Thermodynamics of Indomethacin Adsorption to Phospholipid

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Thermodynamics of Indomethacin Adsorption to Phospholipid Membranes Amanda D Fearon, and Grace Y. Stokes J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.7b08359 • Publication Date (Web): 24 Oct 2017 Downloaded from http://pubs.acs.org on November 2, 2017

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The Journal of Physical Chemistry

Thermodynamics of Indomethacin Adsorption to Phospholipid Membranes Amanda D. Fearon and Grace Y. Stokes* Department of Chemistry and Biochemistry, Santa Clara University, 500 El Camino Real, Santa Clara, CA 95053

ABSTRACT

Using second harmonic generation (SHG), we directly monitored adsorption of indomethacin, a non-steroidal anti-inflammatory drug (NSAID), to supported lipid bilayers composed of phospholipids of varying phase, cholesterol content, and head group charge without the use of extrinsic labels at therapeutically-relevant aqueous concentrations. Indomethacin adsorbed to gel-phase lipids with a high binding affinity, suggesting that like other arylacetic acid-containing drugs, it preferentially interacts with ordered lipid domains. We discovered that adsorption of indomethacin to gel-phase phospholipids was endothermic and entropically-driven while adsorption to fluid-phase phospholipids was exothermic and enthalpically-driven. As temperature increased from 19 °C to 34 °C, binding affinities to gel-phase lipids increased by seven-fold, but relative surface concentration decreased to one-fifth of the original value. We

ACS Paragon Plus Environment

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also compared our results to the entropies reported for indomethacin adsorbed to surfactant micelles, which are used in drug delivery systems, and assert that adsorbed water molecules in the phospholipid bilayer may be buried deeper into the acyl chains and less accessible for disruption. The thermodynamic studies reported here provide mechanistic insight into indomethacin interactions with mammalian plasma membranes in the GI tract and inform studies of drug delivery, where indomethacin is commonly used as a prototypical, hydrophobic smallmolecule drug.

I. INTRODUCTION 4

3.5x10

-1

-1

Molar Extinction (M cm )

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O Cl

3.0 N

2.5

CH 3 H 3CO

2.0

OH

1.5

O

1.0 0.5 0.0 200

300 400 Wavelength (nm)

500

Figure 1. Chemical structure and UV-Vis spectrum of indomethacin in PBS buffer (pH 7.4) where dashed line is used to indicate 266 nm. To better understand the side effects of indomethacin therapy, which include gastrointestinal bleeding and ulcers, studies of nonspecific interactions between indomethacin (Figure 1), a non-steroidal anti-inflammatory drug (NSAID), and phospholipid membranes have been conducted.1-5 In vitro studies of indomethacin as a model drug have previously provided a mechanistic understanding of differing therapeutic effects of structurally diverse NSAIDs in


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vivo.6-8 Natural plasma membranes are compositionally diverse, and membrane model systems composed of synthetic lipids have been used to distinguish the contributions of variables such as acyl chain length, lipid packing density, and head group charge on drug–phospholipid interactions.9,10 However, a comprehensive study of the enthalpies, entropies, and free energies of adsorption for therapeutically-relevant concentrations of aqueous indomethacin adsorbed to phospholipid membranes has not been previously published. In the current study, we characterize the thermodynamic forces governing aqueous-phase indomethacin adsorption to phospholipids of varying charge and fluidity with a direct, label-free detection method. As synthetic phospholipids of varying compositions have been used in drug delivery platforms,11-14 molecular insights gained here may be useful for predicting pH, temperature, and lipid compositions for optimized drug loading in phospholipid-based drug delivery systems. Thermodynamic forces driving indomethacin-lipid interactions are also compared to interactions with other drugencapsulating materials, such as charged surfactants.15 Previous studies of indomethacin–lipid interactions probed phospholipids directly mixed with indomethacin (i.e., indomethacin was co-dissolved in organic solvent and incorporated into the liposomes during the preparation process).16-19 Indomethacin exhibited a significantly greater fluidizing effect on lipid bilayers when the drug was directly mixed with lipids in organic solvent prior to vesicle formation rather than following incubation with aqueous lipid solutions after vesicles were formed.18 Experiments conducted with incubated phospholipids more closely mimic drug adsorption under natural conditions and are the focus of the current studies. We aim to study phospholipids incubated with therapeutically relevant blood plasma concentrations of indomethacin (~7x10-6 M).20 Previous label-free studies utilized aqueous indomethacin concentrations ranging from 2.8x10-4 M to 1.1x10-3 M.18 At these high concentrations,


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indomethacin was reported to adsorb to the interstitial spaces between the flexible acyl chain tails.18,21,22 Lower aqueous drug concentrations (ranging from 4x10-5 M to 6x10-4 M) were accessed in studies utilizing fluorescent probes; indomethacin was thought to adsorb closer to the lipid head group.23 One concern with using extrinsic chemical labels is that the fluorophore tag may modify the structure and fluidity of the lipid bilayer24 and perturb adsorption of small organic molecules. To quantify the thermodynamic forces that drive adsorption of physiologically relevant concentrations of indomethacin incubated in aqueous solution into phospholipid membranes, a label-free approach is preferred. Second harmonic generation (SHG) is a highly sensitive surface analytical technique that does not require an extrinsic chemical tag.25-27 SHG has been previously used to directly monitor small organic molecules adsorbed at the liquid–solid28-30 and liquid–liquid interfaces.31 SHG was also used to monitor adsorption of aqueous solutions of small-molecule drugs,9,10 carbon nanotubes,32 and cationic polymers33 incubated with supported lipid bilayers (SLBs). SLBs are artificial membranes that mimic the 2-D lateral fluidity of natural mammalian plasma membranes.34-36 Due to symmetry considerations, no SHG signal is generated from dissolved indomethacin molecules isotropically distributed within bulk media. Only indomethacin molecules adsorbed to the SLB, where symmetry is necessarily broken, contribute to the SHG signal detected. Therefore, a separation step prior to detection is not required; adsorbed drug molecules can be directly quantified. SHG signal becomes resonantly enhanced when the frequency of the incident (ω) or second harmonic (2ω) light approaches the frequency of an inherent electronic transition of the molecule adsorbed at the interface.37 In the current studies, we show that resonantly enhanced SHG can be used to monitor therapeutically-relevant aqueous indomethacin concentrations adsorbed to SLBs.


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Using SHG, we quantify the binding affinity and maximum surface excess of indomethacin adsorbed to SLBs composed of single-component saturated and unsaturated zwitterionic lipids under steady-state equilibrium conditions. Adsorption of indomethacin has been shown to change the physical structure and fluidity of gel-16-18,23,38 and fluid-phase19,39,40 phosphocholine lipids, as well as gel- and fluid-phase lipids mixed with cholesterol to mimic lipid rafts.40,41 We also study fluid-phase zwitterionic lipids, doped with either cationic or anionic lipids at varying pH and ionic strength conditions to provide insight into the electrostatic forces acting in indomethacin-lipid interactions. Indomethacin has been shown to bind to phospholipids with higher binding affinities and better penetrating capabilities compared to other small molecule drugs that do not contain the arylacetic acid functional group, resulting in high gastric toxicity.18,42 The equilibrium binding constants, free energies, enthalpies and entropies of indomethacin adsorption to phospholipids reported here test our hypothesis that indomethacin’s strong lipophilic character may be attributed to the drug’s capacity to bind interfacial water and localize water molecules into the hydrophobic acyl chain region, destabilizing and decreasing the hydrophobicity of the bilayer structure.

II. EXPERIMENTAL SECTION II. A. Indomethacin solution preparation. Indomethacin (≥99%) was purchased from Sigma Aldrich and used as received. Phosphate buffered saline (PBS buffer) was prepared with 50 mM sodium phosphate (Fisher Scientific) and 100 mM sodium chloride (Alfa Aesar) using ultrapure 18 M*ohm water (ThermoScientific Barnstead Micropure). The pH of the PBS buffer was adjusted to 7.4 with sodium hydroxide (Macron) or hydrochloric acid (EMD). PBS buffers were stored at 4 °C. Due to its low aqueous solubility, a stock solution of 3x10-2 M indomethacin was


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prepared in HPLC grade 99.8% methanol (Alfa Aesar) and stored at 4 °C. Aqueous dilutions of concentrations ranging from 1x10-7 M to 1x10-3 M were prepared at the beginning of each adsorption isotherm experiment by the addition of appropriate volumes of stock indomethacin solution to PBS or acetate buffer so that final methanol content always remained below 3.5%. Acetate buffer (pH 5.0) was prepared with 50 mM sodium acetate (EM Science) and sodium chloride (183 mM) to maintain total ionic strength at 215 mM. II. B. Lipid bilayer preparation. 1,2-dioleoy-sn-glycero-3-phosphocholine (DOPC), 1,2dimyristoyl-sn-glycero-3-phosphocholine

(DMPC),

1,2-dipalmitoyl-sn-glycero-3-

phosphocholine (DPPC), 1,2-dioleoyl-sn-glycero-3-phospho-(1’-rac-glycerol) (DOPG), and 1,2dioleoyl-3-trimethylammonium-propane (chloride salt) (DOTAP) were purchased from Avanti Polar Lipids. Chloroform (HPLC grade, Burdick and Jackson) was dried over 4 Angstrom molecular sieves (Alfa Aesar) prior to use. Cholesterol (≥99%) was purchased from Sigma Aldrich and used as received. Chemical structures of lipid components used in this study are shown in Figure S1. Dried lipid films were prepared by combining the appropriate volumes of stock lipids, as received from supplier in chloroform, and mixed by vortexing. The lipid mixture solutions were evaporated under a gentle stream of nitrogen gas (ultra high purity, Matheson) and vacuum dried overnight to remove residual chloroform. Dried lipid aliquots (1 mg) were stored at -20 °C. Supported lipid bilayers (SLB) were prepared by spontaneous rupture of small unilamellar vesicles (SUVs) and vesicle fusion following established protocols.43 SUVs were prepared by reconstituting dried lipids with 2 mL PBS buffer (final lipid concentration of 0.5 mg/mL) followed by vortexing (to mix) and bath sonication for 20 minutes until solutions were clear. Saturated lipids required heating above the phase transition temperature (Tm) before depositing the lipids at room temperature.44 SUVs were injected into the flow cell (volume ~0.5


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mL) and allowed to equilibrate with the silica prism for 20 minutes. At least 10 mL of PBS buffer was injected into the flow cell to remove unbound lipids. Fluorescence microscopy was used to ensure uniform SLBs composed of DOPC, DMPC and DPPC were prepared with this protocol (Supporting Information). II. B. Flow cell and silica prism cleaning procedures. All components of the main flow cell body and silica prism, injection ports and o-rings were cleaned in piranha etch, a 70:30 v:v solution of 18 M sulfuric acid (VWR) and 30% hydrogen peroxide (VWR) overnight. (CAUTION: This solution is a strong oxidant and reacts violently with organic solvents. Extreme caution must be taken when handling this solution). Prior to each experiment, the silica prism and flow cell were removed from piranha etch and rinsed with copious amounts of ultrapure water. Silica prisms were plasma cleaned (Harrick Scientific) for 3 minutes immediately before they were mounted to the flow cell. II. C. Isotherm collection procedure. To ensure that indomethacin concentrations in the bulk phase above the lipid bilayer were not depleted by adsorption to lipids, at least five 10 mL injections of the same indomethacin solution was introduced into the flow cell at concentrations below 1x10-4 M and equilibrated with the lipid bilayer for a minimum of 25 minutes. At the three highest concentrations (at or above 2x10-4 M), two 5 mL injection of indomethacin were equilibrated with the lipid bilayer for ten minutes. These equilibration times were sufficient to detect a steady-state SHG response. Unless otherwise indicated, temperatures inside the flow cell were maintained at 19 °C. II.D. SHG experimental setup. A Type 1 (SD-1) doubling crystal was used to generate 532 nm light from a Q-switched Nd:YAG laser with a 7 ns pulse width at a repetition rate of 10 Hz


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(Surelite III-EX, Continuum). The 532 nm beam was directed through a series of polarizers, waveplates and lenses to obtain a collimated beam with a final diameter of 4 mm and intensity of 20 mJ/pulse. This light was directed onto the surface of a fused silica prism (Almaz Optics, UVgrade SiO2, KU-1) at an angle of 67° under total internal reflection and steered onto the surface of a high energy Nd:YAG mirror (Thorlabs, KB1-K12). The reflected beam was spatially overlapped with the incident laser light to generate a second harmonic signal perpendicular to the silica surface. SHG signal was detected using a photomultiplier tube (Hamamatsu R7154) after passage through single-band bandpass filters (Semrock, FF01-260 and LL01-266). A schematic of the counter-propagating SHG setup (Figure S2) and a diagram and detailed description of the flow cell setup are found in the Supporting Information. II. E. Normalization procedure. Prior to collecting SHG intensities for normalization, we injected 10 mL of methanol into the flow cell and waited 15 minutes to ensure removal of the lipid bilayer and all adsorbed species from the silica substrate. SHG intensities were corrected for changes in collection efficiency and day-to-day laser fluctuations by a two-point normalization method. We used the SHG signal intensities observed at the end of each laser experiment from 1) a solution of 0.01 M potassium hydroxide (Macron) and 2) PBS buffer (pH 7.4) adsorbed to bare silica. II. F. SHG theory. SHG theory is described in detail elsewhere.25,27,37,45 Briefly, a second harmonic photon with frequency 2ω is generated when two photons of frequency ω are spatially and temporally overlapped at an interface. SHG intensity, ISHG, is proportional to the macroscopic (2) second-order susceptibility tensor χ ijk , squared, which can be expressed as the sum of !

(2) (2) nonresonant and resonant contributions, χ NR and χ R , respectively, as shown in eq 1. ! !


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2

(2) (2) I SHG ∝ χ ijk ∝ χ NR + χ R(2) !

2

(1)

(2) As shown in eq 2, χ ijk is related to the product of N, the number of molecules adsorbed to the !

interface, and the orientational average of the molecules’ corresponding molecular hyperpolarizability tensors, βijk . (2)

!

(2) (2) χ ijk = N βijk !

(2)

In the case of resonantly-enhanced SHG, utilized here, the second order susceptibility of the resonant contribution is significantly larger than the nonresonant contribution, and can be considered the dominant contributor to!χ

(2)

. Resonance enhancement occurs when the frequency

of the incident ( ω ) or second harmonic (!2ω ) light approaches the frequency of an inherent electronic transition in an adsorbed indomethacin molecule.37

(χ )

!

(2) ijk R

∝N∑

a,b,c

a µi c a µ j b b µk c

(2hω − E

ca

)(

− iΓ ca hω − Eba − iΓ bc

)

(3)

In eq 3, h is Planck’s constant, µ is the Cartesian coordinate dipole operator, Γ is the transition line width, and the indices i,j, and k represent the output (i) and input (j,k) fields which can assume any of the three Cartesian coordinates (x,y,z).46 Subscripts a, b, and c are the initial, (2) intermediate and final states, respectively. Within the electric dipole approximation,26,47 !χ

equals zero when molecules are isotropically distributed within bulk media. However, at the


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liquid/solid interface, where symmetry is necessarily broken, oriented indomethacin molecules contribute to!χ

(2)

which may result in increased SHG intensities.

III. RESULTS AND DISCUSSION III.A. SHG allows direct, label-free detection of adsorbed indomethacin. Indomethacin is lipophilic, with a liposome–water partition coefficient (log Plip) of 3.16.48 As shown in Figure 1, aqueous solutions of indomethacin in PBS buffer exhibit strong UV transitions around 266 nm (ε=16700±200 M-1 cm-1), the SHG wavelength used in these studies, suggesting that SHG signal may be resonantly enhanced. This signal enhancement allows us to directly monitor indomethacin adsorbed to supported lipid bilayers (SLBs) at the liquid/solid interface for aqueous indomethacin concentrations as low as 1x10-6 M without the use of fluorophores. Based on previous studies of the intrinsic aqueous solubility limit of indomethacin,49,50 aqueous drug concentrations ranging from 1x10-7 M up to 1x10-3 M were prepared and studied. SHG signal intensity (ISHG) depends not only on the number of adsorbed indomethacin molecules, but also correlates with the orientation of the adsorbed molecules. Polarizationdependent SHG experiments were conducted to confirm that the orientation of adsorbed indomethacin is the same at both low (1x10-5 M) and high (1x10-4 M) aqueous drug concentrations (Supporting Information). These results suggest that variations in SHG intensities were mostly attributed to changes in number of adsorbed indomethacin molecules instead of orientation. At the ionic strength conditions used in our current studies (215 mM), no evidence of (2) optical mixing of a nonresonant χ(3) response with the resonantly enhanced !χ signal were

detected.51


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III.B. Adsorption of indomethacin to SLBs follows the Langmuir model. The Langmuir model assumes fully reversible adsorption, which was supported by SHG desorption studies (Figure S4). Binding isotherms are shown in Figure 2 and fit coefficients for the equilibrium adsorption constant, Ka, and the square root of SHG intensity at saturation, determined from a simplified form of the Langmuir equation. In eq 4,

, were

represents the

aqueous indomethacin concentration. The derivation of eq 4 assumes the non-resonant SHG signal intensity is negligible compared to the resonant contribution,27,46 as detailed in the Supporting Information.

⎛ I max K [drug]⎞ ⎟ I SHG ∝ ⎜ SHG a ⎜⎝ 1+ K a[drug] ⎟⎠ ! 3.0

2

(4)

DOPC DOPC w/CHO DMPC DPPC

2.5 SHG Intensity (a.u.)

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2.0 1.5 1.0 0.5 0.0 0.0

0.2 0.4 0.6 0.8 1.0x10-3 [indomethacin] (M)

Figure 2. SHG signal intensity observed for indomethacin in PBS buffer (pH 7.4) adsorbed to SLBs composed of DOPC (filled circles), DOPC with 30% cholesterol (DOPC w/CHO) (open triangles), DMPC (filled triangles), and DPPC (empty circles) at room temperature. Fits to the Langmuir model are shown. Data shown are averages from at least three separate adsorption experiments.


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Table 1. Langmuir fit coefficients of adsorption data of indomethacin adsorbed to DOPC, DOPC+30% cholesterol (CHO), DMPC, and DPPC in PBS buffer pH 7.4 at room temperature. DOPC + 0% CHO DOPC + 30% CHO DMPC + 0% CHO DPPC + 0% CHO

Tm (°C) -17 24 41

Kax104(M-1) 2.1±0.3 0.93±0.11 0.69±0.08 5.0±0.5

maximum s molec/cm2)

(x1013

1.84±0.08 2.13±0.02 2.39±0.12 0.91±0.04

III.C. High binding affinity of indomethacin adsorbed to gel-phase DPPC. Previous studies suggest that lipophilic drugs bind most strongly to lipids in the liquid-crystalline (or fluid) phase, and that with increasing length of acyl chain hydrocarbons, the number of voids which accommodate drug molecules decrease.52 Most small molecule drugs interact more strongly with fluid-phase DOPC or POPC lipids compared to gel-phase DPPC lipids.9,53 Therefore, indomethacin was predicted to adsorb to phospholipids with the following trend in binding affinities: DOPC > DMPC > DPPC. However, as shown in Table 1, the binding constants (Ka) obtained from the Langmuir model for indomethacin adsorbed to SLBs followed this trend: DPPC > DOPC > DMPC. Control experiments described in the Supporting Information ensured that 1) SHG signal was not generated by indomethacin adsorbed to the silica substrate via defects in the SLB and 2) that aqueous indomethacin concentrations up to 1x10-3 M did not cause removal or destruction of SLBs. The values of Ka determined for indomethacin adsorbed to DOPC and DMPC were comparable to those determined from nonlinear optical studies of small-molecule drugs such as the NSAID ibuprofen and the anesthetic tetracaine. For example, Ka for ibuprofen adsorbed to DOPC (4.37±0.33x104 M-1)54 was two times higher than Ka for indomethacin adsorbed to DOPC. Ka for indomethacin adsorbed to DMPC was nearly the same as Ka for tetracaine adsorbed to DMPC (0.53±0.07 x 104 M-1).9 However, the Ka for indomethacin adsorbed to gel-phase DPPC at


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19 °C (5.0±0.5 x104 M-1) was twenty times higher than Ka for tetracaine adsorbed to fluid-phase DPPC at 46 °C (0.24± 0.08 x 104 M-1).9 One reason for this difference may be that arylacetic acid-containing molecules such as indomethacin have been shown to “specifically target ordered domains”40 resulting in unusually strong interactions with gel-phase DPPC lipid bilayers.18 Similar behavior was observed for ibuprofen, which contains a phenylacetic acid functional group. Ibuprofen adsorbed to DOPC (18:1) with lower affinity compared to DLPC (12:0) even though DLPC has a 15 °C higher phase transition temperature (Tm).55 DLPC was thought to be the “most suitable membrane”55 composition for ibuprofen, because it matched the size and hydrophobicity of the adsorbed drug, resulting in stronger van der Waals interactions. Indomethacin has 1.7 times higher molecular weight and larger aromatic substituents (i.e., indole versus benzene) compared to ibuprofen. Thus, it is not surprising that indomethacin adsorbs with high affinity to a lipid with a 1.3 times longer acyl chain, such as DPPC (16:0). An alternative explanation for the higher affinity to DPPC is that at 19 °C, the DMPC lipid bilayer may be in the ripple phase, not in the gel phase. The ripple phase is an intermediate phase between the fluid and gel phase, where “the surface of the bilayer is rippled and presents a wave-like appearance.”56 Lipids at a temperature that is slightly below the phase transition temperature may be in this phase. Dulfer and Govers suggested that at 37 °C, a temperature which is slightly below the Tm of DPPC, DPPC was in the ripple phase. Adsorption to ripplephase lipids may involve a different mechanism compared to adsorption to gel-phase lipids. Another reason why indomethacin interactions with gel-phase DPPC lipids do not follow expected trends may be the drug’s capacity to significantly alter the physical structure of the DPPC lipid bilayer, facilitating adsorption of subsequent indomethacin molecules. Fluorescence anisotropy studies have indicated that at pH 7.4, aqueous indomethacin solutions lowered the


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phase transition temperature of DPPC liposomes by ~2 °C.23 To our knowledge, the effect of indomethacin on the phase transition temperature of pure DOPC or DMPC lipids has not been reported quantitatively.40 However, in related studies, more significant changes in phase transition temperatures are reported for DPPC compared to DMPC lipids.40,57 This trend aligns well with the data reported in Table 1, which indicate that at room temperature, indomethacin adsorbs more strongly to (and perhaps perturbs) DPPC lipids more strongly than DOPC or DMPC lipids. III.D. Maximum surface excess of indomethacin is higher than most small molecule drugs. To calibrate SHG signal intensities to absolute surface concentrations, we followed previouslyestablished methods,10,54 which utilized published liposome membrane partition coefficients48 and assumed no competition for binding sites in the linear region of the binding isotherm at low surface densities.15 Partitioning of the drug to the lipid membrane was equated with the membrane concentrations of indomethacin found in solution-phase liposomes, as detailed in the Supporting Information. As shown in Table 1, maximum surface excess for DOPC and DMPC were similar, and twice as high as for DPPC. Compared to DOPC and DPPC, higher maximum surface excess for indomethacin adsorbed to DMPC at 19 °C was observed. This result suggests that indomethacin may prefer to incorporate into the “transient defects between disordered and ordered domains” in the lipid bilayer; a similar trend was observed for dopamine agonists, which exhibited high partitioning to lipid bilayers at temperatures where gel and fluid domains coexisted.52 Maximum surface excess for indomethacin adsorbed to DOPC was two times higher than maximum surface excess for tolnaftate adsorbed to DOPC (1.00±0.03 x1013 molecules/cm2).54 Maximum surface excess values for ibuprofen, tetracaine and azithromycin adsorbed to SLBs composed of DOPC were 5, 11, 633 times lower, respectively, than maximum


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surface excess for indomethacin adsorbed to DOPC.54,58 The maximum surface excess reported here confirm that indomethacin incorporates in high number into the lipid bilayer. III.E. Indomethacin adsorption to DOPC decreases in the presence of cholesterol. Cholesterol (CHO) is found in plasma membranes of mammalian cells in concentrations as high as 40% to 90% and alters the physical structure of lipid bilayers.59-62 SLBs composed of DOPC + 30% CHO were prepared to ensure that CHO remains soluble in the lipid membrane with minimal phase segregation.63 For fluid-phase lipids such as DOPC, CHO has a condensing effect, inducing ordering of the acyl chains,64-66 and providing less interstitial space for indomethacin to intercalate. It is not surprising that in the presence of CHO, Ka for adsorption of indomethacin to DOPC decreases by half. CHO has also been shown to reduce adsorption of ibuprofen,55,67 tetracaine,9 and raloxifene10 to DOPC, DMPC, and DPPC lipids. The resulting Ka and maximum surface excess for indomethacin adsorbed to DOPC + 30% CHO are nearly identical to values reported for indomethacin adsorbed to DMPC lipids. This result aligns well with MD simulations of “severely compressed” DOPC carbon chains which resemble shorter-chain lipids when CHO was present at similar concentrations.68 III.F. Electrostatic interactions impact adsorption of charged drug. At circumneutral pH conditions found in the bloodstream, indomethacin (pKa = 4.46) has a net negative charge, but within the acidic gastrointestinal tract, a higher concentration of indomethacin is protonated and neutral in charge. Natural plasma membranes often include charged lipids in the extracellular leaflet of the bilayer.65 To better understand the impact of lipid charge on drug–lipid interactions, we prepared SLBs composed of 1) DOPC doped with 10% DOTAP (+) and 2) DOPC doped with 10% DOPG (-). At pH 7.4, with a buffer total ionic strength maintained at 215 mM, a twofold higher Ka value was determined for anionic indomethacin adsorbed to SLBs containing


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DOTAP (+) compared to DOPG (-), as shown in Figure 3A and Table 2 (yellow shaded region). Under 215 mM ionic strength conditions, lipid charge had negligible impact on maximum surface excess. However, when total ionic strength of the buffer was decreased to 115 mM using 50 mM phosphate buffer at pH 7.4 (0 mM NaCl), a 2.7x higher Ka and 1.6x higher maximum surface excess was observed for indomethacin adsorbed to cationic versus anionic lipids, as shown in Figure 3B and Table 2 (blue shaded region). Under lower ionic strength conditions where charge screening is lower, interfacial charges of lipids located in the SLB are felt more strongly by solution-phase indomethacin molecules. A linear correlation between surface charge density and the experimentally-determined indomethacin surface concentration was made following established methods69 (Supporting Information).

Table 2. Langmuir fit coefficients for adsorption data collected at room temperature of indomethacin adsorbed to pure DOPC, 90% DOPC with 10% DOTAP (+), 90% DOPC with 10% DOPG (-). indomethacin solutions in pH 7.4 buffer were prepared in two different ionic strength (with 100 mM and 0 mM NaCl) pH 7.4. At pH 5.0, 50 mM acetate was used and total ionic strength was maintained at 215 mM with NaCl. charge

Ka x104 (M-1)

maxISHG

I.S. = 215 mM 9:1 DOPC: DOTAP pure DOPC 9:1 DOPC: DOPG

+ 0 -

2.90±0.34 2.07±0.27 1.40±0.06

2.50±0.09 2.53±0.11 2.54±0.05

+ 0 -

1.79±0.18 1.59±0.11 0.67±0.06

2.31±0.08 2.11±0.05 1.49±0.06

pH 5.0

Ka x104 (M-1)

maxISHG

1.92±0.60 5.55±0.96 2.03±0.87

2.67±0.50 2.16±0.15 1.66±0.42

I.S. = 115 mM 9:1 DOPC: DOTAP pure DOPC 9:1 DOPC: DOPG


16

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SHG Intensity (a.u.)

(A)

3.0 2.5

pH 7.4 (I.S. = 215 mM) DOTAP (+) DOPC (0) DOPG (-)

2.0 1.5 1.0 0.5 0.0 0.0


(B)

3.0 2.5

0.2 0.4 0.6 0.8 1.0x10-3 [Indomethacin] (M)

pH 7.4 (I.S. = 115 mM) DOTAP (+) DOPC (0) DOPG (-)

2.0 1.5 1.0 0.5 0.0 0.0

(C)

3.0 2.5


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


0.2 0.4 0.6 0.8 1.0x10-3 [Indomethacin] (M)

pH 5.0 (I.S. = 215 mM) DOPC (0) DOTAP (+) DOPG (-)

2.0 1.5 1.0 0.5 0.0 0.0

0.2 0.4 0.6 0.8 1.0x10-3 [indomethacin] (M)

Figure 3. Adsorption data for indomethacin adsorbed to DOPC doped with 10% cationic DOTAP (blue), zwitterionic DOPC (black), and DOPC doped with 10% anionic DOPG (red) lipids where indomethacin was dissolved in A) PBS buffer at pH 7.4 (I.S. = 215 mM), B) 50 mM phosphate buffer at pH 7.4 (I.S. = 115 mM), and C) 50 mM acetate buffer at pH 5.0 (I.S. = 215 mM). Due to solubility limitations, indomethacin concentrations above 2x10-4 M were not monitored in pH 5.0 acetate buffer.


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To mimic the acidic gastrointestinal environment,17 adsorption of indomethacin to DOPC or to DOPC doped with DOTAP (+) or DOPG (-) was monitored at pH 5.0 at total ionic strength of 215 mM. Neutral small molecules are predicted to adsorb more strongly to zwitterionic lipid bilayers compared to charged small molecules.9,10,70 A higher Ka is observed for neutral indomethacin adsorbed to DOPC (0) and DOTAP (+), as shown in Figure 3C and Table 2 (green shaded region). Neutral drugs adsorb closer to the hydrophobic acyl chains and further from the polar head groups.71 Thus, it is not surprising that charge on the lipid head group has less of an impact on indomethacin adsorption at pH 5.0 than at pH 7.4. III.G. Impact of temperature on Ka depends on lipid composition.

To evaluate the

thermodynamics driving indomethacin adsorption, we determined equilibrium adsorption constants, Keq, by referencing Ka to 55.5 M water, following eq 5.

⎛ I max K [drug]/55.5M ⎞ ⎟ I SHG ∝ ⎜ SHG eq ⎜⎝ 1+(K eq[drug]/55.5M) ⎟⎠ !

2

(5)

Gibbs free energies of adsorption of indomethacin from the water phase to the lipid phase, ΔadsG (in kJ mol-1), included the energy required to form a cavity within the lipid bilayer combined with the interaction energy between the drug and lipid molecules.53 At equilibrium under standard state conditions, ΔadsG=0, and ΔadsG0 values were determined for indomethacin adsorbed to DOPC, DMPC, and DPPC (Table 3) from eq 6 where R is the ideal gas constant and T is the temperature (in Kelvin).

Δ adsG 0 = −RT lnK eq !

(6)


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Following the precedent set in previous publications33,53,58,77 and because Keq is not always measured at standard conditions, we use the term ΔadsG instead of ΔadsG0 to represent the Gibbs energies determined experimentally from eq 6. For aqueous-phase indomethacin adsorbed to lipid bilayers composed of DOPC at 25 °C, ΔadsG is 9.8 kJ/mol higher than the ΔadsG for the anesthetic tetracaine9 (ΔadsG = -23.6 kJ/mol) and 16 kJ/mol higher the plant hormone, abscisic acid72 (ΔadsG = -17.4 kJ/mol) adsorbed to DOPC. Indomethacin is about 1.4 times larger in molecular weight compared to either tetracaine or abscisic acid. The liposome–water partition coefficient (log Plip) for indomethacin is 1.5x higher than log Plip for tetracaine and nearly the same as log Plip reported for abscisic acid. As temperature increases, the relative surface concentration of indomethacin adsorbed to DOPC, DMPC or DPPC decreases. Increased indomethacin aqueous solubility with temperature (Figure S8) may lower the drug’s preferential partitioning into the hydrophobic lipid bilayer at surface saturation conditions. As shown in Table 3 and Supporting Information Figure S11, as temperature increased from 19 °C to 33 °C, Keq for indomethacin adsorbed to DPPC increased, which may be attributed to the preference of hydrophobic drugs for a more fluid environment.73 As temperature increased up to 41 °C, which is the phase transition temperature of DPPC, lipids becomes more fluid with more space available between acyl chains, allowing indomethacin to intercalate more easily and form stronger non-covalent interactions with the acyl chain hydrocarbons. At 43 °C, Keq decreases to values that are lower than those reported for DOPC or DMPC at similar temperatures, and the sign for Δ

G changes to a small positive value (0.68±0.10 kJ/mol).

Other small molecule drugs which similarly exhibit stronger interactions with DPPC with increasing temperature are also hydrophobic (including the dopamine antagonist domperidone52 and 17-β estradiol53).


19


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Page 20 of 32

Table 3. Langmuir fit coefficients (normalized for 55.5 M concentration of water, following eq 5) for indomethacin in PBS buffer (pH 7.4) adsorbed to DOPC, DMPC and DPPC at varying temperatures. (ΔadsH and TΔadsS were calculated without DPPC data at 43 °C.) DOPC: temp (°C) 19 26 31 35

Keq x105 (M-1) 17.4±2.96 6.81±0.90 4.81±1.11 3.72±0.46

Ι 1.50±0.06 1.81±0.07 1.77±0.12 1.74±0.07

Δ G (kJ/mol) -34.9±5.9 -33.4±16.6 -33.1±7.6 -32.9±4.1

Δ

-72±9

-38.3±8.8

Keq x105 (M-1) 5.7±1.3 7.6±0.9 4.9±1.1 3.0±1.3

Ι 1.63±0.11 1.66±0.06 1.46±0.11 1.48±0.23

Δ G (kJ/mol) -33.1±7.6 -33.6±4.0 -33.4±7.5 -33.2±14.4

Δ

ΤΔ

-24±11

+8.9±11.0

Keq x105 (M-1) 64.3±10.3 268±201 463±371 0.772±0.111

Ι 0.628±0.017 0.280±0.026 0.133±0.007 0.246±0.192

Δ G (kJ/mol) -38.1±6.1 -42.5±31.9 -43.6±35.0 0.68±0.10

Δ

ΤΔ

H (kJ/mol)

ΤΔ

S (kJ/mol)

DMPC: temp (°C) 19 25 33 43

H (kJ/mol)

S (kJ/mol)

DPPC: temp (°C) 19 26 34 43*

H (kJ/mol)

+69±43

S (kJ/mol)

+110±43

Keq for indomethacin adsorbed to DOPC and DMPC decreases with temperature. In the case of DOPC, a phase transition does not occur across the physiologically-relevant temperatures probed in this study and changes in surface pressure may be less significant. For fluid-phase lipids such as DOPC and DMPC, other factors may have a greater impact on binding such as a change in the conformation of the lipid head group, which may result as temperature increases. As shown in Figure 4, when temperature increases the conformation of the choline group changes. At lower temperatures, choline is oriented parallel to the lipid bilayer surface, but as temperature increases, it may move deeper into the hydrophobic core of the bilayer.73 Weaker electrostatic attractions result when this cationic functional group is buried deeper into the lipid bilayer. An example of another hydrophobic small molecule drug which exhibited composition-


20

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dependent partitioning to lipid membranes is the anti-cancer drug teniposide, which exhibited lower adsorption to DOPC and higher adsorption to DPPC with increasing temperature.73

O

O O

O O

H

P O

H 3C N

O

ΔT

CH 3

O

O O

CH 3

O O

O

O

-

-

-

+

+

+

-

-

-

+

-

H 3C N CH 3 CH 3

+

+

+

+

P O OH

indomethacin (-)

-

indomethacin (-)

-

H

higher temperature

lower temperature

+

-

-

+

+

+

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 4. Re-orientation of the amine in phosphocholine as temperature increases results in decreased electrostatic interactions between anionic indomethacin at pH 7.4 and cationic choline headgroups. III.H. Adsorption to fluid-phase lipids is exothermic and driven by enthalpy while adsorption to gel-phase lipids is endothermic and driven by increased entropy. We determined enthalpies (Δ

H) and entropies (Δ

S) of adsorption by monitoring adsorption at

varying temperatures. In Figure 5, the natural log of the Keq versus inverse temperature is plotted and a linear fit to these data following the Van’t Hoff equation (eq 7) was used to determine the enthalpy of adsorption (Δ

H) from the slope, and entropy of adsorption (Δ

S) from the y-

intercept, which are reported in Table 3.

lnK eq = − !

Δ ads H 1 Δ ads S ⋅ + R T R

(7)


21


18 17 16

ln Keq

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 32

DOPC DMPC DPPC

15 14 13 12 3.1

3.4 3.5x10-3 -1 Inverse temperature (K ) 3.2

3.3

Figure 5. Van’t Hoff plot of ln Keq (normalized to molar concentration of water) versus inverse temperature (in Kelvin-1) for DOPC (open triangles), DMPC (filled circles), and DPPC (open circles) with corresponding fits. As the ln Keq values deviated significantly in both sign and magnitude, DPPC data at 43 °C were not included in Van’t Hoff plot, but are reported in the Supporting Information (Figure S11). As indomethacin adsorbs to a lipid bilayer, a two-step process occurs: first, a cavity is formed within the lipid bilayer where the drug can intercalate, and second, non-covalent drug-lipid interactions are formed. Enthalpy of adsorption (ΔadsH) values are used to compare the relative energetics in these two steps (i.e., energy expended to form a cavity within the lipid bilayer compared to the energy released when a new interaction is established). As shown in Table 3, adsorption of indomethacin to DOPC and DMPC are associated with negative changes in enthalpy (ΔadsH=-72 and -24 kJ/mol, respectively) while adsorption to DPPC is associated with a positive change in enthalpy (ΔadsH =+69 kJ/mol). For both DOPC and DMPC, energy gained from drug–lipid interactions offsets the energy expended (ΔadsH >0) to form a cavity in the lipid bilayer.9,53 To disrupt the better-ordered acyl chains in DPPC, more energy was expended, and this energy expenditure was not completely offset by the energy gained from drug—DPPC lipid interactions.


22

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These results also suggest that more energy is released when indomethacin adsorbs to DOPC compared to DMPC due to stronger hydrophobic interactions because more space between the acyl chains in DOPC allows more indomethacin to intercalate deeper into the hydrophobic region. Kwon and co-workers also observed negative ΔadsH values for adsorption of endocrinedisrupting small molecules to fluid-phase 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) lipid vesicles and positive ΔadsH values for adsorption to gel phase DPPC lipid vesicles.53 Indomethacin adsorption to DOPC resulted in higher ΔadsH values compared to the

ΔadsH for abscisic acid to DOPC (ΔadsH = -22.6 kJ/mol)72 or the adsorption of estrone to fluidphase POPC liposomes (ΔadsH = -42.6±18.8

kJ/mol).

53

ΔadsH values we determined for

indomethacin adsorbed to DMPC at 25 °C is intermediate between ΔadsH for tetracaine adsorbed to SLBs composed of 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine, or SOPC, (ΔadsH = 31.9 ±10.5 kJ/mol) and to SOPC + 28% CHO (ΔadsH = -4.58 ±0.89 kJ/mol).9 At 25 °C, DMPC has a lipid packing density which is higher than SOPC but lower than SOPC + 28% CHO. Adsorption to fluid-phase lipids (DOPC and DMPC) was an exothermic process while adsorption to gel-phase lipids (DPPC) was an endothermic process. Differences in lipid composition and packing density also impacted the signs for entropies of adsorption (ΔadsS)—a positive ΔadsS was determined for indomethacin adsorption to DMPC and DPPC while a negative ΔadsS was determined for indomethacin adsorption to DOPC at 26 °C. Insertion of indomethacin into SLBs composed of closely-packed, gel-phase lipids result in higher entropy as the drug molecules disrupt the ordered acyl chains and reduce van der Waals forces between the lipid acyl chains.74 To explain the negative ΔadsS for indomethacin adsorption to DOPC at 26 °C, we suggest that indomethacin, which possesses a negative charge under circumneutral pH conditions, may behave like an anionic surfactant.

Adsorption of


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charged surfactants to lipid bilayers can also decrease water entropy (ΔadsS |TΔadsS | and suggests that indomethacin may behave like abscisic acid, which adsorbed to DOPC through an enthalpically-driven process.72 Similar to the results reported here, adsorption of endocrine-disrupting chemicals from the aqueous phase to saturated, gel-phase DPPC lipids was driven by increased entropy while adsorption from aqueous phase to fluid-phase lipids (DOPC and DMPC) was driven by enthalpic forces.53 Binding constants and thermodynamic data indicated that indomethacin–surfactant interactions were weaker than indomethacin–liposome interactions. Previous studies suggested that water molecules associated with phospholipid bilayers were “buried” between the hydrophobic acyl chain and the polar head group while water molecules in simple surfactant systems were located near the charged head groups with opposite orientation.76 The difference in location of adsorbed water molecules may explain differences in the energetics of drug–lipid versus drug–surfactant interactions. Adsorption of indomethacin to both cationic and anionic surfactant micelles were entropically driven and resulted in ΔadsS that were more than three times higher than ΔadsS for indomethacin adsorbed to DPPC.77 As water molecules in the phospholipid bilayer studied here may be “buried” and less accessible for disruption, a smaller increase in water entropy was expected compared to the water molecules in the surfactant system, which may be located near the surfactant/aqueous interface and can be more easily disrupted because they were more accessible. The larger ΔadsS for adsorption to charged surfactants was attributed


24

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to larger number of disrupted water molecules due to the higher surface area available on a surfactant micelle compared to a planar SLB.

CONCLUSIONS In this study, we directly monitored indomethacin adsorption under physiologicallyrelevant conditions and quantified thermodynamics of adsorption using the label-free detection method, SHG. Ka values obtained from the Langmuir model for indomethacin adsorbed to SLBs indicated that arylacetic acid-containing indomethacin preferentially interacted with ordered lipid domains, such as those found in DPPC at low indomethacin concentrations. Our studies indicated that indomethacin adsorbed to DPPC at elevated temperatures with higher binding affinities but lower relative surface concentrations. Under saturation conditions, the highest maximum surface excess was observed for indomethacin adsorbed to DMPC because indomethacin, like haloperidol, may concentrate in lipid bilayers where gel and fluid domains coexist. Our studies confirmed previous assertions that strength of drug adsorption and relative surface concentrations vary with the physical state of the membrane, as well as drug and lipid geometries. Our results suggested that indomethacin may have the capacity to bind and localize water molecules into the hydrophobic acyl chain region of gel-phase DPPC, but not fluid-phase DOPC or DMPC. This destabilization of the bilayer structure may result in higher binding affinity. Guiding principles for the design of liposomes used in drug delivery systems were gained from the equilibrium binding constants, free energies, enthalpies and entropies of indomethacin adsorption to gel- and fluid-phase phospholipids. The current studies indicate that phospholipid-


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Page 26 of 32

based drug delivery systems will exhibit the highest drug loading at temperatures where gel and fluid domains coexist, in the presence of cationic lipids, and in the absence of cholesterol. These insights will inform molecular mechanisms of drug accumulation and retention in lipid bilayers. We will build upon the current work by incorporating cyclooxygenase-2 (COX-2)78 into a lipid membrane and monitor specific interactions between indomethacin and its target membrane receptor within its native lipid environment.

ASSOCIATED CONTENT Chemical structures of the lipid components used in this study, detailed descriptions and diagrams of the SHG setup and flow cell, polarization-dependent SHG experimental details and results, derivation of simplified Langmuir model, studies of indomethacin desorption, fluorescence microscopy characterization of SLBs in the presence and absence of indomethacin, maximum surface excess and limit of detection calculations, correlations between surface charge and maximum surface excess, UV-Vis spectra and SHG adsorption isotherms of indomethacin collected at varying temperatures are available in the Supporting Information. AUTHOR INFORMATION Corresponding Author *Email: [email protected] ACKNOWLEDGMENT We thank two anonymous reviewers, who provided suggestions to improve this paper. GYS gratefully acknowledges startup funds from the College of Arts and Sciences at Santa Clara


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University. We thank Dr. Richard Bastiani for providing ADF with a Summer Research Award. GYS acknowledges funds from a Clare Boothe Luce (CBL) professor award. The authors thank fellow Stokes lab research students for help with lipid and buffer preparation and occasional data collection. Many thanks to Gary Sloan for construction of the flow cells and optical mounts used in the SHG setup and Dr. Linda Brunauer for the donation of the Lauda immersion thermostat used in this study. REFERENCES (1) Shen, T.-Y.; Winter, C. A. Chemical and biological studies on indomethacin, sulindac and their analogs. Adv. Drug Res. 1977, 12, 89-245. (2) Zhou, Y.; Plowman, S. J.; Lichtenberger, L. M.; Hancock, J. F. The anti-inflammatory drug indomethacin alters nanoclustering in synthetic and cell plasma membranes. J. Biol. Chem. 2010, 285, 35188-35195. (3) Lichtenberger, L. M. Where is the evidence that cyclooxygenase inhibition is the primary cause of nonsteroidal anti-inflammatory drug (NSAID)-induced gastrointestinal injury? Biochem. Pharmacol. 2001, 61, 631-637. (4) Yamada, T.; Deitch, E.; Specian, R. D.; Perry, M. A.; Sartor, R. B.; Grisham, M. B. Mechanisms of acute and chronic intestinal inflammation induced by indomethacin. Inflammation 1993, 17, 641-662. (5) Lichtenberger, L. M.; Romero, J. J.; Dial, E. J. Surface phospholipids in gastric injury and protection when a selective cyclooxygenase-2 inhibitor (Coxib) is used in combination with aspirin. Br. J. Pharmacol. 2007, 150, 913-919. (6) Bimbo, L. M.; Makila, E.; Raula, J.; Laaksonen, T.; Laaksonen, P.; Strommer, K.; Kauppinen, E. I.; Salonen, J.; Linder, M. B.; Hirvonen, J.; Santos, H. A. Functional hydrophobin-coating of thermally hydrocarbonized porous silicon microparticles. Biomaterials 2011, 32, 9089-9099. (7) Sarparanta, M. P.; Bimbo, L. M.; Makila, E. M.; Salonen, J. J.; Laaksonen, P. H.; Helariutta, A. M.; Linder, M. B.; Hirvonen, J. T.; Laaksonen, T. J.; Santos, H. A.; Airaksinen, A. J. The mucoadhesive and gastroretentive properties of hydrophobin-coated porous silicon nanoparticle oral drug delivery systems. Biomaterials 2012, 33, 3353-3362. (8) Orlando, B. J.; McDougle, D. R.; Lucido, M. J.; Eng, E. T.; Graham, L. A.; Schneider, C.; Stokes, D. L.; Das, A.; Malkowski, M. G. Cyclooxygenase-2 catalysis and inhibition in lipid bilayer nanodiscs. Arch. Biochem. Biophys. 2014, 546, 33-40. (9) Nguyen, T. T.; Conboy, J. C. High-throughput screening of drug–lipid membrane interactions via counter-propagating second harmonic generation imaging. Anal. Chem. 2011, 83, 5979-5988. (10) Stokes, G. Y.; Conboy, J. C. Measuring selective estrogen receptor modulator (SERM)– membrane interactions with second harmonic generation. J. Am. Chem. Soc. 2014, 136, 14091417.


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Thermodynamics of Indomethacin Adsorption to Phospholipid

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