ibuprofen on the membrane as a function of pHcannot be fully explained by pH-partition theory. For example,most organic acids such as salicylic acid (similar to anotherNSAID Aspirin) with pKa ∼ 3.0 are absorbed quickly in smallintestine after oral administration, despite being completelyionized at the pH of the intestinal tract.36

The role of pH and drug incorporated on the thickness of thelipid bilayer has been addressed by several complementarymethods using SANS measurements. For the pure DMPC, thebilayer is thicker at lowpHwhen compared to the values observedat high pHand is consistent with increase in themelting transitiontemperature at low pH values. The values of the bilayer thicknessat high pH agree quite well with the previous results ofKnoll et al.,23 Balgavy et al.,37 and Nagle et al.27 on small andlarge unilamellar vesicles of DMPC and those of Mortensen

Figure 6. Plots of fitted parameters from the SFF model as afunction of pH shown for (a) thickness values of the headgroup(dH) and the tail (dC), (b) bilayer thickness, i.e., twice the sumof thehead and tail thickness (2dHþ 2dC), and (c) the headgroup area perlipid (AL) (the area of tail and head are assumed to be equal).

Figure 7. SANS data shown for different drug-to-lipid ratios(shown at top left) at pH values of (a) ∼2 and (b) ∼8. The dataat ibuprofen-to-DMPC ratios of 0/1, 0.06/1, 0.19/1, 0.31/1, and0.62/1 mol/mol were displaced from each other by multiplying theoriginal data by 1, 10, 100, 1000, and 10000, respectively. Solidlines are fits to the data using the SFF model (see Materials andMethods).

(35) Hata, T.; Sakamoto, T.; Matsuki, H.; Kaneshina, S. Colloids Surf., B 2001,22, 77–84.

(36) Takagi, M.; Taki, Y.; Sakane, T.; Nadai, T.; Sezaki, H.; Oku, N.;Yamashita, S. J. Pharmacol. Exp. Ther. 1998, 285, 1175–1180.

(37) Balgav�y, P.; Dubni�ckov�a, M.; Ku�cerka, N.; Kiselev, M. A.; Yaradaikin, S.P.; Uhrıkov�a, D. Biochim. Biophys. Acta, Biomembr. 2001, 1512, 40–52.

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and co-workers38 where they examinedmultilamellar vesicles andinterpreted the data in the context of the paracrystalline model.Moreover, in the study of Mortensen and co-workers,38 theyexamined the bilayer thickness at temperatures below the phase-transition temperature of the lipid and observed a significantthickening of the bilayer, consistent with the conclusions of thelow pH experiments performed here. In this context, we note thatthe D54-DMPC system does not demonstrate a thickening ofthe bilayer at pH of ∼ 2 (T = 30 �C). We attribute this to thesignificantly lowered melting transition temperature for thedeuterated DMPC, reported previously for a pD of 7.2 to be∼17 �C as compared toDMPCwith aTm of∼23 �C, that rendersthe vesicle at pH ∼ 2 and T = 30 �C to be in the fluid state.

On the other hand, the introduction of ibuprofen to DMPCbilayers results in a systematic decrease in bilayer thickness, andthe bilayer thinning persists formole ratios of 0.62 drugmoleculesper lipid molecule. The bilayer thickness for such ibuprofen-DMPC adducts (at a fixed drug-to-lipid ratio) is largely indepen-dent of pH, except for perhaps at the very lowest values of pH.

The adducts of ibuprofen with DMPC and D54-DMPC, as aresult of thedifferences in contrast, allow for a systematic evaluationof the influence of the drug on the thickness of the headgroup andtail regions effectively. For the D54-DMPC, the SLD of the acylchain is similar to that of D2O, the dispersing solvent, and the SLDof the headgroup region is significantly different. Thus, the max-imum contrast in the case ofD54-DMPC is between the headgroupregion and water. On the other hand, for the DMPC case, the SLDof the D2O and the acyl chain are significantly different, and theheadgroup region lies between these two extremes. In this case, themaximum contrast in the case of DMPC is between the acyl chainsand the D2O. The structural details of the acyl chain packing arebetter resolved from the DMPC-D2O systems, while the head-group-to-headgroup correlations are best quantified using theD54-DMPC-D2O systems. From these we note that most of the bilayerthinning effect reported here results from a thinning of the head-group region, and a relatively unchanged tail thickness. These alongwith the increased headgroup area with increased drug incorpora-tion into the bilayer and the significant decrease in the hydration ofthe headgroup, suggests that the drug causes significant changes tothe nature of the headgroup region.

The high partition coefficient of ibuprofen in lipid membranesobserved in octanol-water partitioning measurements andfrom other experimental measures29,39,40 as well as by MD

Table 4. Fitted Parameters As a Function of Increasing Drug-to-Lipid Ratio for Two Extreme pH Values (∼2 and ∼8)

thickness (nm)

vesicle radius (R, nm) headgroup (dH) tail (dC) bilayer (2dH þ 2dC) headgroup area/lipid (AL, nm2)

ibuprofen-DMPCmol/mol pH ∼ 2 pH ∼ 8 pH ∼ 2 pH ∼ 8 pH ∼ 2 pH ∼ 8 pH ∼ 2 pH ∼ 8 pH ∼ 2 pH ∼ 8

0.00 69.0 ( 0.4 51.9 ( 1.0 0.89 ( 0.04 0.86 ( 0.04 1.45 ( 0.02 1.31 ( 0.02 4.69 ( 0.11 4.34 ( 0.11 0.538 ( 0.006 0.599 ( 0.0070.06 56.7 ( 1.5 57.1 ( 2.1 0.69 ( 0.03 0.68 ( 0.02 1.49 ( 0.01 1.38 ( 0.01 4.37 ( 0.07 4.13 ( 0.07 0.536 ( 0.003 0.577 ( 0.0040.19 55.0 ( 0.4 60.0 ( 3.1 0.59 ( 0.02 0.61 ( 0.03 1.47 ( 0.01 1.43 ( 0.02 4.12 ( 0.07 4.07 ( 0.09 0.564 ( 0.005 0.582 ( 0.0060.31 51.6 ( 0.4 54.4 ( 4.8 0.54 ( 0.01 0.62 ( 0.04 1.45 ( 0.01 1.40 ( 0.02 3.98 ( 0.04 4.03 ( 0.10 0.594 ( 0.006 0.616 ( 0.0080.62 52.0 ( 0.5 51.7 ( 5.3 0.49 ( 0.01 0.49 ( 0.04 1.44 ( 0.02 1.44 ( 0.02 3.86 ( 0.06 3.86 ( 0.07 0.653 ( 0.009 0.654 ( 0.011

Figure 8. Plots of fitted parameters from the SFF model as afunction of increasing drug-to-lipid ratio at extreme values of pH∼2 andpH∼8 shown for (a) bilayer thickness, i.e., twice the sumofthe head and tail thickness (2dHþ 2dC), (b) thickness values of theheadgroup (dH) and the tail (dC), and (c) the headgroup area perlipid (AL) (the area of tail and head are assumed to be equal).

(38) Lemmich, J.; Mortensen, K.; Ipsen, J. H.; Hoenger, T.; Bauer, R.; Mour-itsen, O. G. Phys. Rev. E 1996, 53, 5169–5180.

(39) Avdeef, A.; Box, K. J.; Comer, J. E. A.; Hibbert, C.; Tam, K. Y. Pharm.Res. 1998, 15, 209–215. Perlovich, G. L.; Kurkov, S. V.; Kinchin, A. N.; Bauer-Brandl,A. AAPS J. 2004, 6, 22–33. Pehourcq, F.; Matoga, M.; Bannwarth, B. Fund. Clin.Pharmacol. 2004, 18, 65–70.

(40) Wenkers, B. P.; Lippold, B. C. J. Pharm. Sci. 1999, 88, 1326–1331. Hadgraft,J.; Plessis, J.; Goosen, C. Int. J. Pharm. 2000, 207, 31–37. P�ehourcq, F.; Matoga, M.;Jarry, C.; Bannwarth, B. J. Liq. Chromatogr. Relat. Technol. 2001, 24, 2177–2186.

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simulations33 is consistent with the experiments describedhere, and a schematic of the hypothesized molecular originsof this incorporation are shown in Figure 9. At low pH,ibuprofen in its neutral form is more likely to interact stronglywith the alkyl chains than with the headgroup. However,because the membrane is less fluid-like and more gel-like, thedrug molecules are possibly forced closer to the headgroupregion rather than the semicrystalline acyl chain region.Moreover,the introduction of an aromatic drug molecule in the largely acylbilayer structure induces some defects in the chain region, makingthe overall bilayer somewhat more fluid-like. On the other hand,at high pH, ibuprofen in anionic form interacts weakly with thechains as indicated by its partition coefficient29,39 and MD

simulations.33 The drug, however, interacts very strongly withtheheadgroupandmanifested in the current structural studies by asignificant thinning of the headgroup.

The influence of molecules such as trehalose, sucrose, choles-terol, ergosterol, and lanosterol on the structure of lipid bilayershas beenpreviously studied.Both sucrose and trehalose have beenshown to decrease the thickness of the bilayer and to slightlyincrease the melting transition of the DMPC vesicle.41 On the

Figure 9. Schematic showing the hypothesis formolecular insights into the effect of pHand incorporation of ibuprofen into the lipid bilayer.

(41) Kiselev, M. A.; Zbytovska, J.; Matveev, D.; Wartewig, S.; Gapienko, I. V.;Perez, J.; Lesieur, P.; Hoell, A.; Neubert, R. Colloids Surf., A 2005, 256, 1–7.Kiselev, M. A.; Wartewig, S.; Janich, M.; Lesieur, P.; Kiselev, A. M.; Ollivon, M.;Neubert, R. Chem. Phys. Lipids 2003, 123, 31–44. Luzardo, M. C.; Amalfa, F.; Nunez,A. M.; Diaz, S.; Biondi de Lopez, A. C.; Disalvo, E. A. Biophys. J. 2000, 78, 2452–2458.

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other hand, cholesterol, ergosterol, and lanosterol increase thebilayer thickness and increase the order of the fluid bilayer.42

Our results here for the ibuprofen-DMPC adducts indicatethat, unlike the addition of sugars and sterols, the bilayer thinsas a result of the incorporation of the ibuprofen and alsoresults in a lowering of the lipid melting transition tempera-ture. The differences with the sterols can be reconciled bynoting that we observe a decrease in the headgroup hydrationdue to the incorporation of the ibuprofen, whereas with theincorporation of the sterols the headgroup hydration signifi-cantly increases.Colloidal Stability of Vesicle Dispersion. One indirect

consequence of this study is the colloidal stability of vesicledispersion in the range of pH studied (from 2 to 8) and the drug-to-lipid ratios of 0/1 to 0.62/1 mol/mol. Fusion of vesicles, oftenmediated by proteins, is frequently observed in biological systems(e.g., intracellular organelles). However, pH (acidic or basic) playsa significant role in many of these cases.43-46 This makes studyingpH-dependent liposome stability essential to ascertain the use ofliposomes as drug-delivery systems especially into the cytoplasm.45

The change in pH can facilitate fusion either by changingsurface charge density or through the hexagonal II phase.47 Insome fusion processes, absolute requirement of negatively chargedlipids in the bilayer strongly suggests the electrostatic nature of thefusion process.46 For example, chitosan (a gene carrier, bioadhe-sive, and permeabilizer) used as membrane perturbant in drugdelivery is shown to induce fusion of individual multilamellarvesicles. Although biocompatible and biodegradable, pH andsubsequent electrostatic interactions play a critical role in thedegree of membrane destabilization behavior of chitosan (pKa ∼6.5).43

In the entire range of pH conditions as well as the drug-to-lipidratios studied, no vesicle fusion was observed as indicated by thevesicle radii values and SFF fits. Vesicle form factor seems to beintact under all the conditions. While there is a slight increase inpolydispersity from about 20% to about 30%with increasing pHfor both pureDMPCvesicles and ibuprofen-DMPCvesicles, thedrug does not seem to significantly alter the polydispersity of thevesicles. Although, the surface charge density certainly changeswith pH, the effect is restricted to changing bilayer structure andnot on the vesicle size.

Concluding Remarks

Understanding the effect of the incorporation of NSAIDsand changes in pH on the bilayer properties and vesicle stabilityare important aspects of the vesicle-based drug-delivery systemfor NSAIDs. Since the pKa of ibuprofen is ∼4.6, in the middleof the physiologically relevant pH range of 2 to 8, a systematicstudy of pure DMPC and ibuprofen-DMPC adducts (DMPCvesicles preassociated with NSAIDs) was performed as afunction of both pH and drug-to-lipid ratios of 0/1 to0.62/1 mol/mol. An SFF model22,25,26 was successfully used

to fit the SANS data from such SUVs. Vesicle dispersions ofpure DMPC and ibuprofen-DMPCmixtures were found to bestable at all pH values and in the entire range of drug-to-lipidratios studied. This suggests that PC-NSAID adducts havethe potential of being very good candidates for drug deliveryof NSAIDs subject to in vivo stability and specific inter-actions between NSAIDs and other targeting molecules onthe bilayer.

The addition of ibuprofen to DMPC-based vesicles has sig-nificant effects on the local bilayer structure. Using SANS,ibuprofen was shown to cause significant thinning of the head-group region, by 12-15% at all pH values, and consequentlyresult in an overall thinning of the bilayer. The presence of drugmolecules in the bilayer seems to exclude the water from theheadgroup region, suggesting strong interactions for drug-lipidcompared to lipid-water interactions at all pH values leading tothe headgroup thinning.

The form factor of all the systems under all the conditions(temperature of 30 �C, overall lipid content of 1 wt%, a pH rangefrom∼2 to∼8, and amaximumdrug-to-lipid ratio of 0.62/1 mol/mol) studied corresponded to that of unilamellar vesicles. Never-theless, at higher ibuprofen-to-lipid ratios or, for the case ofmuchbigger drugs such as Celebrex being added to the lipid orextending to pH values, where the phosphate group is completelyprotonated or where the choline pKa is exceeded, morphologicaltransition to bicelles or other complex structures might occur andneed to be investigated separately.

We anticipate that the pH as well as the addition of drug canaffect the mechanical properties of the membrane by either(1) inducing change in the local bending modulus of the mem-brane to accommodate the drug or (2) affecting the lipid head-group area expansion modulus. The bending modulus (κ) is astrong function of the bilayer thickness and scales roughlyas ∼d2.48 Since the addition of ibuprofen results in a thinning ofthe bilayer, we anticipate that the bending modulus of thedrug-lipid adduct vesicle would be significantly reduced. Onthe other hand, the overall bendingmodulus of themembranewilldependonother competing effects such as changes in hydration ofthe headgroup, the lipid packing in the bilayer, and the introduc-tion of defects in the bilayer structure. This is the focus of ourwork49 that examines the dynamics of DMPC and ibupro-fen-DMPC-based vesicles as a function of pH using quasielasticneutron scattering.

With regards to the lipid headgroup area expansion modulus,since the headgroup area of the pure DMPC lipid membranedecreases at a pH value of∼2, it suggests that the area expansionmodulus of the membrane is affected. Zhou and Raphael15

observed that, in case of 1-stearoyl-2-oleoyl-phosphatidyl-choline (SOPC) lipid membranes, the elastic area compressibilitymodulus is unaffected from pH ∼3 to 9 but is reduced by ∼30%at pH∼ 2, while the apparent compressibility modulus (measureof fraction area change vs membrane tension) drops by ∼35%at pH ∼ 2.

Acknowledgment. We would like to acknowledge the partialfinancial support of the Texas Higher Education CoordinatingBoard via the Advanced Technology Program and the National

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DOI: 10.1021/la903854s 5745Langmuir 2010, 26(8), 5734–5745

Boggara and Krishnamoorti Article

Science Foundation (CMMI-0708096). We thank the NISTCenter for Neutron Research (NCNR) for neutron beam timeand acknowledge that thiswork utilized facilities supported by theNSF under Agreement No. DMR-0454672.

Supporting Information Available: Details of fitting meth-od, additional figures of data fits, and a table of all the fittedparameters. This material is available free of charge via theInternet at http://pubs.acs.org.