Addition of Cleaved Tail Fragments during Lipid Oxidation Stabilizes

Article pubs.acs.org/Langmuir

Addition of Cleaved Tail Fragments during Lipid Oxidation Stabilizes Membrane Permeability Behavior Kristina A. Runas,† Shiv J. Acharya,‡ Jacob J. Schmidt,‡ and Noah Malmstadt*,† †

Mork Family Department of Chemical Engineering and Materials Science University of Southern California, Los Angeles, California 90089, United States ‡ Department of Bioengineering University of California, Los Angeles, Los Angeles, California 90095, United States S Supporting Information *

ABSTRACT: Lipid oxidation has been linked to plasma membrane damage leading to cell death. In previous work, we examined the effect of oxidation on bilayer permeability by replacing defined amounts of an unsaturated lipid species with the corresponding phospholipid product that would result from oxidative tail scission of that species. This study adds the cleaved tail fragment, better mimicking the chemical results of oxidation. Permeability of PEG12-NBD, a small, uncharged molecule, was measured for vesicles with oxidation concentration corresponding to between 0 and 18 mol % of total lipid content. Permeability was measured using a microfluidic trap to capture the vesicles and spinning disk confocal microscopy (SDCM) to measure the transport of fluorescent PEG12-NBD at the equatorial plane. The thicknesses of lipid bilayers containing oxidized species were estimated by measuring capacitance of a black lipid membrane while simultaneously measuring bilayer area. We found that relative to chemically modeled oxidized bilayers without tail fragments, bilayers containing cleaved tail groups were less permeable for the same degree of oxidation. Curiously, membrane capacitance measurements indicated that the addition of tail fragments to chemically modeled oxidized bilayers also thinned these bilayers relative to samples with no tail fragments; in other words, the more permeable membranes were thicker. Above 12.5% chemically modeled oxidation, compositions both with and without the cleaved tail groups showed pore formation. This work highlights the complexity of the relationship between chemically modeled lipid bilayer oxidation and cell membrane properties.

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INTRODUCTION Lipid oxidation is associated with numerous physiological conditions including atherosclerosis1 and aging.2 A key set of lipid oxidation reactions include those involving an unsaturated lipid tail group and a reactive oxygen species. The presence of reactive oxygen species in biological systems have been linked to exposure to radiation,3 air pollutant inhalation,3 signaling processes,4 and cell respiration and metabolism.5 Significant membrane effects such as increases in membrane surface area,6,7 the formation of tubules and membrane budding,8 the promotion of phase separation,9,10 and decreased membrane fluidity11 have also been noted. However, the impact of lipid oxidation on passive membrane permeability has not been extensively studied. As passive transport is a generic pathway by which drugs and environmental toxins can enter a cell,12,13 the impact of lipid oxidation on passive permeability is significant to understanding how this process affects membrane barrier function. We previously showed by chemically modeling lipid oxidation with the replacement of an unsaturated lipid with its corresponding oxidation product that a radical increase in membrane permeability occurs with only a 2.5% increase in chemically modeled oxidation.14 The previous study used a © 2015 American Chemical Society

known pair of unsaturated lipidPLinPC (1-palmitoyl-2linoleoyl-sn-glycero-3-phosphocholine)and its corresponding oxidized productPOxnoPC (1-palmitoyl-2-(9′-oxo-nonanoyl)-sn-glycero-3-phosphocholine).3,15 This technique is an effective method of controlling membrane composition to correlate oxidation to permeability, however, the previously mimicked pathway was incomplete. The oxidation pathway of PLinPC has been shown to result in lipid tail scission yielding POxnoPC as well as a short chain aldehyde as the cleaved tail group.16 This aldehyde is a demonstrated product of the autoxidation of polyunsaturated lipids,17,18 and can be further oxidized to a carboxylic acid. A representative example schematic of the reaction process is shown in Figure 1. Both of the cleaved tail fragments are expected to be surface-active. As the presence of surfactants in model membranes has been associated with the formation of both transient and stable pores,19 phase destabilization20 or transition,21 and the formation of micelles,20 the introduction of these cleaved tail groups are integral to understanding the effects of lipid Received: August 10, 2015 Revised: November 13, 2015 Published: December 24, 2015 779

DOI: 10.1021/acs.langmuir.5b02980 Langmuir 2016, 32, 779−786

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Langmuir

Figure 1. Reaction schematic of the oxidation process of (1) PLinPC into (2) POxnoPC and (3) hexanal, then the second oxidation step of (3) hexanal into (4) hexanoic acid.

step results in termination of the tail group, releasing the tail fragment. This study focuses on evaluating the effect of mimicking lipid oxidation with the addition of the cleaved tail fragments on passive transport. A fluorescent short chain poly(ethylene glycol) molecule (PEG12-NBD) was chosen to measure permeability. PEG12-NBD is an uncharged, hydrophilic molecule which was previously characterized by Li et al. in 2010 using SDCM to measure its octanol/water partition coefficient (0.20 ± 0.02), molecular weight (821.4 g/mol), and diffusivity (3.6 × 10−6 cm2/s).31 Choosing an uncharged species simplifies the transport process, as a charged molecule can interact with lipid headgroups and demonstrate a pH- or charge-dependent permeability.33 By selecting a molecule with these simplifying properties, we can focus on determining the effect of the cleaved tail fragments formed during lipid oxidation.

oxidation on membrane permeability. In our previous work examining the effects of mimicked oxidation on bilayer permeability, only the POxnoPC product was included;14 here, we examine the effect on permeability of including the oxidation products corresponding to the cleaved tail group. It is expected that bilayer permeability should be related to bilayer thickness. Oxidation has been linked with both decreased bilayer thickness and increased lipid tail group interdigitation.22−24 The difference between a noninterdigitated bilayer and an interdigitated one can be examined by looking at the amount of bilayer thickness corresponding to the tail groups of each leaflet.25 For a noninterdigitated bilayer, the tails of each leaflet are half of the bilayer thickness, with no overlap between leaflets. In interdigitated bilayers, the tail groups from each leaflet overlap, where the amount of overlap corresponds to the amount of interdigitation. Previous studies have shown that increasing bilayer thickness yields a linear decrease in permeability.26 The relationship between level of interdigitation and permeation has also been examined, and demonstrated that increasing interdigitation in gel-phase membranes leads to increased permeability.27 In the present report, we used an electrophysiological technique to observe changes in bilayer thickness upon chemically modeled oxidation.28−30 Giant unilamellar vesicles (GUVs) were used as model membranes to measure passive transport. The test system is based on the use of spinning disk confocal microscopy (SDCM) to measure permeation of a fluorescent test solute in a microfluidic channel.14,31,32 The GUVs were immobilized in a simple microfluidic Y-channel using a biotin−avidin interaction, and the test species was added to the channel using a rapid microfluidic buffer exchange. The transport process was then imaged until it reached equilibrium. The oxidation pathway was mimicked by varying the molar ratio of PLinPC to its oxidation products (POxnoPC and one of the two tail fragments, hexanal or hexanoic acid) in GUVs.16 The molecular structures are shown in Figure 1. POxnoPC and the tail fragment were added in equimolar amounts. By varying the ratio of the unsaturated lipid to the oxidation products, we can simulate different points along the oxidation pathway. It has previously been demonstrated that POxnoPC is a major termination product of PLinPC after three oxidative steps.3 The first two steps involve abstracting a hydrogen next to the double bond, then a reaction between the carbon-centered radicals with singlet oxygen to form peroxyl radicals. The third and final

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EXPERIMENTAL SECTION

Reagents Used. 1,2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine (PLinPC), cholesterol (Chol), 1-palmitoyl-2-(9′-oxo-nonanoyl)-snglycero-3-phosphocholine (POxnoPC), 1,2-dihexadecanoyl-sn-glycero3-phosphoethanolamine-N-(cap biotinyl) (biotin-DPPE), and 1,2dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (rhodamine-DPPE) were purchased from Avanti Polar Lipids. Amino-dPEG12-alcohol was purchased from Quanta Biodesign, and poly(dimethylsiloxane) (PDMS) was purchased from Dow Chemical. Succinimidyl 6-(N-(7-nitrobenz-3-oxa-1,3-diazol-4yl)amino)hexanoate (NBD NHS-ester) and avidin were obtained from Invitrogen. All other chemicals were purchased from SigmaAldrich. Preparation of Test Molecule. The reaction protocol is discussed in Runas and Malmstadt.14 Briefly, 1:1 molar ratio of amine-terminated poly(ethylene glycol) alcohol to NBD NHS-ester was reacted in chloroform to form the PEG12-NBD. The reaction was run at 45 °C for 2 h, then at room temperature overnight. High performance liquid chromatography was used to separate the reaction products. PEG12NBD structure was then confirmed using NMR spectroscopy. Preparation of Microfluidic Devices. Coverslips were cleaned and devices were formed in a manner similar to that described previously.14 Coverslips were cleaned by sonication in Milli-Q water (Millipore) at 80 °C for 30 min, then submerged in sulfuric acid with NoChromix (Sigma-Aldrich) overnight. The Y-channel mold was fabricated using a 3D printed acrylonitrile butadiene styrene. The channel depth is 1 mm, with a width of 1 mm, and a length of 1 cm. The microfluidic channel was formed from PDMS using standard 780

DOI: 10.1021/acs.langmuir.5b02980 Langmuir 2016, 32, 779−786

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Langmuir polymer molding techniques.34 The PDMS was then bonded to a #1 coverslip with a corona treatment (BD-20AC, Electro-Technic Products). The coverslip surface was functionalized using a biotinylated-PEG-Silane solution. A 1 mg/mL solution of avidin in water was added to the channel for 30 min, then flushed with 200 mM glucose and 4 mM HEPES buffer at pH 7.0 prior to addition of the GUVs. The avidin bonded to the PEG-Silane, and vesicles containing 6 mol % biotin-DPPE were captured using the biotin−avidin interaction at the coverslip surface. A schematic of the microfluidic device is shown in Figure 2.

Transport Experiments. After all unbound vesicles were flushed from the channel, a single unilamellar vesicle of diameter greater than 10 μm was selected for observation. A buffer solution of 200 mM glucose, 4 mM HEPES, and 5 μM PEG12-NBD at pH 7.0 was added to the channel using a syringe pump. A flow rate of 5 mL/h was chosen as the maximum flow rate that consistently did not detach or damage the GUVs. Spinning disk confocal microscopy with a Yokogawa CSUX confocal head on a Nikon TI-E inverted microscope was used to observe the transport process. Illumination sources were 50 mW solid-state lasers at either 491 or 561 nm. To select a unilamellar GUV for observation, the rhodamine-DPPE was excited at 561 nm with emission centered at 595 nm. PEG12-NBD transport was imaged with excitation at 491 nm and emission centered at 525 nm. While imaging the transport process, the samples were only illuminated with the 491 nm laser. Images were collected at a regular interval during the buffer exchange to visualize the fluorescent species crossing the GUV membrane. Measurement of Bilayer Thickness. Lipid bilayer membranes were formed on clear acrylic chips containing three collinear wells, as described previously.37 The outer wells were connected to each other through a channel on the bottom of the chip, and the center well was connected to this channel through a 150−250 μm circular aperture in a 75 μm thick sheet of delrin (McMaster-Carr). The chip was designed such that the aperture in the delrin sheet, and therefore the lipid bilayer, were oriented horizontally and easily imaged through the clear acrylic with an inverted microscope. All lipids were prepared at 20 mg/mL in decane and allowed to shake vigorously for 1 h prior to use. 1 M KCl, 10 mM TRIS-HCl, pH 8 was used as the aqueous phase in all experiments. Lipid bilayer formation was adapted from Mueller et al.38 Lipid in decane was applied to the aperture before filling the chambers with aqueous solution. The organic solvent coating is then physically manipulated with a glass rod until a bilayer is formed, as observed optically or electrically. Ag/AgCl electrodes were placed in one outer well and the center well and connected to a custom current to voltage amplifier,39 made from an OPA111 op-amp (Texas Instruments) and a 1 GΩ feedback resistor. The output of this circuit was captured by a LabVIEW (National Instruments) computer data acquisition system. Bilayer capacitance was determined by applying a 40 mV 8 Hz triangle wave to the electrodes while measuring the resultant current. The entire apparatus was placed in a small aluminum box with 4 in. square central cutouts in the top and bottom planes and mounted on a Leica DMIRBE inverting microscope. The box was used as a Faraday cage, to eliminate background noise. Images were recorded with an Optronics TEC-470 CCD camera and bilayer area was quantified through image analysis using ImageJ. In the case of higher levels of oxidation, the bilayers were only stable for a short period of time. In these cases, the Faraday cage could not be replaced prior to bilayer rupture. For these samples, filtering the background noise was necessary to analyze membrane capacitance. Further discussion of the filtering techniques used can be found in the Supporting Information (SI). Pore Formation Experiments. GUVs were imaged in a solution of 1 mg/mL fluorescein-dextran in 200 mM glucose, 4 mM HEPES buffer at pH 7.0 to examine the formation of pores in the membrane, as fluorescein dextran cannot cross the membrane without the presence of pores. Two molecular weights of fluorescein-dextran, 40 kDa and 2000 kDa, were used to estimate the size of the pores. Image Processing. Image analysis was performed using Matlab (The MathWorks, Inc.). Images were analyzed by first removing background signal due to dark current noise, then each image was flat fielded according to the procedure detailed by Runas and Malmstadt.14 Data Analysis. To account for the imperfect exclusion of light from outside the focal plane, the technique established by Runas and Malmstadt was used to remove the out of plane light contribution for a vesicle with varying internal concentration.14 As shown in Figure 3, prior to permeation, the intensity profile inside of the vesicle is not flat. Pinhole crosstalk causes a curved intensity profile by increasing measured intensity values around the edges of the vesicle, where the

Figure 2. Schematic of the microfluidic device, a simple Y-channel design with two inlets and a single outlet. The first inlet is for 200 mM glucose, 4 mM HEPES buffer at pH 7.0, the second is for the glucose buffer containing 5 μM PEG12-NBD. The glass surface was functionalized with biotinylated-PEG-silane. After channel preparation, a 1 mg/mL avidin solution was added to the channel for 30 min. As shown in the inset, vesicles are captured in this device using a biotin− avidin interaction. GUV Preparation and Observation. The electroformation technique introduced by Angelova et al. was used to prepare GUVs.35,36 Prior to oxidation, vesicle composition was chosen to be 42.5:42.5:15 molar ratio of DMPC/PLinPC/Chol. Cholesterol was included in the membrane to decrease permeability to a measurable rate, even in cases where oxidized species are present. 0.5 mol % αtocopherol was added to the stock solution of PLinPC to act as an oxygen scavenger and prevent additional oxidation during electroformation and imaging. 0.01 mol % rhodamine-DPPE was added to visualize the membrane. To attach the vesicles to the coverslip, we added 6 mol % biotin-DPPE. The lipids were dissolved in chloroform, then a thin film was spread on the ITO-coated surface of the glass. The film was dried in a vacuum for at least 2 h prior to rehydration in 200 mM sucrose, 4 mM HEPES buffer at pH 7.0. Electroformation was performed with an oscillating signal of 1 V at 10 Hz for 2 h at room temperature. Vesicle compositions are shown in Table 1. GUVs were immediately transferred to the microfluidic channel after electroformation. Uncaptured vesicles were gently flushed from the system using 200 mM glucose, 4 mM HEPES buffer at pH 7.0 using a syringe pump.

Table 1. Vesicle Compositions Studied, In Terms of Mole Ratioa

a

mol DMPC

mol PLinPC

mol POxnoPC

mol chol

mol tail

42.5 42.5 42.5 42.5 42.5 42.5 42.5 42.5

42.5 40 37.5 35 32.5 30 27.5 24.5

0 2.5 5 7.5 10 12.5 15 18

15 15 15 15 15 15 15 15

0 2.5 5 7.5 10 12.5 15 18

Tail group is either hexanal or hexanoic acid. 781

DOI: 10.1021/acs.langmuir.5b02980 Langmuir 2016, 32, 779−786

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Figure 3. Intensity profile across a vesicle immersed in 5 μM PEG12NBD. This image has been corrected for background light and flatfielded to correct for uneven illumination. Prior to permeation, the intensity profile inside of the vesicle should be flat. Pinhole crosstalk causes increased intensity measurements near the edges of the vesicle due to a shorter light path. Data analysis techniques discussed by Runas and Malmstadt were used to account for the intensity contribution due to pinhole crosstalk.14 Figure 4. Permeability vs percent chemically modeled oxidation for (a) POxnoPC and hexanal and (b) POxnoPC and hexanoic acid as the two oxidation products of PLinPC. For both cleaved tail products, there is no difference between 0 and 2.5 mol % chemically modeled oxidation. However, there is an increase in permeability between 2.5 and 7.5 mol % chemically modeled oxidation, with a slight decrease in permeability at 10 mol % chemically modeled oxidation for both cleaved tail species. Each data point represents the permeability of a single GUV. Each cluster of data points represents a single molar composition, with several vesicles measured with the same percentage of oxidized lipid and tail fragment. The uncertainty on each data point was determined from a 95% confidence interval in the best fit of the finite difference model.

light path is shorter. It was previously established that the relationship between pinhole crosstalk and internal fluorophore concentration is linear for a constant vesicle size.14 The data were corrected accordingly. Permeability was calculated from the intensity vs time images based on a finite difference model of Fick’s Second Law in spherical coordinates. Assuming no reaction, symmetry in angular dimensions, a spherical vesicle, and that diffusion is significantly higher contributor than convection, the equation was solved in the time and space using the finite difference approach discussed by Runas and Malmstadt.14 The boundary conditions were set at three points: the vesicle center, the membrane, and an exterior point. The experimental data were fit to the model results by performing a χ2 minimization between the model output and the measured intensity data with permeability as the only free parameter. A χ2 landscape with a confidence interval of 95% was used to determine uncertainties for the permeability of each measured vesicle.

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RESULTS AND DISCUSSION As established in Runas and Malmstadt, vesicle composition was chosen to prevent phase separation prior to and after the addition of the oxidized species.14 PLinPC and POxnoPC were chosen as a commercially available unsaturated and oxidized pair. DMPC was added to provide a nonoxidizing background, and cholesterol was added to decrease membrane permeability such that the transport process was sufficiently slow to measure precisely. The cholesterol content was tailored such that vesicles did not show phase separation after addition of POxnoPC. Membrane structure and lack of phase separation for each GUV was confirmed prior to each transport experiment. GUV composition was varied to model the oxidation process between 0 and 18 mol % total oxidation, to remain below the previously established poration limit.40 To mimic lipid

Figure 5. Average permeability for vesicles that modeled oxidation using POxnoPC only (squares), POxnoPC with hexanal (circles), and POxnoPC with hexanoic acid (triangles). Each data point represents a weighted average and uncertainty from the data presented in Figure 4.

oxidation, portions of the PLinPC were replaced with the corresponding oxidized product (POxnoPC), and one of the 782

DOI: 10.1021/acs.langmuir.5b02980 Langmuir 2016, 32, 779−786

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Figure 6. Specific capacitance vs percent chemically modeled oxidation for bilayers containing POxnoPC alone (squares), POxnoPC and hexanoic acid (triangles), or hexanoic acid alone (diamonds) as the oxidation product. To determine specific capacitance, a time series of capacitance points were measured using the apparatus. These time points were averaged for a single membrane to determine the specific capacticance, with its uncertainty reported as the standard deviation of the time series data. This experiment was repeated for three membranes of identical composition. For a given membrane composition, the data collected for the three membranes were combined using a weighted average and uncertainty. The averaged data for each percent oxidation are shown.

Figure 7. SDCM images showing vesicle formation for 12.5−18% chemically modeled oxidation. These images, taken with the 561 nm laser, excite the rhodamine-DPPE present in the membrane. As such, these images show vesicle structure and formation at each composition. In addition to small spherical vesicles (