Oxidative Damage to Biomimetic Membrane Systems: In Situ Fe(II

Article pubs.acs.org/Langmuir

Oxidative Damage to Biomimetic Membrane Systems: In Situ Fe(II)/ Ascorbate Initiated Oxidation and Incorporation of Synthetic Oxidized Phospholipids Jacqueline J. Knobloch,† Andrew R. J. Nelson,‡ Ingo Köper,§ Michael James,‡,∥ and Duncan J. McGillivray*,†,⊥ †

School of Chemical Sciences, The University of Auckland, Private Bag 92019, Auckland 1142, New Zealand Australian Nuclear Science and Technology Organisation, Locked Bag 2001, Kirrawee DC, NSW 2232, Australia § Centre for Nanoscale Science and Technology, School of Chemical and Physical Sciences, Flinders University, GPO 2100, Adelaide 5001, Australia ∥ Australian Synchrotron, 800 Blackburn Road, Clayton, Victoria 3168, Australia ⊥ MacDiarmid Institute for Advanced Materials and Nanotechnology, P.O. Box 600, Wellington 6140, New Zealand ‡

ABSTRACT: Damage to cellular membranes from oxidative stress has been implicated in aging related diseases. We report the effects of oxidative damage on the structure and properties of biomimetic phospholipid membrane systems. Two oxidation methods were used, in situ oxidation initiated using Fe(II) and ascorbate, and the incorporation of a synthetic “oxidized” phospholipid, PoxnoPC, into biomimetic membranes. The biomimetic systems employed included multibilayer stacks, tethered bilayers, and phospholipid monolayers studied using a combination of reflectometry, attenuated total reflection infrared spectroscopy, electrochemical impedance spectroscopy, and neutron diffraction. We show that oxidation with Fe(II) and ascorbate caused an increase in the order of the membrane, attributed to cross-linking of the phospholipids, and a change in the electrical permeability of the membrane, but no significant impact on the thickness or completeness of the membrane. Incorporation of PoxnoPC, on the other hand, had a larger impact on the structure of the membrane. Inversion of the aldehydeterminated truncated sn-2 chain of PoxnoPC into the head group region was observed, along with a slight decrease in the thickness and order of the membrane.

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INTRODUCTION Oxidative stress has a significant impact on cellular membranes, causing changes in structure, lipid order and mobility, electrical permeability and domain organization.1,2 Changes to these properties affect normal cellular functioning including those mediated by membrane proteins.3 Oxidative stress has also been linked to both aging and the pathologies of neurodegenerative diseases, atherosclerosis, diabetes, ischemia reperfusion injury and rheumatoid arthritis.4,5 The link between oxidative stress and disease may be the presence of oxidized phospholipids, which have been shown to play a role in inflammation and apoptosis, and which may contribute to disease.5,6 Although phospholipid oxidation has been widely reported in literature,7 the outcomes of such studies are often contradictory and the effects of oxidative stress on cellular membranes remain unclear. For example, oxidized phospholipids have been shown to both increase8 and decrease9 the lipid order in biomimetic membrane systems. This discrepancy is more than likely due to the wide range of different methods used to initiate lipid oxidation, different biomimetic membrane systems, different membrane compositions and different characterization techniques employed to examine the effect of oxidative stress on the membrane properties. Traditionally, phospholipid oxidation was initiated in situ using either a chemical oxidant or a physical method such as © 2015 American Chemical Society

UV light. This approach is widely believed to proceed via a free radical mechanism generating a large and diverse array of oxidation products.10 For this reason, in situ oxidized biomimetic membranes have a complex and poorly defined composition. This complicates the characterization of the membrane and makes it challenging to attribute changes observed in the properties of the membrane following oxidation to a single oxidized species. More recently, the effects of lipid oxidation on membrane structure and function have been studied by incorporation of a synthetic “oxidized” phospholipid into biomimetic membrane systems.11,12 This method allows the effect of a single oxidized phospholipid species to be studied in a membrane with known composition. One of the main synthetic “oxidized” phospholipid is 1-palmitoyl-2-(9′-oxo-nonanoyl)-sn-glycero-3-phosphocholine (PoxnoPC), Figure 1, which contains a truncated sn-2 hydrocarbon chain with a terminal aldehyde group. PoxnoPC is one of the main oxidation products of phosphatidylcholine lipids with a saturated sn-1 hydrocarbon chain and an unsaturated sn-2 hydrocarbon chain (the most common class of phospholipids in mammalian cells), the simplest of which is 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), Received: July 25, 2015 Revised: October 30, 2015 Published: October 30, 2015 12679

DOI: 10.1021/acs.langmuir.5b02458 Langmuir 2015, 31, 12679−12687

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volume (24 h. Rapid solvent exchange was used to complete the bilayer.17 A 10 mM ethanolic solution of phospholipid was incubated over the self-assembled monolayer for ∼15 min prior flushing through a large volume of aqueous buffer. NR data were measured on the Platypus time-of-flight reflectometer at the Bragg Institute, Australian Nuclear Science and Technology Organisation (ANSTO), Australia.18 Three incident angles, θ = 0.5°, 2°, and 6°, were used to cover a momentum transfer range 0.005 Å−1 < Qz < 0.4 Å−1 with a cold neutron wavelength spectrum (2.8 Å ≤ λ ≤ 18.0 Å). Each sample was measured under three solvent contrasts, D2O, H2O, and a mix of D2O/H2O with a neutron scattering length density (nSLD) of 3 × 10−6 Å−2 (CM3). Oxidation was achieved by exchanging the buffer for an Fe(II)/ascorbate containing buffer. After 1 h, oxidation was quenched by flushing through an excess volume of buffer to remove the purple oxidizing solution and the oxidized bilayer was measured. Data reduction and analysis were performed using MOTOFIT.19 A slab layer model describing the tethered bilayer was used to fit to the reflectivity data. Each slab layer (silicon oxide, Cr, Au, tether region, inner leaflet, outer leaflet, head groups) was described by four parameters: thickness, neutron scattering length density (nSLD), volume fraction of solvent, and interfacial roughness. The reflectivity of the model was calculated using the Abelès matrix method and refined using genetic optimization based on a minimization of χ2 as previously reported.20 Monte Carlo error analysis was used to estimate the uncertainties of the fitted parameters.21 EIS measurements were conducted using a BioLogic SP-300 potentiostat/frequency response analyzer. Spectra were measured from 1 to 105 Hz with 10 logarithmically spaced measurements per decade. A 5 mV RMS voltage was applied with 0 V bias potential against a Ag/AgCl reference electrode (eDAQ). A three electrode system was used with a platinum counter electrode and the Au coating as the working electrode. The spectra were normalized to the area of the working electrode (0.33 cm2). Data analysis was performed using ZVIEW (version 3.2, Scribner Associates, Southern Pines, NC.). Uncertainties were calculated from a least-squares minimization using calc-modulus data weighting, in which the difference between the model and the fit is divided by the value of the calculated model at each point. Samples were oxidized using the same method as for reflectometry samples. EIS spectra were measured continuously for 1 h following addition of the oxidizing solution. Neutron diffraction measurements were performed on multibilayer phospholipid stacks prepared using the “rock’n’roll” method by Tristram-Nagle et al.22 The phospholipids were dissolved in 2:1 chloroform to methanol and deposited dropwise onto a glass coverslip with a rolling motion to ensure even coverage. Oxidation using Fe(II) and ascorbate was achieved using liposomes as described for the ATR measurements. After 1 h oxidation was quenched by adding a 5-fold

Figure 1. Structures of unmodified POPC (A) and the synthetic oxidized phospholipid PoxnoPC (B).

Figure 1,13 known to be a byproduct of the oxidation of lung surfactant, and thought to be linked to the death of lung cells.14 In this study, we have characterized the physical properties and structure of biomimetic membranes that have been oxidized in situ using Fe(II) and ascorbate, and those of biomimetic membranes incorporating PoxnoPC, in comparison with unmodified membranes. A direct comparison between these two oxidation methods has not yet been reported. However, as we report herein, these two methods generate membranes with distinct physical properties. This study is important for comparing findings using one oxidation method with the other and may help toward understanding some of the previously reported contradictory results regarding the oxidation of membranes. Furthermore, as the use of synthetic oxidized phospholipids becomes more frequent due to both their availability and ability to form oxidized membranes simply, it is important to investigate whether these systems are representative of in vivo oxidized membranes. Attenuated total reflectance infrared spectroscopy (ATR), electrochemical impedance spectroscopy (EIS), neutron reflectometry (NR), and neutron diffraction (ND) have been used to probe the structure, composition, hydrocarbon chain order and motion, electrical permeability and homogeneity of biomimetic membranes. This multitechnique approach not only allows for different properties of the biomimetic systems to be characterized but also allows a direct comparison between the sensitivity of the different techniques. Multiple biomimetic membrane systems including physisorbed liposomes, tethered phospholipid bilayers, multibilayer stacks, and Langmuir monolayers have also been used to ensure that changes to the properties of the oxidized membranes are not due to inherent characteristics of either the technique or biomimetic membrane system.

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MATERIALS AND METHODS

POPC, 1-palmitoyl-d31-2-oleoyl-sn-glycero-3-phosphocholine (d31POPC), and PoxnoPC were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL) and used without further purification. The tether lipids WC14 (20-tetradecyloxy-3,6,9,12,15,18,22- heptaoxahexatricontan e -1-thiol ) a nd HC18 ( Z -20 -( Z -o cta d e c- 9 - e n yl ox y )3,6,9,12,15,18,22-heptaoxatetracont-31-ene-1-thiol) used for construction of the tethered phospholipid bilayers were the kind gift of David J. Vanderah.15,16 Iron(II) sulfate heptahydrate and (+)-sodium-Lascorbate were purchased from Sigma-Aldrich and used without further purification. For all systems, fresh Fe(II) and ascorbate solutions were prepared separately and mixed in a 1:10 ratio immediately prior to oxidation. All experiments were performed in a buffer consisting of 10 mM tris(hydroxymethyl)aminomethane) (Tris), 150 mM NaCl at pH 7.4 unless otherwise stated. ATR measurements were performed on freshly prepared 10 mM small unilamellar liposomes prepared by extrusion through a 100 nm polycarbonate membrane. Oxidation was achieved by adding a small 12680

DOI: 10.1021/acs.langmuir.5b02458 Langmuir 2015, 31, 12679−12687

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Figure 2. Hydrocarbon stretching region of the ATR spectra of POPC physisorbed liposomes. (A) POPC physisorbed liposomes before (solid black line) and 20 min (dashed green line), 40 min (dotted blue line), and 60 min (dash-dotted red line) following oxidation with 0.5 mM Fe(II)/5 mM ascorbate. (B) POPC physisorbed liposomes containing 0 (solid black line), 30 (dashed red line), and 100 mol % (dash-dotted blue line) PoxnoPC. excess of 2.5:1 chloroform to methanol and extracting the phospholipid-containing organic phase from the oxidant-containing aqueous phase.23 The volume of organic solvent was reduced before preparing the multibilayer stacks as described above. The coverslips were left under vacuum overnight to ensure complete solvent removal. Neutron diffraction data was collected on the OFFSPEC neutron reflectometer at ISIS, SFTC Rutherford Appleton Laboratory.24,25 The samples were mounted vertically in a sealed humidity chamber that was purged with argon. A relative humidity of 86% achieved using a saturated solution of KCl placed inside the chamber. Data analysis was carried out using a method previously described.26 For phase determination, each sample was measured with an aqueous KCl solution containing 0, 25, and 50 mol % D2O. The data was background subtracted and corrected for detector efficiency before being integrated across the diffraction peaks. The peak positions and intensities (integrated counts under each peak) were determined by a Gaussian fit to the one-dimensional data. The repeat spacing of the multibilayer stacks, d, was calculated as the gradient of the linear fit of the peak position, Qn, versus peak order, n.

d=

profiles were scaled to have identical nSLD values at both the center of the bilayer and in the aqueous layer between the bilayers.

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RESULTS Attenuated Total Reflectance Infrared Spectroscopy. ATR was used to examine changes in the composition and physical state of physisorbed POPC liposomes after oxidation with Fe(II) and ascorbate and physisorbed POPC liposomes containing PoxnoPC. Figure 2A shows the hydrocarbon stretching region of the ATR spectra of the physisorbed POPC liposomes before and during oxidation with 0.5 mM Fe(II)/5 mM ascorbate. The intensity of the absorbances for the CH2 symmetric (2853 cm−1) and antisymmetric (2924 cm−1) stretching bands decrease following oxidation with Fe(II) and ascorbate indicating a loss of hydrocarbon material from the physisorbed POPC liposomes on the surface of the IRE. As the spectra have been normalized to the choline antisymmetric stretching band (970 cm−1), this indicates selective loss of the hydrocarbon chains of POPC following oxidation with Fe(II) and ascorbate, not desorption of the liposomes from the IRE surface. Figure 2B shows the hydrocarbon stretching region of the ATR spectra of physisorbed POPC liposomes containing 0, 30, and 100 mol % PoxnoPC. The intensity of the absorbances for the CH2 symmetric and antisymmetric stretching bands also decreases with increasing incorporation of PoxnoPC. This caused by the truncated sn-2 hydrocarbon chain of PoxnoPC which has a smaller amount of hydrocarbon material on the surface of the germanium IRE. This is clear evidence for the formation of oxidized phospholipids with truncated hydrocarbon chains (similar to PoxnoPC) after oxidation of POPC with 0.5 mM Fe(II)/5 mM ascorbate. The rate of change in the intensity of the absorbances for the CH2 symmetric and antisymmetric stretching bands, proportional to the rate of loss of hydrocarbon from the surface, during oxidation with different concentrations of Fe(II) and ascorbate are shown in Table 1. The rate of oxidation of the physisorbed POPC liposomes oxidized using 0.5 mM Fe(II)/5 mM ascorbate and 5 mM Fe(II)/50 mM ascorbate agree within error, whereas the rate of oxidation using 50 mM Fe(II)/500 mM ascorbate is significantly higher. Shifts in the wavenumber of the CH2 symmetric and antisymmetric stretching bands were analyzed to examine changes in the order of the hydrocarbon chains following

2nπ Qn

The one-dimensional structure factor was obtained by correcting the peak intensity, In, using the Lorentz factor (sin(2θn)) and absorption correction, An

|Fn|2 = [sin(2θn)]A nIn where An is calculated using the linear absorption coefficient, μ (assumed to be wavelength independent over the range of wavelengths used), and the thickness of the multibilayer stacks, τ.

⎛ 2μτ ⎞ An = ⎜ ⎟(1 − e−2μτ /sinθn)−1 ⎝ sin θn ⎠ The phases of the structure factors were determined by plotting the structure factors with both positive and negative phases against the percentage of D2O. The correct structure factor phase was chosen by assuming that the structure factors increase as the percentage of D2O increases in the aqueous layer between the bilayers.27 Since the bilayer structure is centro-symmetric changing the aqueous layer from H2O to D2O will result in a linear increase in structure factor. The nSLD profile, ρ(x), was calculated using

⎛ 2πnz ⎞ ⎟ ρ(x) = Σn(± )|Fn|cos⎜ ⎝ d ⎠ where ± indicates the phase, d is the bilayer unit cell length, and z is the distance perpendicular to the multibilayer stacks. The nSLD 12681

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translational and rotational motions as well as the librational and torsional motions of the hydrocarbon chains.28,31 An increase in the peak width of the CH2 symmetric stretching band from 11.2(3) cm−1 for the POPC liposome to 13(1) cm−1 for the pure PoxnoPC sample indicating an increase in the motion of the hydrocarbon chains. No change in peak width was observed for the 5 mol % or 10 mol % PoxnoPC containing POPC liposomes or for the Fe(II)/ascorbate oxidized POPC liposomes. The appearance of new bands or the disappearance of existing bands after oxidation with Fe(II) and ascorbate were not observed in the fingerprint region (1800 to 900 cm−1) of the ATR spectra. The assignment of bands in this region was difficult due to subtraction of the strong O−H bending band of H2O (∼1595 cm−1) and therefore changes to the CC stretching band (∼1660 cm−1) were unable to be observed. This is not surprising as the CC stretching band is likely to be weak as POPC is a monounsaturated phospholipid. The appearance of new bands or the disappearance of existing bands with incorporation of PoxnoPC were not observed in the fingerprint region (1800−900 cm−1) of the spectra indicating that ATR was not sensitive enough after background subtraction to detect the presence or loss of the alkene group on the sn-2 hydrocarbon chain of POPC. Neutron Reflectometry. NR was used to analyze the effect of oxidative damage on the structure of tethered phospholipid bilayers. The structures of the unoxidised d31-POPC tethered bilayers (nSLD profiles shown in Figure 3 and model parameters in Table 3) both agreed with previous NR measurements.32 Oxidation of the d31-POPC tethered bilayer with 0.5 mM Fe(II) and 5 mM ascorbate resulted in an increase in the nSLD of the inner hydrocarbon chains from 1.3(1) × 10−6 Å−2 to 2.2(3) × 10−6 Å−2 and outer hydrocarbon chains from 2.6(1) × 10−6 Å−2 to 2.8(2) × 10−6 Å−2, consistent with truncation and subsequent loss of the protonated sn-2 chain as well as incorporation of oxygen containing moieties in the hydrocarbon chain. An increase in the nSLD of the hydrocarbon chains was also observed after oxidation of an h-POPC tethered bilayer (using the same oxidation conditions). This confirms that some of the increase in the nSLD observed on oxidation is due to the incorporation of oxygen containing moieties into the hydrocarbon chains, as no change in nSLD would occur on truncation of the fully protonated h-POPC. The nSLD increase for the h-POPC tethered bilayer was smaller than for the d31-POPC tethered bilayer showing that there is also hydrocarbon chain truncation. There was no change in the volume fraction of water in the hydrocarbon chains or in the thickness of the tethered bilayer following oxidation with 0.5 mM Fe(II) and 5 mM ascorbate, showing that the film remains complete within the resolution of the reflectometry. The effect of PoxnoPC incorporation on the structure of d31-POPC tethered bilayers was also measured using NR (see Figure 3 and Table 3). The amount of d31-POPC incorporated into the bilayer was calculated from the nSLD of the outer hydrocarbon chains assuming nSLDd31‑POPC= 2.7 × 10−6 Å−2 and nSLDPoxnoPC = −0.35 × 10−6 Å−2. It is possible that there is an increase (although the uncertainty is large) in the volume fraction of water in the hydrocarbon chains as the amount of PoxnoPC incorporated in the bilayer increases; 2(2)% for the normal bilayer, 4(3)% for 6 mol % PoxnoPC, and 6(2)% for the 10 mol % bilayer. A decrease in the head group nSLD was also observed as the amount of PoxnoPC in the d31-POPC tethered

Table 1. Rate of Change of Absorbance from the Initial Absorbance of the Antisymmetric and Symmetric Stretching Bands as a Percentage of the Original Intensity, after Oxidation with Different Concentrations of Fe(II) and Ascorbatea rate of % change of absorbance/min−1

a

concn of Fe(II)/ascorbate

antisymmetric stretch

symmetric stretch

0.5 mM/5 mM 5 mM/50 mM 50 mM/500 mM

−5(1) −4.0(3) −20(1)

−5.3(7) −5.0(4) −15(2)

Uncertainty in the last figure given in parentheses.

oxidation with Fe(II) and ascorbate. A shift to lower wavenumbers of the CH2 stretching bands indicates an increase in the proportion of trans conformers in the hydrocarbon chains and therefore an increase in the order of the hydrocarbon chains, whereas a shift to higher wavenumbers indicates a higher proportion of gauche conformers and therefore a decrease in order of the hydrocarbon chains.28−30 As seen in Table 2, no shift in the wavenumbers of the CH2 Table 2. Wavenumbers of the CH2 Symmetric and Antisymmetric Bands before and after Oxidation of Physisorbed POPC Liposomes with Different Concentrations of Fe(II) and Ascorbate, and Physisorbed POPC lipid Containing Different Amounts of PoxnoPCa CH2 antisymmetric Fe(II)/ascorbate oxidized POPC liposomes 0.5 mM/5 mM before oxidation 2853.4(1) after oxidation 2853.3(1) 5 mM/50 mM before oxidation 2853.5(1) after oxidation 2853.5(1) 50 mM/500 mM before oxidation 2853.4(1) after oxidation 2852.6(1) PoxnoPC incorporated POPC liposomes POPC liposome 2853.6(1) 30 mol % PoxnoPC 2853.1(1) PoxnoPC liposome 2853.9(2)

CH2 symmetric

2923.8(1) 2923.7(1) 2924.1(1) 2924.1(1) 2924.0(1) 2923.0(1) 2924.2(1) 2923.9(1) 2925.6(2)

a

Uncertainties in the last digit (one standard deviation) are reported in parentheses.

stretching bands were observed following oxidation with either 0.5 mM Fe(II)/5 mM ascorbate or 5 mM Fe(II)/50 mM ascorbate. At higher oxidant concentrations, 50 mM Fe(II)/500 mM ascorbate, a decrease in the CH2 symmetric stretching band absorbance from 2853.4(1) cm−1 to 2852.6(1) cm−1 and antisymmetric stretching band absorbance from 2924.0(1) cm−1 to 2923.0(1) cm−1 was observed, indicating an increase in the order of the hydrocarbon chains. An increase in the wavenumber of the CH2 symmetric stretching band for the 100 mol % PoxnoPC physisorbed liposomes (2925.6(2) cm−1) compared to the 0 and 30 mol % PoxnoPC incorporated POPC liposomes (2924.2(1) and 2923.9(1) cm−1 respectively) indicated a decrease in the order of the hydrocarbon chains. There was no clear trend observed for the CH2 antisymmetric stretching bands with increasing PoxnoPC. The peak widths of the CH2 stretching bands were also analyzed to determine the relative rate and amplitude of the 12682

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Figure 3. Neutron reflectivity (A) and the corresponding nSLD profiles (B) for a d31-POPC tethered bilayer before and after oxidation with 0.5 mM Fe(II) and 5 mM ascorbate. (A) Filled circle, before oxidation; *, after oxidation; solid lines, fits to the data. Data are offset for clarity. (B) Dashed lines before oxidation and solid lines after oxidation. Fitted neutron reflectivity (C), and the corresponding modeled nSLD profiles (D), for a normal d31-POPC tethered bilayer and ones incorporating 6 and 10 mol % PoxnoPC. (A) Filled circle, 6 mol % PoxnoPC; *, 10 mol % PoxnoPC (normal bilayer data not shown); solid lines, fits to the data. Data are offset for clarity. (B) Dotted line, normal bilayer, solid line, 6 mol % PoxnoPC; dashdotted line, 10 mol % PoxnoPC. In all figures D2O (blue), CM3 (green), and H2O (red). The vertical lines represent the layer boundaries of the normal bilayer.

bilayer was increased from 1.2(2) × 10−6 Å−2 for the normal bilayer, 1.0(3) × 10−6 Å−2 for the 6 mol % bilayer and 0.7(3) × 10−6 Å−2 for the 10 mol % bilayer. A weak decreasing trend in the thickness of the head groups may be observed from 9.1(7) Å for the normal bilayer, 8.2(9) Å for the 6 mol %, and 7.9(6) Å for the 10 mol % d31-POPC tethered bilayer. Electrochemical Impedance Spectroscopy. EIS was used to analyze the effect of oxidative damage on the dielectric properties of tethered bilayers. The EIS data were modeled using an electrical equivalent circuit (EEC) composed of resistors (R) and constant phase elements (CPE) shown in the inset of Figure 4. CPEs are used to describe nonideal capacitors, where the reported CPE coefficient describes the capacitance and α describes the homogeneity of the bilayer and can vary between 1 (for an ideal capacitor) and 0 (for an ideal resistor). The EEC has been used previously to model tethered bilayers32 where the CPEbilayer describes the bilayer as a near ideal insulating dielectric layer and the Rdefects and CPEdefects account for any defects in the bilayer. Rsolution (the resistance of the electrolyte and electrical leads) and Cstray (capacitance at the Au−tether interface) are not reported herein. A typical EIS Bode plot of a POPC tethered bilayer before and after oxidation with 0.5 mM Fe(II) and 5 mM ascorbate is shown in Figure 4 with the EIS model parameters (averaged

Table 3. Fitted Parameter Values for a d31-POPC Tethered Bilayer, and the Same Bilayer after Oxidation with 0.5 mM Fe(II)/5 mM Ascorbatea

thickness/Å hydrocarbon chains outer head groups nSLD/10‑6 Å‑2 inner chains outer chains outer head groups volume fraction of hydrocarbon chains outer head groups

unoxidized bilayer

after manual oxidation

6 mol % PoxnoPC

10 mol % PoxnoPC

31(2)

29(2)

31(2)

30(2)

9.1(7)

8.2(8)

8.2(9)

7.9(6)

1.3(1) 2.6(1) 1.2(1)

2.2(3) 2.8(2) 1.2(2)

0.6(1) 2.5(1) 1.0(3)

0.5(2) 2.4(2) 0.7(3)

water/% 2(2)

6(4)

4(3)

6(2)

46(7)

60(10)

60(10)

51(4)

a

d31-POPC tethered bilayers containing 6 and 10 mol % PoxnoPC are also shown. Uncertainties (one standard deviation) in the last digit are reported in parentheses.

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ascorbate, or containing different amounts of PoxnoPC, are shown in Figure 5 with the corresponding values of the dspacings in Table 5. These show that while the Fe(II)/ ascorbate oxidized bilayers are minimally affected in terms of structure there is a significant decrease in the d-spacing observed with increasing amount of PoxnoPC from 52.1(1) Å for the unoxidized POPC multibilayer stack to 49.1(1) Å for the 30 mol % PoxnoPC containing POPC multibilayer stack. An increase in the nSLD of the head group layer (including water) of the multibilayer stacks is also observed with incorporation of PoxnoPC.

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DISCUSSION The structural and functional properties of model phospholipid bilayer membranes are clearly affected by oxidative damage using both of the methods of simulating oxidative damage probed here, namely the incorporation of synthetic oxidized phospholipids or the use of chemical oxidation. The oxidation was assessed using tethered membranes, membrane bilayer stacks, and physisorbed lipid bilayers, and consistent trends have been found among them. Following oxidation of a tethered monounsaturated phospholipid bilayer with Fe(II) and ascorbate, a loss of hydrocarbon material was observed using ATR from physisorbed liposomes that was related to the concentration of the chemical oxidant. The rate of this hydrocarbon loss was also related to the concentration of the oxidant, with the low chemical oxidant samples significantly slower to change than the 50 mM/500 mM Fe(II)/ascorbate sample. Similarly, the modeled nSLD of the partially deuterated hydrocarbon chain layers of tethered bilayers increased after oxidation, implying loss of material from the hydrogenous unsaturated chain. Although the mechanism of phospholipid oxidation is still debated, this is consistent with models of bilayer oxidation via radical chain reactions leading to chain truncation at the site of the unsaturation,13 with loss of the short hydrophilic truncated section into the aqueous solution. A similar cleavage of the sn-2 hydrocarbon chain of POPC below the alkene group has been shown by Thompson et al.,33 who observed a decrease in the neutron reflectivity after exposure to ozone of a POPC monolayer, which had the terminal C9 portion of the sn-2 hydrocarbon chain deuterated. PoxnoPC is a known product of Fe(II) and ascorbate initiated phospholipid oxidation,34 and is used here as a biologically relevant14 damaged phospholipid with a truncated sn-2 hydrocarbon chain. PoxnoPC or similar oxidized products with are likely to be present in the Fe(II) and ascorbate oxidized POPC physisorbed liposomes that we have examined here. Increasing the amount of PoxnoPC present in physisorbed liposomes also decreases the amount of hydrocarbon material present in the bilayer, as observed in the chemically oxidized samples above. Note that the ATR spectra of the physisorbed liposomes containing PoxnoPC do not show evidence of the molecules aldehyde groups, highlighting the insensitivity of these measurements to the oxygen-containing moieties also expected to be present in the Fe(II) and ascorbate oxidized liposomes. A decrease in the modeled nSLD, reduction in the water content of the head group region, and slight decrease in the thickness of the head groups of the PoxnoPC bilayer was observed compared to a pure POPC bilayer. Concurrently, a decrease in the d-spacing of the multibilayer stacks was observed. These observations are consistent with previous

Figure 4. Representative Bode plots of an unoxidized POPC tethered bilayer (black), a POPC tethered bilayer after oxidation with 0.5 mM Fe(I) and 5 mM ascorbate (red), and a 30 mol % PoxnoPC containing POPC tethered bilayer (blue). Filled circles represent the absolute impedance (|Z|) and open squares represent the phase shift (θ). The EEC model fits are shown as solid lines.

over seven samples) shown in Table 4. The unoxidized POPC tethered bilayers had similar properties to those previously Table 4. Average EIS Model Parameter Values for Seven Repeats Each of Unoxidized POPC Tethered Bilayers, 0.5 mM Fe(II) and 5 mM Ascorbate Oxidized POPC Tethered Bilayers, and a POPC Tethered Bilayer Containing 30 mol % PoxnoPCa unoxidized POPC bilayer Rdefect/kΩ cm2 CPEdefect/ μ F cm−2 s(α−1) αdefect CPEbilayer/ μ F cm−2 s(α−1) α bilayer a

Fe(II) and ascorbate oxidized POPC bilayer

30 mol % PoxnoPC POPC bilayer

60(20) 6(1)

80(40) 5(1)

33(8) 9(1)

0.5(1) 1.0(1)

0.6(1) 1.3(5)

0.8(1) 1.3(1)

0.98(1)

0.97(2)

0.97(1)

Uncertainties in the last digit are reported in parentheses.

reported indicating a complete bilayer.32 Following oxidation, it is clear from the measured spectrum that a simple EEC is no longer a good representation of the oxidized bilayer−indicative of the inhomogeneity of the surface. When modeled with the simple EEC, the best model showed increases in the Rdefects to 80(40) kΩ cm2, αdefects to 0.6(1), CPEbilayer to 1.3(5) μF cm−2 s(α‑1) and a decrease in the αbilayer to 0.97(2). An EIS Bode plot of a POPC tethered bilayer containing 30 mol % PoxnoPC is shown in Figure 4 with the corresponding model parameter values in Table 4. Incorporation of 30 mol % PoxnoPC into the tethered bilayer caused a decrease in the Rdefects to 33(8) kΩ cm2, and the αbilayer to 0.97(1), with an increase in αdefects to 0.8(1) and in the CPEbilayer to 1.3(1) μF cm−2 s(α‑1). In contrast to the case with the Fe/ascorbate oxidation, the bilayer is still well-modeled by the EEC. Neutron Diffraction. ND was employed to give structural characterization of the effect of oxidative damage on the structure of multibilayer stacks at higher resolution than available from reflectometry. The nSLD profiles of multibilayer stacks oxidized with different concentrations of Fe(II) and 12684

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Figure 5. Neutron diffraction from hydrated multi-bilayer stacks (only the 100% H2O hydrated samples are shown for clarity) and the corresponding nSLD profiles. (A and C) Unoxidized POPC multi-bilayer stacks (black) and POPC multibilayer stacks oxidized with 0.5 mM/5 mM (red), 5 mM/ 50 mM (blue), and 50 mM/500 mM (green) Fe(II) and ascorbate. (B and D) POPC multibilayer stacks containing 0 mol % (black), 5 mol % (red), 10 mol % (blue), and 30 mol % (green) PoxnoPC.

However, no equivalent decrease in the thickness of the bilayer was observed for chemically oxidized bilayers, either with NR or ND, despite similar phospholipid truncation. A potential explanation of this can be seen in the significant increase in the order of the hydrocarbon chains of POPC physisorbed liposomes observed with ATR after oxidation with the highest tested concentrations of Fe(II) and ascorbate. The relative order of the liposomes determined by ATR is a measure of the rate and amplitude of motion of the hydrocarbon chains.28,31 An increase in order following oxidation similar to that observed here has previously been attributed to “solidification” of the hydrocarbon chains possibly due to crosslinking of the oxidized phospholipids.8 The formation of crosslinked oxidized species would hinder inversion of the sn-2 hydrocarbon chain of PoxnoPC (or other similar oxidized species) as the cross-linking occurs between the oxidized alkene position on the sn-2 chain of neighboring phospholipids. The formation of cross-linked species would also prevent interdigitation of the sn-1 hydrocarbon chains and therefore prevent a decrease in the thickness of the bilayers. Similarly, no increase in the volume fraction of water was observed in the hydrocarbon chains using NR, as cross-linking would prevent water penetrating the bilayer. Conversely, incorporation of PoxnoPC into POPC physisorbed liposomes resulted in a decrease in the order of the hydrocarbon chains determined by ATR. This implies a more fluid bilayer with an increase in the motion of the hydrocarbon chains, as would be expected following inversion of the sn-2 hydrocarbon chains and disruption of the packing of the sn-1 hydrocarbon chains. This increased disorder in the hydro-

Table 5. d-Spacing Values of an Unoxidized POPC MultiBilayer Stack, Fe(II) and Ascorbate Oxidized Multi-Bilayer Stacks, and PoxnoPC Containing Multi-Bilayer Stacksa oxidant concentration

d-spacing/Å

unoxidized POPC multibilayer stack average of three samples 52.1 Fe(II)/ascorbate oxidized POPC multibilayer stacks 0.5 mM/5 mM 52.2 5 mM/50 mM 52.1 50 mM/500 mM 52.3 PoxnoPC incorporated POPC multibilayer stacks 5 mol % PoxnoPC 51.4 10 mol % PoxnoPC 50.1 30 mol % PoxnoPC 49.1 a

All values have an uncertainty of ±0.1 Å.

reports that the truncated aldehyde terminated sn-2 chain of PoxnoPC inverts such that the polar aldehyde group is located near the hydrated head group region displacing some water from this area.11,35,36 This inverted conformation also creates a void in the hydrocarbon chains resulting in interdigitation of the sn-1 chains decreasing the bilayer thickness, as predicted with molecular dynamic simulations.35 Overall, the bilayer is less tightly packed than the undamaged membrane. This is consistent with the previously observed large increase in the passive permeability of phospholipid vesicle membranes on introduction of PoxnoPC,37 or the increase in lipid flip-flop between layers.12 12685

DOI: 10.1021/acs.langmuir.5b02458 Langmuir 2015, 31, 12679−12687

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Langmuir

biological mechanisms of response to oxidative stress and repair, to give insight into some of the implications of lipid oxidation at a molecular level.

carbon change region also leads to an increase in the volume fraction of water and a decrease in the nSLD of the hydrocarbon chains observed with NR. This agrees with previous findings that have reported an increase in the volume fraction of water in the hydrocarbon chains and an increase in the average area per phospholipid with increasing amount of PoxnoPC incorporated.36 Based on the findings of this study, although both types of membrane are quite different from unperturbed phospholipid membranes, biomimetic membranes that have been oxidized in situ using Fe(II) and ascorbate have vastly different properties to biomimetic membranes containing PoxnoPC. Oxidation using 0.5 mM Fe(II) and 5 mM ascorbate had a mild effect on the properties of the membranes whereas incorporation of 5 to 30 mol % PoxnoPC had a greater and more systematic effect. The differences affect both the head and chain group regions of the lipid membranes, and are expected to mean that the membranes function quite differently in their interactions with membrane-active proteins. The difference in the observed properties between the types of oxidized membranes may be explained by several factors. First, it is clear that as a monounsaturated phospholipid POPC is not as susceptible to oxidation as polyunsaturated phospholipids. Several studies have reported small and even no changes to POPC after various oxidation techniques.38 Our findings show that indeed, except at the highest concentration of oxidant added, the changes in the physical properties of the Fe(II)/ascorbate oxidized layers are very subtle and challenging to resolve. Although the concentration of Fe(II) and ascorbate chosen for this study was at the higher end of the reported oxidations in the literature, it had a mild effect on the membrane properties compared to incorporation of PoxnoPC, and concentrations of PoxnoPC as high as the 6 mol % used are almost certainly not found in the chemically modified membranes. Nevertheless, there are resolvable changes in the physical properties of the membrane, even at the lowest chemical oxidation concentration in our measurements, which raises the question of what is the smallest level of oxidative stress for which the changes are significant in biological systems. A second explanation for the difference between the model membrane systems is that in situ oxidation is known to produce a wide range of oxidized phospholipids, which may show synergistic effects such as the cross-linking observed for the chemically oxidized bilayers. While these effects will never be observed for biomimetic systems with pure additives, such as PoxnoPC, there are clear benefits to being able to observe the individual impacts of each component. Indeed, as shown in the measurements of PoxnoPC-containing bilayers, very high concentrations of the additive can be added to the bilayer to augment the additives signal, while still maintaining the integrity of the bilayer. A fundamental question that remains from this study is how the observed physical and structural changes of the bilayer on oxidation, or addition of oxidation-products, affects the functioning of the membrane in a biological role. It has previously been shown in many studies that biomimetic membrane systems can support normal biological functioning of membrane-active proteins. We are extending our work to determine how the membrane perturbations observed due to oxidative damage to lipids changes the interactions of the membrane with a selection of membrane-active proteins. We will particularly focus on proteins thought to be connected to

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CONCLUSIONS We have measured the structural and functional properties of phospholipid membranes under the impact of damage due to oxidation, where a normal biomimetic phospholipid membrane is compared with one exposed to phospholipid oxidation due to a commonly used Fe(II)/ascorbate chemical oxidation system or one incorporating the synthetic oxidized phospholipid, PoxnoPC. We have found that oxidative damage changes the structure and electrical characteristics of the damaged biomimetic membranes compared to the undamaged biomimetic membrane. Moreover, we conclude that the use of synthetic “oxidized” phospholipids such as PoxnoPC to examine the properties of chemically oxidized cellular membranes (as would be found in vivo) requires the amount of synthetic phospholipid added to be carefully considered to match the effects of chemical oxidation. They are, however, useful for determining the effect of individual oxidized phospholipids on the properties of the oxidized bilayer which may collectively help decipher the properties of oxidized membranes in vivo. Oxidized phospholipids are also thought to play important roles in various biological activities, such as mediators of disease. We have shown that the membrane properties are significantly modified due the presence of oxidative stress compared to a normal biomimetic membrane, and predict that this will also impact the functioning of membrane-active proteins. Pure synthetic “oxidized” phospholipids will be important in these tests to determine the importance of specific, rather than general, interactions mediating protein behavior.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by a Fellowship (D.J.M.) and a Post Graduate Research Award (J.J.K.) from the Australian Institute of Nuclear Science and Engineering and by the Marsden Fund of the Royal Society of NZ. Travel was supported in part by an AINSE Research Award. Neutron beam time was awarded under an ANSTO program proposal (PP1594). We wish to gratefully acknowledge Dr David Vanderah (NIST) for the supply of WC14 and HC18, and Dr. Frank Heinrich (NIST), Dr. Michel Nieuwoldt (University of Auckland), and Dr Rob Dalgeish (ISIS) for assistance and valuable discussions.

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ABBREVIATIONS ATR, attenuated total reflectance infrared spectroscopy; EIS, electrochemical impedance spectroscopy; d31-POPC, 1-palmitoyl-d31-2-oleoyl-sn-glycero-3-phosphocholine; NR, neutron reflectometry; POPC, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine; PoxnoPC, 1-palmitoyl-2-(9′-oxo-nonanoyl)-snglycero-3-phosphocholine; Tris, tris(hydroxymethyl)-aminomethane 12686

DOI: 10.1021/acs.langmuir.5b02458 Langmuir 2015, 31, 12679−12687

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Langmuir

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DOI: 10.1021/acs.langmuir.5b02458 Langmuir 2015, 31, 12679−12687